Mammalian Cytochrome P450 Enzymes Catalyze the Phenol-coupling Step in Endogenous Morphine Biosynthesis

A cytochrome P450 (P450) enzyme in porcine liver that catalyzed the phenol-coupling reaction of the substrate (R)-reticuline to salutaridine was previously purified to homogeneity (Amann, T., Roos, P. H., Huh, H., and Zenk, M. H. (1995) Heterocycles 40, 425–440). This reaction was found to be catalyzed by human P450s 2D6 and 3A4 in the presence of (R)-reticuline and NADPH to yield not a single product, but rather (−)-isoboldine, (−)-corytuberine, (+)-pallidine, and salutaridine, the para-ortho coupled established precursor of morphine in the poppy plant and most likely also in mammals. (S)-Reticuline, a substrate of both P450 enzymes, yielded the phenol-coupled alkaloids (+)-isoboldine, (+)-corytuberine, (−)-pallidine, and sinoacutine; none of these serve as a morphine precursor. Catalytic efficiencies were similar for P450 2D6 and P450 3A4 in the presence of cytochrome b₅ with (R)-reticuline as substrate. The mechanism of phenol coupling is not yet established; however, we favor a single cycle of iron oxidation to yield salutaridine and the three other alkaloids from (R)-reticuline. The total yield of salutaridine formed can supply the 10 nm concentration of morphine found in human neuroblastoma cell cultures and in brain tissues of mice.Cytochrome P450 (P450)² enzymes catalyze the most versatile chemical reactions in nature (1). There is, however, a discrepancy between the plant and the animal kingdoms with regard to the sheer number of these biocatalysts. Whereas in a single model plant, Arabidopsis thaliana, there are to date 273 P450 proteins, in the human genome, only 57 of these proteins are present. Whereas plants and animals share a multitude of highly regio- and stereospecific O-demethylation reactions, more complex reactions such as phenol coupling are much more abundant in plants than in animals, especially in the alkaloid field (2–11). The proposal of Barton and Cohen (12) correlated the structure of specific plant alkaloids in terms of this reaction mechanism and gave mechanistic proposals of how these phenol-coupled products may possibly be biosynthesized in nature. The oxidation of phenols by one-electron transfer affords radicals, which, by radical pairing, form new C-C or C-O bonds either by intra- or intermolecular coupling. The first two examples that unequivocally demonstrated the formation of C-C and C-O bonds in a stereo- and regioselective manner in plant metabolism are catalyzed by specific P450-linked microsomal-bound plant enzymes (13). One of these enzymes was salutaridine synthase from Papaver somniferum (opium poppy) (14). This synthase catalyzes the intramolecular formation of the critical C12-C13 carbon bridge and is a key enzyme in morphine biosynthesis.The groups of Goldstein (15) and Spector (16) have published a number of reports over the past 25 years claiming that mammals are capable of synthesizing de novo traces of endogenous morphine. However, no convincing experimental data have been presented regarding the enzymes. Phenol-coupling reactions in mammals are extremely rare, and the only example described thus far is the formation of thyroxine, by radical pairing, in humans. If the key step of morphine synthesis, the formation of phenol-coupled salutaridine from (R)-reticuline, occurs in mammals then in analogy to plants, a P450 enzyme must be present (in mammals) to catalyze this reaction. In 1987, the first experiments were conducted in an attempt to discover the reaction by supplying uniformly labeled racemic [³H]reticuline in the presence of rat microsomes and NADPH to examine whether [³H]salutaridine can be formed under these conditions (15). A radioactive compound was formed in 1% yield and assumed to be the phenol-coupled product salutaridine. We later repeated this experiment using (R)-[N-¹⁴CH₃]reticuline and NADPH-fortified microsomes from pig, rat, cow, and sheep. We observed the formation of [N-¹⁴CH₃]salutaridine with the correct stereochemistry at carbon 9 (17). The enzyme from porcine liver was subsequently purified to homogeneity and the reaction product was characterized by mass spectrometry and physical parameters to be a product of a P450 enzyme, which we provisionally named “salutaridine synthase” (18). The aim of this report is the identification of the homogenous porcine P450 enzyme, its equivalent in humans, and the mechanism of the phenol-coupling reaction in the formation of precursors of morphine.     

Removal of Substrate Inhibition and Increase in Maximal Velocity in the Short Chain Dehydrogenase/Reductase Salutaridine Reductase Involved in Morphine Biosynthesis

Salutaridine reductase (SalR, EC 1.1.1.248) catalyzes the stereospecific reduction of salutaridine to 7(S)-salutaridinol in the biosynthesis of morphine. It belongs to a new, plant-specific class of short-chain dehydrogenases, which are characterized by their monomeric nature and increased length compared with related enzymes. Homology modeling and substrate docking suggested that additional amino acids form a novel α-helical element, which is involved in substrate binding. Site-directed mutagenesis and subsequent studies on enzyme kinetics revealed the importance of three residues in this element for substrate binding. Further replacement of eight additional residues led to the characterization of the entire substrate binding pocket. In addition, a specific role in salutaridine binding by either hydrogen bond formation or hydrophobic interactions was assigned to each amino acid. Substrate docking also revealed an alternative mode for salutaridine binding, which could explain the strong substrate inhibition of SalR. An alternate arrangement of salutaridine in the enzyme was corroborated by the effect of various amino acid substitutions on substrate inhibition. In most cases, the complete removal of substrate inhibition was accompanied by a substantial loss in enzyme activity. However, some mutations greatly reduced substrate inhibition while maintaining or even increasing the maximal velocity. Based on these results, a double mutant of SalR was created that exhibited the complete absence of substrate inhibition and higher activity compared with wild-type SalR.The benzylisoquinoline alkaloids (BIAs)³ comprise a large and diverse group of nitrogen-containing secondary metabolites with about 2500 compounds identified in plants (1). Among them are several important pharmaceuticals, such as the antimicrobials berberine and sanguinarine, and the vasodilator papaverine. The most prominent compounds of this class are the antitussive codeine, the analgesic morphine, and their biosynthetic precursor thebaine. The latter is used as the starting molecule for the production of a variety of semi-synthetic analgesics including oxycontin and buprenorphine. Pentacyclic morphinan alkaloids possess several chiral centers, which preclude chemical synthesis as an option for the efficient production of these widely used pharmaceuticals. Therefore, the worldwide supply of these narcotic compounds is still achieved by their isolation mainly from the opium poppy, Papaver somniferum L. With the availability of an increasing number of isolated genes encoding several pathway enzymes, recent interest has focused on the qualitative and quantitative modulation of the alkaloid profile in transgenic opium poppy plants (2–6), the production of BIAs in microbes (7, 8), and de novo synthesis by a combination of chemical and biochemical conversions. BIA biosynthesis begins with the condensation of the tyrosine-derived precursors dopamine and p-hydroxyphenylacetaldehyde to (S)-norcoclaurine (see Fig. 1) (1). Subsequent regiospecific O- and N-methylations and aromatic ring hydroxylation lead to (S)-reticuline, which is the central intermediate for almost all BIAs. For morphinan alkaloid biosynthesis, (S)-reticuline undergoes an inversion of stereochemistry to (R)-reticuline, followed by C-C phenol coupling catalyzed by a unique cytochrome P450-dependent monooxygenase to yield salutaridine. Subsequent stereospecific reduction to 7(S)-salutaridinol is required for the attachment of an acetyl moiety to produce salutaridinol-7-O-acetate, which spontaneously rearranges to thebaine (9). The O-demethylation of thebaine and the reduction of codeinone to codeine represent the penultimate steps in morphine biosynthesis. Cognate cDNAs have been isolated for all of the enzymes leading to (S)-reticuline, as well as those involved in the conversion of (R)-reticuline to salutaridine-7-O-acetate (1). Salutaridine reductase (SalR, EC 1.1.1.248) catalyzes the stereospecific, NADPH-dependent reduction of salutaridine to 7(S)-salutaridinol and is a member of the classical subgroup of the short chain dehydrogenase/reductase (SDR) protein family (10, 11). The main characteristics of this category of SDRs are the largely conserved TGXXXGhG motif for cofactor binding and the YXXXK motif, which together with an upstream Ser residue represent the catalytic center (12). In this catalytic triad, Lys forms hydrogen bonds with the ribose moiety of the cofactor, which itself is hydrogen bonded to Tyr. This hydrogen bond network is presumed to lower the pK_a of the Tyr hydroxyl group, which functions as the catalytic base. Ser has been suggested to either stabilize the substrate (13, 14) or to interact with Tyr (15). Additionally, an Asn residue has been proposed to stabilize the position of the Lys residue, thereby forming a proton relay system involving water (16). Most other members of the SDR protein family are categorized into three additional subgroups (i.e. divergent, intermediate, or complex) exhibiting different overall sizes and slight amino acid sequence variations in conserved regions (17). Non-classical SDRs predominantly consist of isomerases (EC 5.-.-.-), such as galactose epimerase, and lyases (EC 4.-.-.-), such as glucose dehydratase, whereas classical SDRs encompass oxidoreductases (EC 1.-.-.-), such as SalR. Although classical SDRs are typically multimeric, SalR is a monomer because of an additional stretch of 40 amino acids preceding the YXXXK catalytic motif. In porcine testicular carbonyl reductase, these amino acids form a helix blocking the dimer interface (18) and homology modeling revealed a similar feature in SalR (19). In contrast with porcine testicular carbonyl reductase, SalR exhibits an additional stretch of 40 amino acids that have only been detected in some SDRs from plants (11, 20–22). Although attempts to obtain a crystal structure for SalR have so far been unsuccessful, homology modeling using porcine testicular carbonyl reductase as a template produced a tertiary structure in which the additional amino acids form an additional helix (19). The subsequent docking of salutaridine into the active site of this model suggested the involvement of this structural element in substrate binding, which was supported by preliminary site-directed mutagenesis.Open in a separate windowFIGURE 1.Selected steps in morphine biosynthesis. Double arrows indicate the involvement of more than one enzyme. The SalR reaction is highlighted. HPAA, p-hydroxyphenylacetaldehyde; SalR, salutaridine reductase.SalR from the Persian poppy Papaver bracteatum L. shows strong substrate inhibition with a K_i around 150 μm (19). Substrate inhibition can substantially and negatively impact chemical engineering strategies by limiting the quantity of substrate that can be fed into a system, which reduces overall efficiency. The strong substrate inhibition exhibited by SalR could limit its biotechnological application in plant, microbial, or enzyme-based systems. To investigate the structural basis of substrate inhibition, we substituted all amino acids putatively involved in salutaridine binding and analyzed various kinetic parameters. Over the course of these experiments, substrate docking required modification because some mutations had unexpected consequences that did not fully agree with the original docking. The discrepancy was mainly due to the side chain arrangements of amino acids residing in the new helix. Precise prediction of this domain is difficult because of the lack of an equivalent crystal structure. In this report, we present a revised substrate docking for salutaridine into SalR, supported by comprehensive site-directed mutagenesis, which facilitated the creation of an enzyme variant devoid of substrate inhibition.     

Evidence for an Ionic Intermediate in the Transformation of Fatty Acid Hydroperoxide by a Catalase-related Allene Oxide Synthase from the Cyanobacterium Acaryochloris marina

Allene oxides are reactive epoxides biosynthesized from fatty acid hydroperoxides by specialized cytochrome P450s or by catalase-related hemoproteins. Here we cloned, expressed, and characterized a gene encoding a lipoxygenase-catalase/peroxidase fusion protein from Acaryochloris marina. We identified novel allene oxide synthase (AOS) activity and a by-product that provides evidence of the reaction mechanism. The fatty acids 18.4ω3 and 18.3ω3 are oxygenated to the 12R-hydroperoxide by the lipoxygenase domain and converted to the corresponding 12R,13-epoxy allene oxide by the catalase-related domain. Linoleic acid is oxygenated to its 9R-hydroperoxide and then, surprisingly, converted ∼70% to an epoxyalcohol identified spectroscopically and by chemical synthesis as 9R,10S-epoxy-13S-hydroxyoctadeca-11E-enoic acid and only ∼30% to the 9R,10-epoxy allene oxide. Experiments using oxygen-18-labeled 9R-hydroperoxide substrate and enzyme incubations conducted in H₂¹⁸O indicated that ∼72% of the oxygen in the epoxyalcohol 13S-hydroxyl arises from water, a finding that points to an ionic intermediate (epoxy allylic carbocation) during catalysis. AOS and epoxyalcohol synthase activities are mechanistically related, with a reacting intermediate undergoing a net hydrogen abstraction or hydroxylation, respectively. The existence of epoxy allylic carbocations in fatty acid transformations is widely implicated although for AOS reactions, without direct experimental support. Our findings place together in strong association the reactions of allene oxide synthesis and an ionic reaction intermediate in the AOS-catalyzed transformation.A diverse spectrum of signaling molecules is biosynthesized in nature from polyunsaturated fatty acids, their peroxides, and further transformations of the fatty acid peroxides. The peroxides are formed by two classes of dioxygenase enzyme. The hemoprotein dioxygenases include prostaglandin H synthase (cyclooxygenase) in animals (1), α-dioxygenase in plants (2), and several linoleate dioxygenases in fungi (3–5). The non-heme iron lipoxygenases are even more widespread, being almost ubiquitous among organisms that contain polyunsaturated fatty acids (6–8). Although further biosynthetic transformation is sometimes accomplished by an additional catalytic activity of the initiating dioxygenase (e.g. leukotriene A₄ synthase (9) or aldehyde-synthesizing hydroperoxide cleaving activity (10) among the LOX² enzymes), commonly another distinct enzyme is used to rearrange or otherwise modify the reactive fatty acid peroxide intermediate. Two hemoprotein types are found that have become specialized for this biosynthetic role: cytochrome P450s and catalase-related enzymes.The fatty acid peroxide-metabolizing P450s are by far the better known and include CYP5 (thromboxane synthase) and CYP8A (prostacyclin synthase) in animals (11), and the entire family of CYP74 in plants (12). The individual CYP74 enzymes include allene oxide synthase (AOS), one of which catalyzes a key step in cyclopentenone synthesis in the jasmonate pathway, hydroperoxide lyase, divinyl ether synthase, and epoxyalcohol synthase (12, 13). The catalase-related enzymes are distinctive in that they have been found naturally as a fusion protein with the LOX enzyme that forms their hydroperoxide substrate (14). The known activities include AOS in Plexaura homomalla and other marine corals (with a different specificity for fatty acid hydroperoxide compared with the plant P450 AOS) (15, 16), and the unique bicyclobutane synthase and other allylic epoxide synthase activities of the enzyme in the cyanobacterium Anabaena PCC-7120 (17, 18). Currently we are trying to understand the scope of the reactions catalyzed by this catalase-related family of enzymes. Our objectives are to help understand the structure-function relationships in the reactions with peroxides, to provide new insights on the mechanism of these hemoprotein-catalyzed transformations, as well as, by reflection, to give a different perspective on the parent hemoprotein, the hydrogen peroxide-metabolizing true catalase.The underlying chemistry of the fatty acid hydroperoxide transformations by the specialized P450 and catalase-related hemoproteins can be written as purely free radical in character, or ionic, or with facets of both (19–22). Reaction is generally considered to be initiated by homolytic cleavage of the peroxide O–O bond (see Fig. 1). While subsequent reactions can be construed as following a radical pathway to product (e.g. in the hydroperoxide lyase reaction (19)), others are considered to involve an electron transfer step, giving a carbocation intermediate in the penultimate steps to product (22) (Fig. 1). The underlying grounds for these mechanisms are more a subject of debate and of comparison to other chemistry than of defining evidence on the reactions in question. The results reported here add a measure of experimental support for ionic events in the peroxide transformation by a catalase-related hemoprotein.Open in a separate windowFIGURE 1.Hemoprotein-catalyzed transformation of fatty acid hydroperoxides via diverging routes. A radical pathway can lead to aldehydes (left side) and an ionic pathway via a putative carbocation intermediate to allene oxides (right side).Recently genome sequencing was completed on the cyanobacterium Acaryochloris marina (23). A. marina is a focus of attention owing to its harboring a light-harvesting complex containing the unusual chlorophyll d, considered a bridge in the evolutionary development of photosynthetic mechanisms (24, 25). BLAST searches of the A. marina genome reveal three individual LOX sequences and additionally, the presence of DNA encoding a putative fusion protein of lipoxygenase and catalase-related hemoprotein, the topic of this report. We find an unexpected by-product in the reaction with one particular fatty acid (C18.2ω6); the partial incorporation of ¹⁸O from water in a newly formed hydroxyl group has implications related to the mechanism of hydroperoxide transformation.     

Hyaluronan Molecular Weight Is Controlled by UDP-N-acetylglucosamine Concentration in Streptococcus zooepidemicus

The molecular weight of hyaluronan is important for its rheological and biological function. The molecular mechanisms underlying chain termination and hence molecular weight control remain poorly understood, not only for hyaluronan synthases but also for other β-polysaccharide synthases, e.g. cellulose, chitin, and 1,3-betaglucan synthases. In this work, we manipulated metabolite concentrations in the hyaluronan pathway by overexpressing the five genes of the hyaluronan synthesis operon in Streptococcus equi subsp. zooepidemicus. Overexpression of genes involved in UDP-glucuronic acid biosynthesis decreased molecular weight, whereas overexpression of genes involved in UDP-N-acetylglucosamine biosynthesis increased molecular weight. The highest molecular mass observed was at 3.4 ± 0.1 MDa twice that observed in the wild-type strain, 1.8 ± 0.1 MDa. The data indicate that (a) high molecular weight is achieved when an appropriate balance of UDP-N-acetylglucosamine and UDP-glucuronic acid is achieved, (b) UDP-N-acetylglucosamine exerts the dominant effect on molecular weight, and (c) the wild-type strain has suboptimal levels of UDP-N-acetylglucosamine. Consistent herewith molecular weight correlated strongly (ρ = 0.84, p = 3 × 10⁻⁵) with the concentration of UDP-N-acetylglucosamine. Data presented in this paper represent the first model for hyaluronan molecular weight control based on the concentration of activated sugar precursors. These results can be used to engineer strains producing high molecular weight hyaluronan and may provide insight into similar polymerization mechanisms in other polysaccharides.Hyaluronan (HA)³ is a linear polymer of a repeating disaccharide, β1–3 d-N-acetylglucosamine (GlcNAc) β1–4 d-glucuronic acid (GlcUA) (1) (see Fig. 1). Ubiquitous in the extracellular matrix in vertebrates, HA is particularly abundant in cartilage, synovial fluid, dermis, and the vitreous humor of the eye, where it serves specialized functions. HA also plays a critical role during fertilization and embryogenesis. In many group A and C streptococci, HA forms a capsule that helps these microbes evade the host immune system (2). HA molecular weight is important for the physiochemical as well as biological properties of HA. High molecular weight is important for HA to exert its unique rheological properties (3), for mucoadherence (4, 5), and anti-inflammatory effects (6, 7), whereas low molecular weight is a potent signaling molecule (8).Open in a separate windowFIGURE 1.Biosynthetic pathway of HA in S. zooepidemicus.HA is produced by a processive synthase (9, 10) from the activated sugar precursors, UDP-glucuronic acid (UDP-GlcUA) and UDP-N-acetylglucosamine (UDP-GlcNAc) (see Fig. 1). In addition to the HA synthase (hasA), streptococcal has operons encode for one or more enzymes involved in biosynthesis of the activated sugars (11). The Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) operon encodes for five genes: HA synthase (EC 2.4.1.212; hasA), UDP-glucose dehydrogenase (EC 1.1.1.22; hasB), UDP-glucose pyrophosphorylase (EC 2.7.7.9; hasC), a glmU paralog encoding for a dual function enzyme acetyltransferase and pyrophosphorylase activity (EC 2.3.1.4/EC 2.7.7.23; hasD), and a pgi paralog encoding for phosphoglucoisomerase (EC 5.3.1.9; hasE).Although the biosynthetic mechanism is well established, little is known about what controls HA molecular weight. This is true not only for HA, but also for the highly abundant β-polysaccharides: cellulose, chitin, and 1,3-betaglucan. Molecular weight is partly an intrinsic parameter of the HA synthase. Weigel and colleagues have demonstrated that, at least in vitro, mutation of conserved cysteine or polar residues in streptococcal HA synthases results in reduced molecular weight with limited effect on biosynthetic rate (12–14). In a vertebrate HA synthase from Xenopus, the mutation of a serine or a cysteine residue yielded HA of higher, lower, or similar molecular weight depending on the amino acid substitution (15).We and others have demonstrated that in vivo molecular weight is also affected by culture parameters, e.g. temperature and aeration (16–20). Although changed culture conditions affect the physiochemical environment of the HA synthase, a more likely explanation is that molecular weight is affected by the availability of activated sugar substrates (UDP-GlcUA and UDP-GlcNAc) as well as the concentration of possible effector molecules, such as free UDP (21). Although such a mechanism has been suggested for several processive synthases (22, 23), there has never been any direct evidence linking molecular weight to the concentration of a substrate.Experimental support for the hypothesis has been obtained for the type 3 polysaccharide of Streptococcus pneumoniae (24). Like HA, the type 3 polysaccharide in S. pneumoniae is synthesized by a processive synthase from alternating addition of activated sugars, in this case UDP-glucose (UDP-Glc) and UDP-GlcUA. Mutants with reduced UDP-glucose dehydrogenase (“hasB”) activity not only produce less polysaccharide, but also polysaccharide with lower molecular weight (24). Although the levels of UDP-GlcUA were below detection in all strains, this supports the idea that UDP-GlcUA concentration controls molecular weight. Moreover, it is consistent with previous in vitro studies showing that low levels of UDP-GlcUA cause chain termination and hence low molecular weight (25). It was proposed that the concentration of UDP-GlcUA is critical for the successful transition from oligosaccharide lipid to highly processive polysaccharide synthesis (26). A similar mechanism is not likely for HA biosynthesis, because there is no indication that the HA synthase needs a primer (27).In the present study, we manipulated metabolite concentrations in the HA pathway by overexpressing the five genes in the has operon of S. zooepidemicus (Fig. 1). Overexpression of these genes had a profound effect on HA molecular weight, which correlated with the levels of UDP-sugars and in particular, UDP-GlcNAc.     

Cardiolipin Molecular Species with Shorter Acyl Chains Accumulate in Saccharomyces cerevisiae Mutants Lacking the Acyl Coenzyme A-binding Protein Acb1p: NEW INSIGHTS INTO ACYL CHAIN REMODELING OF CARDIOLIPIN*

The function of the mitochondrial phospholipid cardiolipin (CL) is thought to depend on its acyl chain composition. The present study aims at a better understanding of the way the CL species profile is established in Saccharomyces cerevisiae by using depletion of the acyl-CoA-binding protein Acb1p as a tool to modulate the cellular acyl chain content. Despite the presence of an intact CL remodeling system, acyl chains shorter than 16 carbon atoms (C16) were found to accumulate in CL in cells lacking Acb1p. Further experiments revealed that Taz1p, a key CL remodeling enzyme, was not responsible for the shortening of CL in the absence of Acb1p. This left de novo CL synthesis as the only possible source of acyl chains shorter than C16 in CL. Experiments in which the substrate specificity of the yeast cardiolipin synthase Crd1p and the acyl chain composition of individual short CL species were investigated, indicated that both CL precursors (i.e. phosphatidylglycerol and CDP-diacylglycerol) contribute to comparable extents to the shorter acyl chains in CL in acb1 mutants. Based on the findings, we conclude that the fatty acid composition of mature CL in yeast is governed by the substrate specificity of the CL-specific lipase Cld1p and the fatty acid composition of the Taz1p substrates.Cardiolipin (CL)⁵ is a unique anionic glycerophospholipid with dimeric structure containing four acyl chains, which is almost exclusively localized to the mitochondrial inner membrane in eukaryotic cells (1, 2). CL has been shown to co-isolate with, and to be required for optimal activity of a number of enzymes in the respiratory chain (3–5), and it has been implicated in the stability and assembly of protein (super)complexes (6–8). In the presence of divalent cations and dependent on the acyl chain composition, CL has a propensity for membrane negative curvature, a property that may be important in, e.g. membrane fusion and fission (9, 10). In addition, CL is thought to serve as a proton trap in oxidative phosphorylation (11). In recent years, CL has also been implicated in apoptosis (12, 13).CL is synthesized in the inner mitochondrial membrane by condensation of PG and CDP-DAG, catalyzed by the cardiolipin synthase Crd1p (see Fig. 1; reviewed in Ref. 4). Compared with the other phospholipid classes, CL is enriched in unsaturated acyl chains, and the molecular species of CL possess a high degree of molecular symmetry (14). The CL-specific acyl chain pattern originates from substrate preferences during biosynthesis and subsequent remodeling by acyl chain exchange (15). The finding of an aberrant CL species profile in patients suffering from Barth syndrome, which results from mutations in the tafazzin gene (16), revealed the importance of CL remodeling, and set the stage for the identification of tafazzin as the acyltransferase involved (17, 18). The Drosophila homologue of tafazzin was shown to be a CoA-independent phospholipid transacylase with substrate preference for CL and PC (19).Open in a separate windowFIGURE 1.The cardiolipin biosynthetic pathway in the context of phospholipid biosynthesis in yeast. The enzymes of the CL biosynthetic pathway identified at the gene level are indicated: Cds1p, CDP-DAG synthase; Pgs1p, phosphatidylglycerolphosphate synthase; Crd1p, CL synthase; Taz1p, Tafazzin; Cld1p, CL-specific deacylase.The biosynthesis and remodeling of CL have been extensively studied in the yeast Saccharomyces cerevisiae. After synthesis by Crd1p, CL is subject to deacylation and reacylation, which involves the yeast homologue of tafazzin encoded by the TAZ1 gene. The yeast taz1 mutant has defects similar to those found in Barth syndrome, including reduced CL content, an aberrant CL species profile, and an accumulation of monolyso-CL (20). The bioenergetic coupling of isolated mitochondria from a taz1 mutant is compromised (21), which may be accounted for by the impaired assembly of the III₂IV₂ supercomplex (22). Recently, the CL-specific phospholipase Cld1p was identified, which functions upstream of Taz1p (23).Because the acyl chain composition of CL is important for its function, we investigated how the molecular species profile of CL is attained by using depletion of the 10-kDa cytosolic acyl-CoA-binding protein Acb1p as a tool to modify the cellular acyl chain content. Deletion of the ACB1 gene increases the cellular levels of C14 and C16 fatty acids at the expense of C18, without having adverse effects on cell growth or on the rate of glycerophospholipid synthesis (24–26). The changes in fatty acid composition are reflected to varying extents in the molecular species profile of phospholipids in Acb1p-depleted cells as determined by electrospray ionization-mass spectrometry (ESI-MS) (27, 28). We first determined by mass spectrometry that in the absence of Acb1p acyl chains shorter than C16 accumulate in CL as in the other phospholipid classes despite the Cld1p-Taz1p remodeling system. Using appropriate mutants and analysis by mass spectrometry, we investigated two possible origins of the shorter acyl chains in CL: (i) remodeling by Taz1p and (ii) de novo synthesis of CL from PG and CDP-DAG.     

A Straight Path to Circular Proteins

Folding and stability are parameters that control protein behavior. The possibility of conferring additional stability on proteins has implications for their use in vivo and for their structural analysis in the laboratory. Cyclic polypeptides ranging in size from 14 to 78 amino acids occur naturally and often show enhanced resistance toward denaturation and proteolysis when compared with their linear counterparts. Native chemical ligation and intein-based methods allow production of circular derivatives of larger proteins, resulting in improved stability and refolding properties. Here we show that circular proteins can be made reversibly with excellent efficiency by means of a sortase-catalyzed cyclization reaction, requiring only minimal modification of the protein to be circularized.Sortases are bacterial enzymes that predominantly catalyze the attachment of surface proteins to the bacterial cell wall (1, 2). Other sortases polymerize pilin subunits for the construction of the covalently stabilized and covalently anchored pilus of the Gram-positive bacterium (3–5). The reaction catalyzed by sortase involves the recognition of short 5-residue sequence motifs, which are cleaved by the enzyme with the concomitant formation of an acyl enzyme intermediate between the active site cysteine of sortase and the carboxylate at the newly generated C terminus of the substrate (1, 6–8). In many bacteria, this covalent intermediate can be resolved by nucleophilic attack from the pentaglycine side chain in a peptidoglycan precursor, resulting in the formation of an amide bond between the pentaglycine side chain and the carboxylate at the cleavage site in the substrate (9, 10). In pilus construction, alternative nucleophiles such as lysine residues or diaminopimelic acid participate in the transpeptidation reaction (3, 4).When appended near the C terminus of proteins that are not natural sortase substrates, the recognition sequence of Staphylococcus aureus sortase A (LPXTG) can be used to effectuate a sortase-catalyzed transpeptidation reaction using a diverse array of artificial glycine-based nucleophiles (Fig. 1). The result is efficient installation of a diverse set of moieties, including lipids (11), carbohydrates (12), peptide nucleic acids (13), biotin (14), fluorophores (14, 15), polymers (16), solid supports (16–18), or peptides (15, 19) at the C terminus of the protein substrate. During the course of our studies to further expand sortase-based protein engineering, we were struck by the frequency and relative ease with which intramolecular transpeptidation reactions were occurring. Specifically, proteins equipped with not only the LPXTG motif but also N-terminal glycine residues yielded covalently closed circular polypeptides (Fig. 1). Similar reactivity using sortase has been described in two previous cases; however, rigorous characterization of the circular polypeptides was absent (16, 20). The circular proteins in these reports were observed as minor components of more complex reaction mixtures, and the cyclization reaction itself was not optimized.Open in a separate windowFIGURE 1.Protein substrates equipped with a sortase A recognition sequence (LPXTG) can participate in intermolecular transpeptidation with synthetic oligoglycine nucleophiles (left) or intramolecular transpeptidation if an N-terminal glycine residue is present (right).Here we describe our efforts toward applying sortase-catalyzed transpeptidation to the synthesis of circular and oligomeric proteins. This method has general applicability, as illustrated by successful intramolecular reactions with three structurally unrelated proteins. In addition to circularization of individual protein units, the multiprotein complex AAA-ATPase p97/VCP/CDC48, with six identical subunits containing the LPXTG motif and an N-terminal glycine, was found to preferentially react in daisy chain fashion to yield linear protein fusions. The reaction exploited here shows remarkable similarities to the mechanisms proposed for circularization of cyclotides, small circular proteins that have been isolated from plants (21–23).     

Limonene-1,2-Epoxide Hydrolase from Rhodococcus erythropolis DCL14 Belongs to a Novel Class of Epoxide Hydrolases

An epoxide hydrolase from Rhodococcus erythropolis DCL14 catalyzes the hydrolysis of limonene-1,2-epoxide to limonene-1,2-diol. The enzyme is induced when R. erythropolis is grown on monoterpenes, reflecting its role in the limonene degradation pathway of this microorganism. Limonene-1,2-epoxide hydrolase was purified to homogeneity. It is a monomeric cytoplasmic enzyme of 17 kDa, and its N-terminal amino acid sequence was determined. No cofactor was required for activity of this colorless enzyme. Maximal enzyme activity was measured at pH 7 and 50°C. None of the tested inhibitors or metal ions inhibited limonene-1,2-epoxide hydrolase activity. Limonene-1,2-epoxide hydrolase has a narrow substrate range. Of the compounds tested, only limonene-1,2-epoxide, 1-methylcyclohexene oxide, cyclohexene oxide, and indene oxide were substrates. This report shows that limonene-1,2-epoxide hydrolase belongs to a new class of epoxide hydrolases based on (i) its low molecular mass, (ii) the absence of any significant homology between the partial amino acid sequence of limonene-1,2-epoxide hydrolase and amino acid sequences of known epoxide hydrolases, (iii) its pH profile, and (iv) the inability of 2-bromo-4′-nitroacetophenone, diethylpyrocarbonate, 4-fluorochalcone oxide, and 1,10-phenanthroline to inhibit limonene-1,2-epoxide hydrolase activity.Epoxides are highly reactive compounds which readily react with numerous biological compounds, including proteins and nucleic acids. Consequently, epoxides are cytotoxic, mutagenic, and potentially carcinogenic, and there is considerable interest in biological degradation mechanisms for these compounds.In bacteria, epoxides are formed during the metabolism of alkenes (23) and halohydrins (15, 26, 34, 49). Enzymes belonging to a large number of enzyme classes, including dehydrogenases (17), lyases (21), carboxylases (1, 43), glutathione S-transferases (6, 8), isomerases (24), and hydrolases (7, 19, 44), are involved in the microbial degradation of epoxides.Epoxide hydrolases are enzymes catalyzing the addition of water to epoxides forming the corresponding diol. This group of enzymes has been extensively studied in mammals, while only limited information is available on bacterial epoxide hydrolases. Three functions for epoxide hydrolases are recognized (42). In bacteria, epoxide hydrolases are involved in the degradation of several hydrocarbons, including 1,3-dihalo-2-propanol (34), 2,3-dihalo-1-propanol (15, 26), epichlorohydrin (46), propylene oxide (16), 9,10-epoxy fatty acids (30, 36), trans-2,3-epoxysuccinate (2), and cyclohexene oxide (14). Other epoxide hydrolases, such as microsomal and cytosolic epoxide hydrolase from mammals (for reviews, see references 4, 8, and 44), are involved in the detoxification of epoxides formed due to the action of P-450-dependent monooxygenases (8). Epoxide hydrolases are also involved in biosynthesis of hormones, such as leukotrienes and juvenile hormone (40, 45), and plant cuticular elements (11). Remarkably, the bacterial and eukaryotic epoxide hydrolases described so far form a homogeneous group of enzymes belonging to the α/β-hydrolase fold superfamily (10, 38).Rhodococcus erythropolis DCL14, a gram-positive bacterium, is able to grow on both (+)- and (−)-limonene as the sole source of carbon and energy (47). Cells grown on limonene contained a novel epoxide hydrolase that does not belong to the α/β-hydrolase fold superfamily. This limonene-1,2-epoxide hydrolase converts limonene-1,2-epoxide to limonene-1,2-diol (p-menth-8-ene-1,2-diol [Fig. 1]). In this report, we describe the purification and characterization of this enzyme and show that limonene-1,2-epoxide hydrolase belongs to a novel class of epoxide hydrolases. Open in a separate windowFIG. 1Reaction catalyzed by limonene-1,2-epoxide hydrolase.     

The Nutrigenetics of Hyperhomocysteinemia: QUANTITATIVE PROTEOMICS REVEALS DIFFERENCES IN THE METHIONINE CYCLE ENZYMES OF GENE-INDUCED VERSUS DIET-INDUCED HYPERHOMOCYSTEINEMIA*

Hyperhomocysteinemia has long been associated with atherosclerosis and thrombosis and is an independent risk factor for cardiovascular disease. Its causes include both genetic and environmental factors. Although homocysteine is produced in every cell as an intermediate of the methionine cycle, the liver contributes the major portion found in circulation, and fatty liver is a common finding in homocystinuric patients. To understand the spectrum of proteins and associated pathways affected by hyperhomocysteinemia, we analyzed the mouse liver proteome of gene-induced (cystathionine β-synthase (CBS)) and diet-induced (high methionine) hyperhomocysteinemic mice using two-dimensional difference gel electrophoresis and Ingenuity Pathway Analysis. Nine proteins were identified whose expression was significantly changed by 2-fold (p ≤ 0.05) as a result of genotype, 27 proteins were changed as a result of diet, and 14 proteins were changed in response to genotype and diet. Importantly, three enzymes of the methionine cycle were up-regulated. S-Adenosylhomocysteine hydrolase increased in response to genotype and/or diet, whereas glycine N-methyltransferase and betaine-homocysteine methyltransferase only increased in response to diet. The antioxidant proteins peroxiredoxins 1 and 2 increased in wild-type mice fed the high methionine diet but not in the CBS mutants, suggesting a dysregulation in the antioxidant capacity of those animals. Furthermore, thioredoxin 1 decreased in both wild-type and CBS mutants on the diet but not in the mutants fed a control diet. Several urea cycle proteins increased in both diet groups; however, arginase 1 decreased in the CBS^+/− mice fed the control diet. Pathway analysis identified the retinoid X receptor signaling pathway as the top ranked network associated with the CBS^+/− genotype, whereas xenobiotic metabolism and the NRF2-mediated oxidative stress response were associated with the high methionine diet. Our results show that hyperhomocysteinemia, whether caused by a genetic mutation or diet, alters the abundance of several liver proteins involved in homocysteine/methionine metabolism, the urea cycle, and antioxidant defense.Homocysteine (Hcy)1 is a thiol-containing amino acid that is produced in every cell of the body as an intermediate of the methionine cycle (Fig. 1, Reactions 1–5) (1). Once formed, the catabolism of homocysteine occurs via three enzymatic pathways. 1) Hcy is remethylated back to methionine using vitamin B₁₂-dependent methionine synthase (Fig. 1, Reaction 4) and/or 2) betaine-homocysteine methyltransferase (BHMT) (Fig. 1, Reaction 5), and 3) Hcy is converted to cysteine via the transsulfuration pathway using CBS and γ-cystathionase (Fig. 1, Reactions 6 and 7). Under normal conditions ∼40–50% of the Hcy that is produced in the liver is remethylated, ∼40–50% is converted to cysteine, and a small amount is exported (1–3). However, when Hcy production is increased (i.e. increased dietary methionine/protein intake) or when Hcy catabolism is decreased (i.e. CBS deficiency or B vitamin deficiencies), excess Hcy is exported into the extracellular space, resulting in hyperhomocysteinemia (1–5).Open in a separate windowFig. 1.Homocysteine metabolism in liver and kidney. In classical homocystinuria, the initial enzyme of the transsulfuration pathway, CBS (Reaction 6), is deficient. MTHF, methylenetetrahydrofolate; THF, tetrahydrofolate; DHF, dihydrofolate; MeCbl, methylcobalamin; DMG, dimethylglycine; PLP, pyridoxal 5′-phosphate.Homocystinuria was first described in the 1960s by Carson et al. (6): they observed 10 pediatric patients with severely elevated levels of Hcy in the urine and hypermethioninemia. Normal concentrations of plasma total homocysteine (tHcy) range from 5 to 12 μm (7); however, in homocystinuria, tHcy levels can exceed 100 μm. Homocystinuric patients present with mental retardation, abnormal bone growth, fine hair, malar flush, and dislocation of the lens of the eye, and most die from premature cardiovascular disease (6, 8). Autopsy findings indicate widespread thromboembolism, arteriosclerosis, and fatty livers (6, 8). Mudd et al. (9, 10) identified the cause of homocystinuria as a defect in the enzyme cystathionine β-synthase. A recent study of newborn infants in Denmark estimated the birth prevalence for CBS heterozygosity to be about 1:20,000 (11).Plasma tHcy concentrations are also directly correlated with dietary methionine/protein intake (12, 13). Guttormsen et al. (13) demonstrated that a protein-rich meal affected tHcy for at least 8–24 h. When normal subjects were fed a low protein-containing breakfast (12–15 g), plasma methionine levels increased slightly after 2 h (22.5–27.5 μm), but tHcy levels did not change significantly. However, when these same subjects were fed a high protein meal (52 g), plasma methionine levels peaked after 4 h (38 μm), and tHcy rose steadily until a maximum level was reached 8 h postmeal (7.6 versus 8.5 μm) (13). Thus, the following questions can be raised. How does the hepatic proteome respond to a hyperhomocysteinemic diet, and are the changes that accompany such a diet the same as or different from those that may be observed in gene-induced hyperhomocysteinemia?Because hyperhomocysteinemia is a strong independent risk factor for cardiovascular, cerebrovascular, and peripheral vascular disease, most of the current research has focused on the mechanisms involved in Hcy-induced endothelial dysfunction (14–24). The results of those studies have concluded that Hcy induces intracellular oxidative stress by generating ROS, which in turn lead to decreased bioavailable nitric oxide (NO), altered gene expression, increased endoplasmic reticulum stress, and activation of cholesterol biosynthesis. Also, several studies have examined the association between hyperhomocysteinemia and alcoholic liver disease, but few have looked at the effect of Hcy on the non-alcoholic liver even though fatty liver is a constant finding in homocystinuria (6, 8), and the liver is the major source of circulating Hcy (4, 5, 10). We hypothesize that 1) the liver proteome will respond to hyperhomocysteinemia by altering the expression of proteins involved in methionine/homocysteine metabolism and antioxidant defense and that 2) the set of proteins that are expressed when hyperhomocysteinemia is induced by CBS deficiency will differ from those expressed as a result of a high methionine diet. In the present study, we use a well established mouse model of CBS deficiency to study the early changes in the liver proteome that accompany hyperhomocysteinemia (25).     

Ceramide Synthesis Is Modulated by the Sphingosine Analog FTY720 via a Mixture of Uncompetitive and Noncompetitive Inhibition in an Acyl-CoA Chain Length-de pend ent Manner

FTY720, a sphingosine analog, is in clinical trials as an immunomodulator. The biological effects of FTY720 are believed to occur after its metabolism to FTY720 phosphate. However, very little is known about whether FTY720 can interact with and modulate the activity of other enzymes of sphingolipid metabolism. We examined the ability of FTY720 to modulate de novo ceramide synthesis. In mammals, ceramide is synthesized by a family of six ceramide synthases, each of which utilizes a restricted subset of acyl-CoAs. We show that FTY720 inhibits ceramide synthase activity in vitro by noncompetitive inhibition toward acyl-CoA and uncompetitive inhibition toward sphinganine; surprisingly, the efficacy of inhibition depends on the acyl-CoA chain length. In cultured cells, FTY720 has a more complex effect, with ceramide synthesis inhibited at high (500 nm to 5 μm) but not low (<200 nm) sphinganine concentrations, consistent with FTY720 acting as an uncompetitive inhibitor toward sphinganine. Finally, electrospray ionization-tandem mass spectrometry demonstrated, unexpectedly, elevated levels of ceramide, sphingomyelin, and hexosylceramides after incubation with FTY720. Our data suggest a novel mechanism by which FTY720 might mediate some of its biological effects, which may be of mechanistic significance for understanding its mode of action.FTY720 (2-amino-(2-2-[4-octylphenyl]ethyl)propane 1,3-diol hydrochloride), also known as Fingolimod, is an immunosuppressant drug currently being tested in clinical trials for organ transplantation and autoimmune diseases such as multiple sclerosis (1). FTY720 is a structural analog of sphingosine, a key biosynthetic intermediate in sphingolipid (SL)² metabolism (see Fig. 1). In vivo, FTY720 is rapidly phosphorylated by sphingosine kinase 2 (2, 3) to form FTY720 phosphate (FTY720-P), an analog of sphingosine 1-phosphate (S1P) (see Fig. 1A). FTY720-P binds to S1P receptors (S1PRs) (4, 5) and thereby induces a variety of phenomena such as T-lymphocyte migration from lymphoid organs (6–9); accordingly, FTY720 treatment results in lymphopenia as lymphocytes (especially T-cells) become sequestered inside lymphoid organs (10–12). The ability of FTY720 to sequester lymphocytes has stimulated its use in treatment of allograft rejection and autoimmune diseases (13), and FTY720 is currently under phase III clinical trials for treatment of relapsing-remitting multiple sclerosis (14).Open in a separate windowFIGURE 1.SL structure and metabolism. A, structures of SLs and SL analogs used in this study. B, metabolic inter-relationships between SLs and the metabolism of FTY720. The enzymes are denoted in italics. LPP3, lipid phosphate phosphatase 3; LPP1α, lipid phosphate phosphatase 1α.Apart from the binding of FTY720-P to S1PRs, the ability of FTY720 to inhibit S1P lyase (15) (see Fig. 1B), and its inhibitory effect on cytosolic phospholipase A2 (16), whose activity can be modulated by ceramide 1-phosphate (17), little is known about whether FTY720 or FTY720-P can modulate the activity of other enzymes of SL metabolism. Because FTY720 is an analog of sphingosine, one of the two substrates of ceramide synthase (CerS) (see Fig. 1), we now examine whether FTY720 can modulate CerS activity. CerS utilizes fatty acyl-CoAs to N-acylate sphingoid long chain bases. Six CerS exist in mammals, each of which uses a restricted subset of acyl-CoAs (18–23). We demonstrate that FTY720 inhibits CerS activity and that the extent of inhibition varies according to the acyl chain length of the acyl-CoA substrate. Surprisingly, FTY720 inhibits CerS activity toward acyl-CoA via noncompetitive inhibition and toward sphinganine via uncompetitive inhibition. Finally, the mode of interaction of FTY720 with CerS in cultured cells depends on the amount of available sphinganine. Together, we show that FTY720 modulates ceramide synthesis, which may be of relevance for understanding its biological effects in vivo and its role in immunomodulation.     

Purification and cDNA Cloning of Isochorismate Synthase from Elicited Cell Cultures of Catharanthus roseus

Isochorismate is an important metabolite formed at the end of the shikimate pathway, which is involved in the synthesis of both primary and secondary metabolites. It is synthesized from chorismate in a reaction catalyzed by the enzyme isochorismate synthase (ICS; EC 5.4.99.6). We have purified ICS to homogeneity from elicited Catharanthus roseus cell cultures. Two isoforms with an apparent molecular mass of 64 kD were purified and characterized. The K_m values for chorismate were 558 and 319 μm for isoforms I and II, respectively. The isoforms were not inhibited by aromatic amino acids and required Mg²⁺ for enzyme activity. Polymerase chain reaction on a cDNA library from elicited C. roseus cells with a degenerated primer based on the sequence of an internal peptide from isoform II resulted in an amplification product that was used to screen the cDNA library. This led to the first isolation, to our knowledge, of a plant ICS cDNA. The cDNA encodes a protein of 64 kD with an N-terminal chloroplast-targeting signal. The deduced amino acid sequence shares homology with bacterial ICS and also with anthranilate synthases from plants. Southern analysis indicates the existence of only one ICS gene in C. roseus.The shikimate pathway is a major pathway in primary and secondary plant metabolism (Herrmann, 1995). It provides chorismate for the synthesis of the aromatic amino acids Phe, Tyr, and Trp, which are used in protein biosynthesis, but also serves as a precursor for a wide variety of aromatic substances (Herrmann, 1995; Weaver and Hermann, 1997; Fig. ​Fig.1a).1a). Chorismate is also the starting point of a biosynthetic pathway leading to phylloquinones (vitamin K₁) and anthraquinones (Poulsen and Verpoorte, 1991). The first committed step in this pathway is the conversion of chorismate into isochorismate, which is catalyzed by ICS (Poulsen and Verpoorte, 1991; Fig. ​Fig.1b).1b). Its substrate, chorismate, plays a pivotal role in the synthesis of shikimate-pathway-derived compounds, and its distribution over the various pathways is expected to be tightly regulated. Elicited cell cultures of Catharanthus roseus provide an example of the partitioning of chorismate. Concurrently, these cultures produce both Trp-derived indole alkaloids and DHBA (Moreno et al., 1994). In bacteria DHBA is synthesized from isochorismate (Young et al., 1969). Elicitation of C. roseus cell cultures with a fungal extract induces not only several enzymes of the indole alkaloid biosynthetic pathway (Pasquali et al., 1992) but also ICS (Moreno et al., 1994). Information concerning the expression and biochemical characteristics of the enzymes that compete for available chorismate (ICS, CM, and AS) may help us to understand the regulation of the distribution of this precursor over the various pathways. Such information is already available for CM (Eberhard et al., 1996) and AS (Poulsen et al., 1993; Bohlmann et al., 1995) but not for ICS. Figure 1a, Position of ICS in the plant metabolism. SA, Salicylic acid, OSB, o-succinylbenzoic acid. b, Reaction catalyzed by ICS.Isochorismate plays an important role in bacterial and plant metabolism as a precursor of o-succinylbenzoic acid, an intermediate in the biosynthesis of menaquinones (vitamin K₂) (Weische and Leistner, 1985) and phylloquinones (vitamin K₁; Poulsen and Verpoorte, 1991). In bacteria isochorismate is also a precursor of siderophores such as DHBA (Young et al., 1969), enterobactin (Walsh et al., 1990), amonabactin (Barghouthi et al., 1991), and salicylic acid (Serino et al., 1995). Although evidence from tobacco would indicate that salicylic acid in plants is derived from Phe via benzoic acid (Yalpani et al., 1993; Lee et al., 1995; Coquoz et al., 1998), it cannot be excluded that it is also synthesized from isochorismate. In the secondary metabolism of higher plants, isochorismate is a precursor for the biosynthesis of anthraquinones (Inoue et al., 1984; Sieweke and Leistner, 1992), naphthoquinones (Müller and Leistner, 1978), catalpalactone (Inouye et al., 1975), and certain alkaloids in orchids (Leete and Bodem, 1976).ICS was first extracted and partially purified from crude extracts of Aerobacter aerogenes (Young and Gibson, 1969). Later, ICS activity was detected in protein extracts of cell cultures from plants of the Rubiaceae, Celastraceae, and Apocynaceae families (Ledüc et al., 1991; Poulsen et al., 1991; Poulsen and Verpoorte, 1992). Genes encoding ICS have been cloned from bacteria such as Escherichia coli (Ozenberger et al., 1989), Pseudomonas aeruginosa (Serino et al., 1995), Aeromonas hydrophila (Barghouthi et al., 1991), Flavobacterium K_3–15 (Schaaf et al., 1993), Hemophilus influenzae (Fleischmann et al., 1995), and Bacillus subtilis (Rowland and Taber, 1996). Both E. coli and B. subtilis have two distinct ICS genes; one is involved in siderophore biosynthesis and the other is involved in menaquinone production (Daruwala et al., 1996, 1997; Müller et al., 1996; Rowland and Taber, 1996). The biochemical properties of the two ICS enzymes from E. coli are different (Daruwala et al., 1997; Liu et al., 1990). Sequence analysis has revealed that the bacterial ICS enzymes share homology with the chorismate-utilizing enzymes AS and p-aminobenzoate synthase, suggesting that they share a common evolutionary origin (Ozenberger et al., 1989).Much biochemical and molecular data concerning the shikimate pathway in plants have accumulated in recent years (Schmid and Amrhein, 1995; Weaver and Hermann, 1997), but relatively little work has been done on ICS from higher plants. The enzyme has been partially purified from Galium mollugo cell cultures (Ledüc et al., 1991, 1997), but purification of the ICS protein to homogeneity has remained elusive, probably because of instability of the enzyme.Our interests focus on the role of ICS in the regulation of chorismate partitioning over the various pathways. Furthermore, we studied ICS in C. roseus to gain insight into the biosynthesis of DHBA in higher plants (Moreno et al., 1994). In this paper we report the first purification, to our knowledge, of ICS to homogeneity from a plant source and the cloning of the corresponding cDNA.     

Mutational Biosynthesis of Novel Rapamycins by a Strain of Streptomyces hygroscopicus NRRL 5491 Disrupted in rapL,Encoding a Putative Lysine Cyclodeaminase

The gene rapL lies within the region of the Streptomyces hygroscopicus chromosome which contains the biosynthetic gene cluster for the immunosuppressant rapamycin. Introduction of a frameshift mutation into rapL by ΦC31 phage-mediated gene replacement gave rise to a mutant which did not produce significant amounts of rapamycin. Growth of this rapL mutant on media containing added l-pipecolate restored wild-type levels of rapamycin production, consistent with a proposal that rapL encodes a specific l-lysine cyclodeaminase important for the production of the l-pipecolate precursor. In the presence of added proline derivatives, rapL mutants synthesized novel rapamycin analogs, indicating a relaxed substrate specificity for the enzyme catalyzing pipecolate incorporation into the macrocycle.Rapamycin is a 31-member macrocyclic polyketide produced by Streptomyces hygroscopicus NRRL 5491 which, like the structurally related compounds FK506 and immunomycin (Fig. ​(Fig.1),1), has potent immunosuppressive properties (24). Such compounds are potentially valuable in the treatment of autoimmune diseases and in preventing the rejection of transplanted tissues (16). The biosynthesis of rapamycin requires a modular polyketide synthase, which uses a shikimate-derived starter unit (11, 20) and which carries out a total of fourteen successive cycles of polyketide chain elongation that resemble the steps in fatty acid biosynthesis (2, 27). l-Pipecolic acid is then incorporated (21) into the chain, followed by closure of the macrocyclic ring, and both these steps are believed to be catalyzed by a pipecolate-incorporating enzyme (PIE) (18), the product of the rapP gene (8, 15). Further site-specific oxidations and O-methylation steps (15) are then required to produce rapamycin. Open in a separate windowFIG. 1Structures of rapamycin, FK506, and immunomycin.The origin of the pipecolic acid inserted into rapamycin has been previously established (21) to be free l-pipecolic acid derived from l-lysine (although the possible role of d-lysine as a precursor must also be borne in mind) (9). Previous work with other systems has suggested several alternative pathways for pipecolate formation from lysine (22), but the results of the incorporation of labelled lysine into the pipecolate moiety of immunomycin (Fig. ​(Fig.1)1) clearly indicate loss of the α-nitrogen atom (3). More recently, the sequencing of the rap gene cluster revealed the presence of the rapL gene (Fig. ​(Fig.2),2), whose deduced gene product bears striking sequence similarity to two isoenzymes of ornithine deaminase from Agrobacterium tumefaciens (25, 26). Ornithine deaminase catalyzes the deaminative cyclization of ornithine to proline, and we have proposed (15) that the rapL gene product catalyzes the analogous conversion of l-lysine to l-pipecolate (Fig. ​(Fig.3).3). Open in a separate windowFIG. 2A portion of the rapamycin biosynthetic gene cluster which contains ancillary (non-polyketide synthase) genes (15, 27). PKS, polyketide synthase.Open in a separate windowFIG. 3(A) The conversion of l-ornithine to l-proline by ornithine cyclodeaminase (17). (B) Proposed conversion of l-lysine to l-pipecolic acid by the rapL gene product.Here, we report the use of ΦC31 phage-mediated gene replacement (10) to introduce a frameshift mutation into rapL and the ability of the mutant to synthesize rapamycins in the absence or presence of added pipecolate or pipecolate analogs.     

Contribution of Flavin Covalent Linkage with Histidine 99 to the Reaction Catalyzed by Choline Oxidase

The FAD-dependent choline oxidase has a flavin cofactor covalently attached to the protein via histidine 99 through an 8α-N(3)-histidyl linkage. The enzyme catalyzes the four-electron oxidation of choline to glycine betaine, forming betaine aldehyde as an enzyme-bound intermediate. The variant form of choline oxidase in which the histidine residue has been replaced with asparagine was used to investigate the contribution of the 8α-N(3)-histidyl linkage of FAD to the protein toward the reaction catalyzed by the enzyme. Decreases of 10-fold and 30-fold in the k_cat/K_m and k_cat values were observed as compared with wild-type choline oxidase at pH 10 and 25 °C, with no significant effect on k_cat/K_O using choline as substrate. Both the k_cat/K_m and k_cat values increased with increasing pH to limiting values at high pH consistent with the participation of an unprotonated group in the reductive half-reaction and the overall turnover of the enzyme. The pH independence of both ^D(k_cat/K_m) and ^Dk_cat, with average values of 9.2 ± 3.3 and 7.4 ± 0.5, respectively, is consistent with absence of external forward and reverse commitments to catalysis, and the chemical step of CH bond cleavage being rate-limiting for both the reductive half-reaction and the overall enzyme turnover. The temperature dependence of the ^Dk_red values suggests disruption of the preorganization in the asparagine variant enzyme. Altogether, the data presented in this study are consistent with the FAD-histidyl covalent linkage being important for the optimal positioning of the hydride ion donor and acceptor in the tunneling reaction catalyzed by choline oxidase.A number of enzymes, including dehydrogenases (1–3), monooxygenases (4–7), halogenases (8–11), and oxidases (7, 12, 13), employ flavin cofactors (FAD or FMN) for their catalytic processes. About a tenth of all flavoproteins have been shown to contain a covalently attached cofactor, which may be linked at the C8M position via histidyl, tyrosyl, or cysteinyl side chains or at the C6M position via a cysteinyl side chain (14). Glucooligosaccharide oxidase (15, 16), hexose oxidase (17), and berberine bridge enzyme (18, 19) are examples of flavoproteins (FAD as cofactor) with both linkages present in one flavin molecule. The covalent linkages in flavin-dependent enzymes have been shown to stabilize protein structure (20–22), prevent loss of loosely bound flavin cofactors (23), modulate the redox potential of the flavin microenvironment (20, 23–27), facilitate electron transfer reactions (28), and contribute to substrate binding as in the case of the cysteinyl linkage (20). However, no study has implicated a mechanistic role of the flavin covalent linkages in enzymatic reactions in which a hydride ion is transferred by quantum mechanical tunneling.The discovery of quantum mechanical tunneling in enzymatic reactions, in which hydrogen atoms, protons, and hydride ions are transferred, has attracted considerable interest in enzyme studies geared toward understanding the mechanisms underlying the several orders of magnitudes in the rate enhancements of protein-catalyzed reactions compared with non-enzymatic ones. Tunneling mechanisms have been shown in a wide array of cofactor-dependent enzymes, including flavoenzymes. Examples of flavoenzymes in which the tunneling mechanisms have been demonstrated include morphinone reductase (29, 30), pentaerythritol tetranitrate reductase (29), glucose oxidase (31–33), and choline oxidase (34). Mechanistic data on Class 2 dihydroorotate dehydrogenases, also with a flavin cofactor (FMN) covalently linked to the protein moiety (35, 36), could only propose a mechanism that is either stepwise or concerted with significant quantum mechanical tunneling for the hydride transfer from C6 and the deprotonation at C5 in the oxidation of dihydroorotate to orotate (37). This leaves choline oxidase as the only characterized enzyme with a covalently attached flavin cofactor (12, 38), where the oxidation of its substrate occurs unequivocally by quantum mechanical tunneling.Choline oxidase from Arthrobacter globiformis catalyzes the two-step FAD-dependent oxidation of the primary alcohol substrate choline to glycine betaine with betaine aldehyde, which is predominantly bound to the enzyme and forms a gem-diol species, as intermediate (Scheme 1). Glycine betaine accumulates in the cytoplasm of plants and bacteria as a defensive mechanism against stress conditions, thus making genetic engineering of relevant plants of economic interest (39–45), and the biosynthetic pathway for the osmolyte is a potential drug target in human microbial infections of clinical interest (46–48). The first oxidation step catalyzed by choline oxidase involves the transfer of a hydride ion from a deprotonated choline to the protein-bound flavin followed by reaction of the anionic flavin hydroquinone with molecular oxygen to regenerate the oxidized FAD (for a recent review see Ref. 50). The gem-diol choline, i.e. hydrated betaine aldehyde, is the substrate for the second oxidation step (49), suggesting that the reaction may follow a similar mechanism. The isoalloxazine ring of the flavin cofactor, which is buried within the protein, is physically constrained through a covalent linkage via the C(8) methyl of the flavin and the N(3) atom of the histidine side chain at position 99 (Fig. 1) (12). Also contributing to the physical constrain are the proximity of Ile-103 to the pyrimidine ring and the interactions of the backbone atoms of residues His-99 through Ile-103 with the isoalloxazine ring. The rigid positioning of the isoalloxazine ring could only permit a solvent-excluded cavity of ∼125 ų adjacent to the re face of the FAD to accommodate a 93-ų choline molecule in the substrate binding domain (12). Mechanistic data thus far obtained on choline oxidase, coupled with the crystal structure of the wild-type enzyme resolved to 1.86 Å, are consistent with a quantum tunneling mechanism for the hydride ion transfer occurring within a highly preorganized enzyme-substrate complex (Scheme 2) (12, 34, 50). Exploitation of the tunneling mechanism requires minimal independent movement of the hydride ion donor and acceptor, with the only dynamic motions permitted being the ones that promote the hydride transfer reaction.Open in a separate windowSCHEME 1.Two-step, four-electron oxidation of choline catalyzed by choline oxidase.Open in a separate windowFIGURE 1.x-ray crystal structure of the active site of wild-type choline oxidase resolved to 1.86 Å (PDB 2jbv). Note the significant distortion of the flavin ring at the C(4a) atom, which is due to the presence of a C(4a) adduct (69).Open in a separate windowSCHEME 2.The hydride ion transfer reaction from the α-carbon of the activated choline alkoxide species to the N(5) atom of the isoalloxazine ring of the enzyme-bound flavin in choline oxidase.In the present study, the contribution of the physically constrained flavin isoalloxazine ring to the reaction catalyzed by choline oxidase has been investigated in a variant enzyme in which the histidine residue at position 99 was replaced with an asparagine. The results suggest that, although not being required per se, the covalent linkage in choline oxidase contributes to the hydride tunneling reaction by either preventing independent movement or contributing to the optimal positioning of the flavin acting as hydride ion acceptor with respect to the alkoxide species acting as a donor. However, the covalent linkage is not required for the reaction.