1,2-Dehydroreticuline synthase, the branch point enzyme opening the morphinan biosynthetic pathway

A synthase which oxidizes (S)-reticuline to 1,2-dehydroreticuline has been found to occur in seedlings of opium poppy (Papaver somniferum L.). Due to its instability, this enzyme could only be partly purified (ca. 5-fold enrichment). Partial characterization at this stage of purification showed that it does not need a redox cofactor and accepts both (S)-reticuline and (S)-norreticuline as substrates. [1-(2)H, (13)C]-(R,S)-reticuline was enzymatically converted into [1-(13)C]-dehydroreticuline, which has been identified by mass spectrometry. Release of the hydrogen atom in position C-1 of the isoquinoline alkaloid during the oxidative conversion, was exploited as a sensitive assay system for this enzyme. The enzyme has a pH optimum of 8.75, a temperature optimum of 37 degrees C and the apparent K(M) value for the substrate reticuline was shown to be 117 microM. Moreover it could be demonstrated by sucrose density gradient centrifugation that the enzyme is located in vesicles of varying size. In combination with the previously discovered strictly stereoselective and NADPH dependent 1,2-dehydroreticuline reductase the detection of this enzyme, the 1,2-dehydroreticuline synthase, provides the necessary inversion of configuration and completes the pathway from two molecules of L-tyrosine via (S)-norcoclaurine to (R)-reticuline in opium poppy involving a total number of 11 enzymes.     

Characterization of olivetol synthase, a polyketide synthase putatively involved in cannabinoid biosynthetic pathway

Alkylresorcinol moieties of cannabinoids are derived from olivetolic acid (OLA), a polyketide metabolite. However, the polyketide synthase (PKS) responsible for OLA biosynthesis has not been identified. In the present study, a cDNA encoding a novel PKS, olivetol synthase (OLS), was cloned from Cannabis sativa. Recombinant OLS did not produce OLA, but synthesized olivetol, the decarboxylated form of OLA, as the major reaction product. Interestingly, it was also confirmed that the crude enzyme extracts from flowers and rapidly expanding leaves, the cannabinoid-producing tissues of C. sativa, also exhibited olivetol-producing activity, suggesting that the native OLS is functionally expressed in these tissues. The possibility that OLS could be involved in OLA biosynthesis was discussed based on its catalytic properties and expression profile.     

Subcellular localization and targeting of N-acetylglucosaminyl phosphatidylinositol de-N-acetylase, the second enzyme in the glycosylphosphatidylinositol biosynthetic pathway

The second step in glycosylphosphatidylinositol biosynthesis is the de-N-acetylation of N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI) catalyzed by N-acetylglucosaminylphosphatidylinositol deacetylase (PIG-L). Previous studies of mouse thymoma cells showed that GlcNAc-PI de-N-acetylase activity is localized to the endoplasmic reticulum (ER) but enriched in a mitochondria-associated ER membrane (MAM) domain. Because PIG-L has no readily identifiable ER sorting determinants, we were interested in learning how PIG-L is localized to the ER and possibly enriched in MAM. We used HeLa cells transiently or stably expressing epitope-tagged PIG-L variants or chimeric constructs composed of elements of PIG-L fused to Tac antigen, a cell surface protein. We first analyzed the subcellular distribution of PIG-L and Glc-NAc-PI-de-N-acetylase activity and then studied the localization of Tac-PIG-L chimeras to identify sequence elements in PIG-L responsible for its subcellular localization. We show that human PIG-L is a type I membrane protein with a large cytoplasmic domain and that, unlike the result with mouse thymoma cells, both PIG-L and GlcNAc-PI-de-N-acetylase activity are uniformly distributed between ER and MAM in HeLa cells. Analyses of a series of Tac-PIG-L chimeras indicated that PIG-L contains two ER localization signals, an independent retention signal located between residues 60 and 88 of its cytoplasmic domain and another weak signal in the luminal and transmembrane domains that functions autonomously in the presence of membrane proximal residues of the cytoplasmic domain that themselves lack any retention information. We conclude that PIG-L, like a number of other ER membrane proteins, is retained in the ER through a multi-component localization signal rather than a discrete sorting motif.     

The second enzyme in pyrrolnitrin biosynthetic pathway is related to the heme-dependent dioxygenase superfamily

Pyrrolnitrin is a commonly used and clinically effective treatment for fungal infections and provides the structural basis for the more widely used fludioxinil. The pyrrolnitrin biosynthetic pathway consists of four chemical steps, the second of which is the rearrangement of 7-chloro-tryptophan by the enzyme PrnB, a reaction that is so far unprecedented in biochemistry. When expressed in Pseudomonas fluorescens, PrnB is red in color due to the fact that it contains 1 mol of heme b per mole of protein. The crystal structure unexpectedly establishes PrnB as a member of the heme-dependent dioxygenase superfamily with significant structural but not sequence homology to the two-domain indoleamine 2,3-dioxygenase enzyme (IDO). The heme-binding domain is also structurally similar to that of tryptophan 2,3-dioxygenase (TDO). Here we report the binary complex structures of PrnB with d- and l-tryptophan and d- and l-7-chloro-tryptophan. The structures identify a common hydrophobic pocket for the indole ring but exhibit unusual heme ligation and substrate binding when compared with that observed in the TDO crystal structures. Our solution studies support the heme ligation observed in the crystal structures. Purification of the hexahistidine-tagged PrnB yields homogeneous protein that only displays in vitro activity with 7-chloro-l-tryptophan after reactivation with crude extract from the host strain, suggesting that an as yet unknown cofactor is required for activity. Mutation of the proximal heme ligand results, not surprisingly, in inactive enzyme. Redox titrations show that PrnB displays a significantly different reduction potential to that of IDO or TDO, indicating possible differences in the PrnB catalytic cycle. This is confirmed by the absence of tryptophan dioxygenase activity in PrnB, although a stable oxyferrous adduct (which is the first intermediate in the TDO/IDO catalytic cycle) can be generated. We propose that PrnB shares a key catalytic step with TDO and IDO, generation of a tryptophan hydroperoxide intermediate, although this species suffers a different fate in PrnB, leading to the eventual formation of the product, monodechloroaminopyrrolnitrin.     

Amorpha-4,11-diene synthase: cloning and functional expression of a key enzyme in the biosynthetic pathway of the novel antimalarial drug artemisinin

The sesquiterpenoid artemisinin, isolated from the plant Artemisia annua L., and its semi-synthetic derivatives are a new and very effective group of antimalarial drugs. A branch point in the biosynthesis of this compound is the cyclisation of the ubiquitous precursor farnesyl diphosphate into the first specific precursor of artemisinin, namely amorpha-4,11-diene. Here we describe the isolation of a cDNA clone encoding amorpha-4,11-diene synthase. The deduced amino acid sequence exhibits the highest identity (50%) with a putative sesquiterpene cyclase of A. annua. When expressed in Escherichia coli, the recombinant enzyme catalyses the formation of amorpha-4,11-diene from farnesyl diphosphate. Introduction of the gene into tobacco (Nicotiana tabacum L.) resulted in the expression of an active enzyme and the accumulation of amorpha-4,11-diene ranging from 0.2 to 1.7 ng per g fresh weight. Received: 8 June 2000 / Accepted: 21 August 2000     

Mechanism of 4-(beta-D-ribofuranosyl)aminobenzene 5'-phosphate synthase, a key enzyme in the methanopterin biosynthetic pathway

The first committed step in methanopterin biosynthesis is catalyzed by 4-(beta-D-ribofuranosyl)aminobenzene 5'-phosphate (RFA-P) synthase. Unlike all known phosphoribosyltransferases, beta-RFA-P synthase catalyzes the unique formation of a C-riboside instead of an N-riboside in the condensation of p-aminobenzoic acid (pABA) and 5-phospho-alpha-D-ribosyl-1-pyrophosphate (PRPP) to produce 4-(beta-D-ribofuranosyl)aminobenzene 5'-phosphate (beta-RFA-P), CO(2), and inorganic pyrophosphate (PP(i)). Here we report the successful cloning, active overexpression in Escherichia coli, and purification of this homodimeric enzyme containing two 36.2-kDa subunits from the methanogen Methanococcus jannaschii. Steady-state initial velocity and product inhibition kinetic studies indicate an ordered Bi-Ter mechanism involving binding of PRPP, then pABA, followed by release of the products CO(2), then beta-RFA-P, and finally PP. The Michaelis parameters are as follows: K(m)pABA, 0.15 mm; K(m)PRPP, 1.50 mm; V(max), 375 nmol/min/mg; k(cat), 0.23 s(-1). CO(2) showed uncompetitive inhibition, K(i) = 0.990 mm, under varied PRPP and saturated pABA, and a mixed type of inhibition, K(1) = 1.40 mm and K = 3.800 mm, under varied pABA and saturated PRPP. RFA-P showed uncompetitive inhibition, K(i) = 0.210 mm, under varied PRPP and saturated pABA, and again uncompetitive, K(i) = 0.300 mm, under saturated PRPP and varied pABA. PP(i) exhibits competitive inhibition, K(i) = 0.320 mm, under varied PRPP and saturated pABA, and a mixed type of inhibition, K(1) = 0.60 mm and K(2) = 1.900 mm, under saturated PRPP and varied pABA. Synthase lacks any chromogenic cofactor, and the presence of pyridoxal phosphate and the mechanistically related pyruvoyl cofactors has been strictly excluded.     

Crystal structure of Bacillus anthracis phosphoglucosamine mutase, an enzyme in the peptidoglycan biosynthetic pathway

Phosphoglucosamine mutase (PNGM) is an evolutionarily conserved bacterial enzyme that participates in the cytoplasmic steps of peptidoglycan biosynthesis. As peptidoglycan is essential for bacterial survival and is absent in humans, enzymes in this pathway have been the focus of intensive inhibitor design efforts. Many aspects of the structural biology of the peptidoglycan pathway have been elucidated, with the exception of the PNGM structure. We present here the crystal structure of PNGM from the human pathogen and bioterrorism agent Bacillus anthracis. The structure reveals key residues in the large active site cleft of the enzyme which likely have roles in catalysis and specificity. A large conformational change of the C-terminal domain of PNGM is observed when comparing two independent molecules in the crystal, shedding light on both the apo- and ligand-bound conformers of the enzyme. Crystal packing analyses and dynamic light scattering studies suggest that the enzyme is a dimer in solution. Multiple sequence alignments show that residues in the dimer interface are conserved, suggesting that many PNGM enzymes adopt this oligomeric state. This work lays the foundation for the development of inhibitors for PNGM enzymes from human pathogens.     

Cloning, structure, and expression of the mitochondrial cytochrome P-450 sterol 26-hydroxylase, a bile acid biosynthetic enzyme

The conversion of cholesterol into bile acids in the liver represents the major catabolic pathway for the removal of cholesterol from the body. In this complex biosynthetic pathway, at least 10 enzymes modify both the ring structure and side chain of cholesterol, resulting in the formation of the primary bile acids, cholic acid, and chenodeoxycholic acid. To gain insight into the details and regulation of this pathway, we have used protein sequencing and molecular cloning techniques to isolate and characterize a cDNA encoding the rabbit mitochondrial sterol 26-hydroxylase. This enzyme catalyzes the first step in the oxidation of the side chain of sterol intermediates in the biosynthesis of bile acids. The structure of the sterol 26-hydroxylase, as deduced by both DNA sequence analysis of the cDNA and protein sequence analysis, reveals it to be a mitochondrial cytochrome P-450. A signal sequence of 36 residues precedes a coding region of 499 amino acids, predicting a molecular weight of 56,657 for the mature protein. The identity of the 26-hydroxylase cDNA was further confirmed by expression in monkey COS cells employing a versatile eukaryotic expression vector. Blotting experiments revealed that the mRNA for this enzyme is expressed in many tissues and that it is encoded by a low copy number gene in the rabbit genome.     

Purification of an indole alkaloid biosynthetic enzyme, strictosidine synthase, from a recombinant strain of Escherichia coli.

The gene for the indole alkaloid biosynthetic enzyme, strictosidine synthase, of Catharanthus roseus has been cloned into an inducible Escherichia coli expression vector using an expression cassette polymerase chain reaction technique. Induction of the gene resulted in overexpression of the enzyme which accumulated mainly as insoluble inclusion bodies. Denaturation and refolding of the insoluble protein resulted in the ability to purify up to 6 mg of active enzyme from a single liter of cell culture. The recombinant enzyme has good activity (approximately 30 nkat/mg).     

Dihydrodipicolinate synthase ofnicotiana sylvestris,a chloroplast-localized enzyme of the lysine pathway

The first enzyme of the lysine-biosynthesis pathway, dihydrodipicolinate synthase (DHDPS; EC 4.2.1.52) has been purified and characterized inNicotiana sylvestris Speggazini et Comes. A purification scheme was developed for the native DHDPS that subsequently led to the purification to homogeneity of its subunits using two-dimensional gel electrophoresis. Subsequent elution of the purified polypeptide has opened the way for the production of rabbit polyclonal anti-DHDPS sera. The molecular weight of the enzyme was determined to be 164000 daltons (Da) by an electrophoretic method. By labeling with [¹⁴C]pyruvate, the enzyme was shown to be composed of four identical subunits of 38500 Da. Pyruvate acts as a stabilizing agent and contributes to the preservation of the tetrameric structure of the enzyme. The enzyme ofN. sylvestris is strongly inhibited by lysine with anI _0.5 of 15 μM; S-(2-aminoethyl)L-cysteine and γ-hydroxylysine, two lysine analogs, were found to be only weak inhibitors. An analog of pyruvate, 2-oxobutyrate, competitively inhibited the enzyme and was found to act at the level of the pyruvate-binding site. Dihydrodipicolinate synthase was localized in the chloroplast and identified as a soluble stromal enzyme by enzymatic and immunological methods. Its properties are compared with those known for other plant and bacterial DHDPS enzymes.     

Yeast cystathionine beta-synthase is a pyridoxal phosphate enzyme but, unlike the human enzyme, is not a heme protein

Our studies of cystathionine beta-synthase from Saccharomyces cerevisiae (yeast) are aimed at clarifying the cofactor dependence and catalytic mechanism and obtaining a system for future investigations of the effects of mutations that cause human disease (homocystinuria or coronary heart disease). We report methods that yielded high expression of the yeast gene in Escherichia coli and of purified yeast cystathionine beta-synthase. The absorption and circular dichroism spectra of the homogeneous enzyme were characteristic of a pyridoxal phosphate enzyme and showed the absence of heme, which is found in human and rat cystathionine beta-synthase. The absence of heme in the yeast enzyme facilitates spectroscopic studies to probe the catalytic mechanism. The reaction of the enzyme with L-serine in the absence of L-homocysteine produced the aldimine of aminoacrylate, which absorbed at 460 nm and had a strong negative circular dichroism band at 460 nm. The formation of this intermediate from the product, L-cystathionine, demonstrates the partial reversibility of the reaction. Our results establish the overall catalytic mechanism of yeast cystathionine beta-synthase and provide a useful system for future studies of structure and function. The absence of heme in the functional yeast enzyme suggests that heme does not play an essential catalytic role in the rat and human enzymes. The results are consistent with the absence of heme in the closely related enzymes O-acetylserine sulfhydrylase, threonine deaminase, and tryptophan synthase.     

The structure of adenylosuccinate lyase, an enzyme with dual activity in the de novo purine biosynthetic pathway

Background: Adenylosuccinate lyase is an enzyme that plays a critical role in both cellular replication and metabolism via its action in the de novo purine biosynthetic pathway. Adenylosuccinate lyase is the only enzyme in this pathway to catalyze two separate reactions, enabling it to participate in the addition of a nitrogen at two different positions in adenosine monophosphate. Both reactions catalyzed by adenylosuccinate lyase involve the beta-elimination of fumarate. Enzymes that catalyze this type of reaction belong to a superfamily, the members of which are homotetramers. Because adenylosuccinate lyase plays an integral part in maintaining proper cellular metabolism, mutations in the human enzyme can have severe clinical consequences, including mental retardation with autistic features. Results: The 1.8 A crystal structure of adenylosuccinate lyase from Thermotoga maritima has been determined by multiwavelength anomalous dispersion using the selenomethionine-substituted enzyme. The fold of the monomer is reminiscent of other members of the beta-elimination superfamily. However, its active tetrameric form exhibits striking differences in active-site architecture and cleft size. Conclusions: This first structure of an adenylosuccinate lyase reveals that, along with the catalytic base (His141) and the catalytic acid (His68), Gln212 and Asn270 might play a vital role in catalysis by properly orienting the succinyl moiety of the substrates. We propose a model for the dual activity of adenylosuccinate lyase: a single 180 degrees bond rotation must occur in the substrate between the first and second enzymatic reactions. Modeling of the pathogenic human S413P mutation indicates that the mutation destabilizes the enzyme by disrupting the C-terminal extension.     

Structure and mechanism of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase. An enzyme in the mevalonate-independent isoprenoid biosynthetic pathway.

The enzyme 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MECDP) synthase catalyzes the conversion of 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate (CDP-ME2P) to MECDP, a highly unusual cyclodiphosphate-containing intermediate on the mevalonate-independent pathway to isopentenyl diphosphate and dimethylallyl diphosphate. We now report two x-ray crystal structures of MECDP synthase refined to 2.8-A resolution. The first structure contains a bound Mn(2+) cation, and the second structure contains CMP, MECDP, and Mn(2+). The protein adopts a homotrimeric quaternary structure built around a central hydrophobic cavity and three externally facing active sites. Each of these active sites is located between two adjacent monomers. A tetrahedrally arranged transition metal binding site, potentially occupied by Mn(2+), sits at the base of the active site cleft. A phosphate oxygen of MECDP and the side chains of Asp(8), His(10), and His(42) occupy the metal ion coordination sphere. These structures reveal for the first time the structural determinants underlying substrate, product, and Mn(2+) recognition and the likely catalytic mechanism accompanying the biosynthesis of the cyclodiphosphate-containing isoprenoid precursor, MECDP.     

Sulfoquinovose synthase - an important enzyme in the N-glycosylation pathway of Sulfolobus acidocaldarius

Recently, the Surface (S)-layer glycoprotein of the thermoacidophilic crenarchaeote Sulfolobus acidocaldarius was found to be N-glycosylated with a heterogeneous family of glycans, with the largest having a composition Glc(1)Man(2)GlcNAc(2) plus 6-sulfoquinovose. However, genetic analyses of genes involved in the N-glycosylation process in Crenarchaeota were missing so far. In this study we identify a gene cluster involved in the biosynthesis of sulfoquinovose and important for the assembly of the S-layer N-glycans. A successful markerless in-frame deletion of agl3 resulted in a decreased molecular mass of the S-layer glycoprotein SlaA and the flagellin FlaB, indicating a change in the N-glycan composition. Analyses with nanoLC ES-MS/MS confirmed the presence of only a reduced trisaccharide structure composed of Man(1) GlcNAc(2) , missing the sulfoquinovose, a mannose and glucose. Biochemical studies of the recombinant Agl3 confirmed the proposed function as a UDP-sulfoquinovose synthase. Furthermore, S. acidocaldarius cells lacking agl3 had a significantly lower growth rate at elevated salt concentrations compared with the background strain, underlining the importance of the N-glycosylation to maintain an intact and stable cell envelope, to enable the survival of S. acidocaldarius in its extreme environment.     

Crystal structure of Escherichia coli PdxA,an enzyme involved in the pyridoxal phosphate biosynthesis pathway

Pyridoxal 5'-phosphate is an essential cofactor for many enzymes responsible for the metabolic conversions of amino acids. Two pathways for its de novo synthesis are known. The pathway utilized by Escherichia coli consists of six enzymatic steps catalyzed by six different enzymes. The fourth step is catalyzed by 4-hydroxythreonine-4-phosphate dehydrogenase (PdxA, E.C. 1.1.1.262), which converts 4-hydroxy-l-threonine phosphate (HTP) to 3-amino-2-oxopropyl phosphate. This divalent metal ion-dependent enzyme has a strict requirement for the phosphate ester form of the substrate HTP, but can utilize either NADP+ or NAD+ as redox cofactor. We report the crystal structure of E. coli PdxA and its complex with HTP and Zn2+. The protein forms tightly bound dimers. Each monomer has an alpha/beta/alpha-fold and can be divided into two subdomains. The active site is located at the dimer interface, within a cleft between the two subdomains and involves residues from both monomers. A Zn2+ ion is bound within each active site, coordinated by three conserved histidine residues from both monomers. In addition two conserved amino acids, Asp247 and Asp267, play a role in maintaining integrity of the active site. The substrate is anchored to the enzyme by the interactions of its phospho group and by coordination of the amino and hydroxyl groups by the Zn2+ ion. PdxA is structurally similar to, but limited in sequence similarity with isocitrate dehydrogenase and isopropylmalate dehydrogenase. These structural similarities and the comparison with a NADP-bound isocitrate dehydrogenase suggest that the cofactor binding mode of PdxA is very similar to that of the other two enzymes and that PdxA catalyzes a stepwise oxidative decarboxylation of the substrate HTP.     

Yeast Eap1p, an eIF4E-associated protein, has a separate function involving genetic stability

A rate-limiting step during translation initiation in eukaryotic cells involves binding of the initiation factor eIF4E to the 7-methylguanosine-containing cap of mRNAs. Overexpression of eIF4E leads to malignant transformation [1-3], and eIF4E is elevated in many human cancers [4-7]. In mammalian cells, three eIF4E-binding proteins each interact with eIF4E and inhibit its function [8-10]. In yeast, EAP1 encodes a protein that binds eIF4E and inhibits cap-dependent translation in vitro [11]. A point mutation in the canonical eIF4E-binding motif of Eap1p blocks its interaction with eIF4E [11]. Here, we characterized the genetic interactions between EAP1 and NDC1, a gene whose function is required for duplication of the spindle pole body (SPB) [12], the centrosome-equivalent organelle in yeast that functions as the centrosome. We found that the deletion of EAP1 is lethal when combined with the ndc1-1 mutation. Mutations in NDC1 or altered NDC1 gene dosage lead to genetic instability [13,14]. Yeast strains lacking EAP1 also exhibit genetic instability. We tested whether these phenotypes are due to loss of EAP1 function in regulating translation. We found that both the synthetic lethal phenotype and the genetic instability phenotypes are rescued by a mutant allele of EAP1 that is unable to bind eIF4E. Our findings suggest that Eap1p carries out an eIF4E-independent function to maintain genetic stability, most likely involving SPBs.     

6-O-sulfotransferase-1 represents a critical enzyme in the anticoagulant heparan sulfate biosynthetic pathway

Using recombinant retroviral transduction, we have introduced the heparin/heparan sulfate (HS) 3-O-sulfotransferase 1 (3-OST-1) gene into Chinese hamster ovary (CHO) cells. Expression of 3-OST-1 confers upon CHO cells the ability to produce anticoagulantly active HS (HS(act)). To understand how 6-OST and other proteins regulate HS(act) biosynthesis, a CHO cell clone with three copies of 3-OST-1 was chemically mutagenized. Resulting mutants that make HS but are defective in generating HS(act) were single-cell-cloned. One cell mutant makes fewer 6-O-sulfated residues. Modification of HS chains from the mutant with pure 6-OST-1 and 3'-phosphoadenosine 5'-phosphosulfate increased HS(act) from 7% to 51%. Transfection of this mutant with 6-OST-1 created a CHO cell line that makes HS, 50% of which is HS(act). We discovered in this study that (i) 6-OST-1 is a limiting enzyme in the HS(act) biosynthetic pathway in vivo when the limiting nature of 3-OST-1 is removed; (ii) HS chains from the mutant cells serve as an excellent substrate for demonstrating that 6-OST-1 is the limiting factor for HS(act) generation in vitro; (iii) in contradiction to the literature, 6-OST-1 can add 6-O-sulfate to GlcNAc residues, especially the critical 6-O-sulfate in the antithrombin binding motif; (iv) both 3-O- and 6-O-sulfation can be the final step in HS(act) biosynthesis in contrast to prior publications that concluded 3-O-sulfation is the final step in HS(act) biosynthesis; (v), in the presence of HS interacting protein peptide, 3-O-sulfate-containing sugars can be degraded into disaccharides by heparitinase digestion as demonstrated by capillary high performance liquid chromatography coupled with mass spectrometry.