Effect of C-Terminal Protein Tags on Pentitol and l-Arabinose Transport by Ambrosiozyma monospora Lat1 and Lat2 Transporters in Saccharomyces cerevisiae

Functional expression in heterologous hosts is often less successful for integral membrane proteins than for soluble proteins. Here, two Ambrosiozyma monospora transporters were successfully expressed in Saccharomyces cerevisiae as tagged proteins. Growth of A. monospora on l-arabinose instead of glucose caused transport activities of l-arabinose, l-arabitol, and ribitol, measured using l-[1-³H]arabinose, l-[¹⁴C]arabitol, and [¹⁴C]ribitol of demonstrated purity. A. monospora LAT1 and LAT2 genes were cloned earlier by using their ability to improve the growth of genetically engineered Saccharomyces cerevisiae on l-arabinose. However, the l-arabinose and pentitol transport activities of S. cerevisiae carrying LAT1 or LAT2 are only slightly greater than those of control strains. S. cerevisiae carrying the LAT1 or LAT2 gene fused in frame to the genes for green fluorescent protein (GFP) or red fluorescent protein (mCherry) or adenylate kinase (AK) exhibited large (>3-fold for LAT1; >20-fold for LAT2) increases in transport activities. Lat1-mCherry transported l-arabinose with high affinity (K_m ≈ 0.03 mM) and l-arabitol and ribitol with very low affinity (K_m ≥ 75 mM). The Lat2-GFP, Lat2-mCherry, and Lat2-AK fusion proteins could not transport l-arabinose but were high-affinity pentitol transporters (K_ms ≈ 0.2 mM). The l-arabinose and pentitol transport activities of A. monospora could not be completely explained by any combination of the observed properties of tagged Lat1 and Lat2, suggesting either that tagging and expression in a foreign membrane alters the transport kinetics of Lat1 and/or Lat2 or that A. monospora contains at least one more l-arabinose transporter.     

Role of Transmembrane Domain 8 in Substrate Selectivity and Translocation of SteT,a Member of the l-Amino Acid Transporter (LAT) Family

System l-amino acid transporters (LAT) belong to the amino acid, polyamine, and organic cation superfamily of transporters and include the light subunits of heteromeric amino acid transporters and prokaryotic homologues. Cysteine reactivity of SteT (serine/threonine antiporter) has been used here to study the substrate-binding site of LAT transporters. Residue Cys-291, in transmembrane domain 8 (TM8), is inactivated by thiol reagents in a substrate protectable manner. Surprisingly, DTT activated the transporter by reducing residue Cys-291. Cysteine-scanning mutagenesis of TM8 showed DTT activation in the single-cysteine mutants S287C, G294C, and S298C, lining the same α-helical face. S-Thiolation in Escherichia coli cells resulted in complete inactivation of the single-cysteine mutant G294C. l-Serine blocked DTT activation with an EC₅₀ similar to the apparent K_M of this mutant. Thus, S-thiolation abolished substrate translocation but not substrate binding. Mutation of Lys-295, to Cys (K295C) broadened the profile of inhibitors and the spectrum of substrates with the exception of imino acids. A structural model of SteT based on the structural homologue AdiC (arginine/agmatine antiporter) positions residues Cys-291 and Lys-295 in the putative substrate binding pocket. All this suggests that Lys-295 is a main determinant in the recognition of the side chain of SteT substrates. In contrast, Gly-294 is not facing the surface, suggesting conformational changes involving TM8 during the transport cycle. Our results suggest that TM8 sculpts the substrate-binding site and undergoes conformational changes during the transport cycle of SteT.     

Substrate Binding Tunes Conformational Flexibility and Kinetic Stability of an Amino Acid Antiporter

We used single molecule dynamic force spectroscopy to unfold individual serine/threonine antiporters SteT from Bacillus subtilis. The unfolding force patterns revealed interactions and energy barriers that stabilized structural segments of SteT. Substrate binding did not establish strong localized interactions but appeared to be facilitated by the formation of weak interactions with several structural segments. Upon substrate binding, all energy barriers of the antiporter changed thereby describing the transition from brittle mechanical properties of SteT in the unbound state to structurally flexible conformations in the substrate-bound state. The lifetime of the unbound state was much shorter than that of the substrate-bound state. This leads to the conclusion that the unbound state of SteT shows a reduced conformational flexibility to facilitate specific substrate binding and a reduced kinetic stability to enable rapid switching to the bound state. In contrast, the bound state of SteT showed an increased conformational flexibility and kinetic stability such as required to enable transport of substrate across the cell membrane. This result supports the working model of antiporters in which alternate substrate access from one to the other membrane surface occurs in the substrate-bound state.The amino acid/polyamine/organocation (APC)² superfamily comprises about 250 members that occur in all phyla from prokaryotes to higher eukaryotes. These membrane proteins function as solute/cation symporters or solute/solute antiporters (1). One APC subfamily is established by l-amino acid transporters (LATs), which correspond to the light subunits of eukaryotic heteromeric amino acid transporters (2, 3). Heteromeric amino acid transporters are composed of a light subunit that provides transport activity and a disulfide-linked heavy subunit that shows responsibility for plasma membrane targeting. Genetic defects in light and heavy subunits cause a number of inherited human diseases. Mutations in the light as well as the heavy subunit of system b^0,+ lead to cystinuria (4, 5), whereas mutations in the light subunit y⁺LAT1 cause lysinuric protein intolerance (6, 7). Another light subunit, xCT that mediates cysteine uptake and glutamate efflux (8, 9), is involved in vivo in cocaine relapse (10) and maintenance of the plasma redox balance (11). LAT1, the light subunit of system L, is overexpressed in certain primary human tumors. It transports essential neutral amino acids with long, branched, or aromatic side chains required by tumor cells to support their unabated growth (12). Therefore, amino acid transporters like LAT1 are attractive anticancer drug targets.So far a high resolution structure of a eukaryotic LAT family member is not available. However, studies on xCT revealed a membrane topology of 12 transmembrane helices (TMHs) with cytosolic N and C termini and a re-entrant loop structure between TMHs II and III (13). The identified first prokaryotic member of the LAT family, SteT from Bacillus subtilis, is a serine/threonine antiporter, which shows high sequence identity (∼30%) to the light subunits of eukaryotic heteromeric amino acid transporters. Moreover SteT exhibits a similar putative membrane topology and sequential mode of obligate exchange (14). Thus, SteT is an excellent model for studying the structure-function relationship of LAT family members.According to current models, transport proteins undergo functionally related conformational changes. Transporters alternate between two conformations to expose their binding sites to the cytoplasmic and extracellular side (15–22). However, prior to conformational changes substrates have to be recognized and bound. If substrates are amino acids, three main features can be used for specific selection and binding: (i) the negatively charged α-carboxyl group, (ii) the positively charged α-amino group, and (iii) the electrostatic, hydrophobic, or spatial properties of the side chain (22–24). α-Carboxyl and α- amino groups of l-amino acids possess similar structural and chemical characteristics (except for proline); however, their side chains differ in shape, size, and electrostatic properties. Combinations of these features are assumed to establish different interactions within the side chain binding pocket, which determines the substrate specificity of the transporter. The two main substrates of SteT, l-serine and l-threonine, differ by only one methylene group in their side chain; thus they have similar properties. Additionally SteT transports aromatic l-amino acids (Trp, Tyr, and Phe) albeit less efficiently (14).Since its invention, the atomic force microscope (AFM) (25) has evolved from a surface imaging device to a versatile tool for studying interactions of manifold biological systems (26–31). Introduced to characterize interactions between receptor-ligand complexes (32, 33) and complementary DNA strands (34), AFM-based single molecule force spectroscopy (SMFS) has been exploited to explore antibody-antigen recognition (35) and unfolding and refolding of soluble proteins (29, 36) and to probe the adhesion of living cells at molecular resolution (37). Applied to membrane proteins, SMFS uses the AFM stylus to exert a mechanical pulling force to the terminal end of a protein that is embedded and anchored by the lipid membrane (see Fig. 1A) (38). Sufficiently high stretching forces initiate sequential unfolding of the membrane protein with each step indicating the unfolding of a structural segment (39). Recording the applied force over pulling distance results in a force-distance (F-D) curve in which individual force peaks represent the rupture of intra- and intermolecular interactions. The height of a force peak measures the strength of an interaction with piconewton accuracy, and the pulling distance, at which the force peak occurs, allows the interaction within the membrane protein structure to be located (38).Open in a separate windowFIGURE 1.SMFS of SteT. A, pushing the AFM stylus onto the proteoliposomes promotes contacting single transporters to the stylus. This molecular link allows exertion of a mechanical pulling force that initiates stepwise unfolding of SteT. During the experiments, sample and cantilever are immersed in buffer solution. B, F-D curves recorded while unfolding single substrate-free SteT molecules. C, superimpositions of F-D curves recorded while unfolding SteT in buffer lacking any substrate (top) and supplemented with 5 mm l-serine (middle) or 5 mm l-threonine (bottom). Superimpositions are represented as density plots, each calculated from 60 F-D curves. Gray lines represent WLC curves with a persistence length of 0.4 nm and contour length (in amino acids) as indicated by the numbers next to the lines. The contour lengths were obtained from the Gaussian fits shown in D. F-D curves were obtained at room temperature at a pulling velocity of 654 nm/s in buffer solution (150 mm NaCl, 20 mm Tris-HCl, pH 8.0, substrate as indicated). D, frequency of force peaks detected at different positions of the stretched polypeptide. Every force peak detected in individual F-D curves (B) was fitted using the WLC model with the contour length of the stretched polypeptide as the only fitting parameter. The frequency at which the force peaks appeared is plotted in the histogram: substrate-free, n = 132; 5 mm l-serine, n = 128; and 5 mm l-threonine, n = 127. The bin size of the histograms is 3 aa and reflects the accuracy of fitting the WLC model (55) to individual force peaks. Error bars representing the S.E. were calculated using S.E. = (p(1 − p)/n)^0.5 where p is the probability and n is the total number of F-D curves. The width of each force peak distribution is given by the experimental noise, conformational variability of the structural segments, and fitting accuracy of the force peaks (53, 99–102). The gray solid curve represents the sum of seven Gaussian fits to the seven main peaks from the histograms and superimpositions (C). Numbers next to peaks denote peak positions (measured in amino acids) obtained from Gaussian fits.Besides quantification and localization of molecular interactions in membrane proteins, SMFS provides information about their energy landscape. For that purpose, the interactions of membrane proteins are probed over a range of different time scales by dynamic force spectroscopy (DFS). Bell (40) and Evans and co-worker (41, 42) provided the most commonly used theoretical framework to analyze DFS data. Their model describes the deformation of the energy landscape by an externally applied force, F. Such force-induced deformations reduce the energy barriers that separate bound and unbound states (see Fig. 2). Consequently transition rates over such energy barriers are force-dependent. Probing the interactions at different pulling velocities and thus at different force loading rates, r_f, leads to a so-called dynamic force spectrum in which the most probable force, F*, of rupture is plotted versus the logarithm of r_f. In these dynamic force spectra, each linear regime represents an energy barrier. Energy barriers located closer to the bound state are probed at higher pulling velocities because the energy barriers located further from the bound state are suppressed by increasingly applied forces (see Fig. 2) (41). The slope of each linear regime measures the distance from the ground state to the transition state, whereas extrapolation of a linear regime to zero force provides the rate constant of crossing the corresponding barrier in the absence of any load. These two parameters allow an estimate of the rigidity of the probed structure (43, 44).Open in a separate windowFIGURE 2.Energy landscape tilted by force. Schematic representation of the free energy profile along the reaction coordinate and applied force according to the Bell-Evans theory (40–42). The potential along the reaction coordinate (vector of force) in the absence of force (black curve) exhibits two energy barriers separating the folded from the unfolded state. Application of an external force, F, changes the thermal likelihood of reaching the top of the energy barrier(s). Although for a sharp barrier the position, x_u, of the energy barrier relative to the folded state is not changed, the thermally averaged projection of the energy profile along the pulling direction is tilted by the mechanical energy (−F·cos θ)x (long-dashed line). This tilt decreases the energy barriers (short-dashed curve). Consequently the relevant energy barrier that has to be overcome is the outermost barrier. At slow pulling velocities, the thermal contribution is higher, and therefore, the mechanical energy required to overcome the barrier is smaller. With increasing pulling velocities, the barriers are further lowered. At some velocity, the height of the outer barrier will be lower than that of the inner barrier (short-dashed curve), which then becomes the relevant energy barrier to be overcome. Each energy barrier manifests as a linear regime in dynamic force spectra (Fig. 3).In this study, we applied SMFS to characterize molecular interactions that stabilize SteT in the absence and in the presence of its substrates, l-serine and l-threonine. We used DFS to characterize how substrate binding changes the energy landscape and the mechanical properties of the antiporter. It was observed that the structural regions stabilized within SteT did not depend on substrate binding. However, substrate binding dynamically changed the energy landscape of these structures. In the absence of substrate all structural regions within SteT were stabilized by a narrow inner energy barrier and co-stabilized by a second outer energy barrier. The unique properties of these energy barriers restricted the conformation of SteT thereby trapping the antiporter in a kinetically instable and mechanically rigid conformation. In contrast, substrate binding sets SteT into a different energy minimum that significantly increased the kinetic stability and conformational flexibility of the antiporter.     

Regulation of sulfate uptake by amino acids in cultured tobacco cells

The sulfur requirements of tobacco (Nicotiana tabacum L. var. Xanthi) XD cells grown in chemically defined liquid media can be satisfied by sulfate, thiosulfate, l-cyst(e)ine, l-methionine or glutathione, and somewhat less effectively by d-cyst (e) ine, d-methionine or dl-homocyst (e)ine. Sulfate uptake is inhibited after a 2 hr lag by l-cyst (e)ine, l-methionine, l-homocyst(e)ine or l-isoleucine, but not by any of the other protein amino acids, nor by d-cyst(e)ine. l-cyst(e)ine is neither a competitive nor a non-competitive inhibitor of sulfate uptake. Its action most closely resembles apparent uncompetitive inhibition. Inhibition of sulfate uptake by l-cyst(e)ine can be partially prevented by equimolar l-arginine, l-lysine, l-leucine, l-phenylalanine, l-tyrosine or l-tryptophan, but is little affected by any of the other protein amino acids. The effective amino acids are apparent competitive inhibitors of l-cyst(e)ine uptake after a 2 hr lag. Inhibition of sulfate uptake by l-methionine cannot be prevented, nor can uptake of l-methionine be inhibited by any single protein amino acid. The results suggest the occurrence of negative feedback control of sulfate assimilation by the end products, the sulfur amino acids, in cultured tobacco cells.     

Human 60-kDa Lysophospholipase Contains an N-terminal l-Asparaginase Domain That Is Allosterically Regulated by l-Asparagine

The structural and functional characterization of human enzymes that are of potential medical and therapeutic interest is of prime significance for translational research. One of the most notable examples of a therapeutic enzyme is l-asparaginase, which has been established as an antileukemic protein drug for more than four decades. Up until now, only bacterial enzymes have been used in therapy despite a plethora of undesired side effects mainly attributed to the bacterial origins of these enzymes. Therefore, the replacement of the currently approved bacterial drugs by human homologs aiming at the elimination of adverse effects is of great importance. Recently, we structurally and biochemically characterized the enzyme human l-asparaginase 3 (hASNase3), which possesses l-asparaginase activity and belongs to the N-terminal nucleophile superfamily of enzymes. Inspired by the necessity for the development of a protein drug of human origin, in the present study, we focused on the characterization of another human l-asparaginase, termed hASNase1. This bacterial-type cytoplasmic l-asparaginase resides in the N-terminal subdomain of an overall 573-residue protein previously reported to function as a lysophospholipase. Our kinetic, mutagenesis, structural modeling, and fluorescence labeling data highlight allosteric features of hASNase1 that are similar to those of its Escherichia coli homolog, EcASNase1. Differential scanning fluorometry and urea denaturation experiments demonstrate the impact of particular mutations on the structural and functional integrity of the l-asparaginase domain and provide a direct comparison of sites critical for the conformational stability of the human and E. coli enzymes.     

Specificity Determinants for Lysine Incorporation in Staphylococcus aureus Peptidoglycan as Revealed by the Structure of a MurE Enzyme Ternary Complex

Formation of the peptidoglycan stem pentapeptide requires the insertion of both l and d amino acids by the ATP-dependent ligase enzymes MurC, -D, -E, and -F. The stereochemical control of the third position amino acid in the pentapeptide is crucial to maintain the fidelity of later biosynthetic steps contributing to cell morphology, antibiotic resistance, and pathogenesis. Here we determined the x-ray crystal structure of Staphylococcus aureus MurE UDP-N-acetylmuramoyl-l-alanyl-d-glutamate:meso-2,6-diaminopimelate ligase (MurE) (E.C. 6.3.2.7) at 1.8 Å resolution in the presence of ADP and the reaction product, UDP-MurNAc-l-Ala-γ-d-Glu-l-Lys. This structure provides for the first time a molecular understanding of how this Gram-positive enzyme discriminates between l-lysine and d,l-diaminopimelic acid, the predominant amino acid that replaces l-lysine in Gram-negative peptidoglycan. Despite the presence of a consensus sequence previously implicated in the selection of the third position residue in the stem pentapeptide in S. aureus MurE, the structure shows that only part of this sequence is involved in the selection of l-lysine. Instead, other parts of the protein contribute substrate-selecting residues, resulting in a lysine-binding pocket based on charge characteristics. Despite the absolute specificity for l-lysine, S. aureus MurE binds this substrate relatively poorly. In vivo analysis and metabolomic data reveal that this is compensated for by high cytoplasmic l-lysine concentrations. Therefore, both metabolic and structural constraints maintain the structural integrity of the staphylococcal peptidoglycan. This study provides a novel focus for S. aureus-directed antimicrobials based on dual targeting of essential amino acid biogenesis and its linkage to cell wall assembly.     

Regulation of the ald Gene Encoding Alanine Dehydrogenase by AldR in Mycobacterium smegmatis

The regulatory gene aldR was identified 95 bp upstream of the ald gene encoding l-alanine dehydrogenase in Mycobacterium smegmatis. The AldR protein shows sequence similarity to the regulatory proteins of the Lrp/AsnC family. Using an aldR deletion mutant, we demonstrated that AldR serves as both activator and repressor for the regulation of ald gene expression, depending on the presence or absence of l-alanine. The purified AldR protein exists as a homodimer in the absence of l-alanine, while it adopts the quaternary structure of a homohexamer in the presence of l-alanine. The binding affinity of AldR for the ald control region was shown to be increased significantly by l-alanine. Two AldR binding sites (O1 and O2) with the consensus sequence GA-N₂-ATC-N₂-TC and one putative AldR binding site with the sequence GA-N₂-GTT-N₂-TC were identified upstream of the ald gene. Alanine and cysteine were demonstrated to be the effector molecules directly involved in the induction of ald expression. The cellular level of l-alanine was shown to be increased in M. smegmatis cells grown under hypoxic conditions, and the hypoxic induction of ald expression appears to be mediated by AldR, which senses the intracellular level of alanine.     

Structural and functional analysis of gum arabic l-rhamnose-α-1,4-d-glucuronate lyase establishes a novel polysaccharide lyase family

Gum arabic (GA) is widely used as an emulsion stabilizer and coating in several industrial applications, such as foods and pharmaceuticals. GA contains a complex carbohydrate moiety, and the nonreducing ends of the side chains are often capped with l-rhamnose; thus, enzymes that can remove these caps are promising tools for the structural analysis of the carbohydrates comprising GA. In this study, GA-specific l-rhamnose-α-1,4-d-glucuronate lyase from the fungus Fusarium oxysporum 12S (FoRham1) was cloned and characterized. FoRham1 showed the highest amino acid sequence similarity with enzymes belonging to the glycoside hydrolase family 145; however, the catalytic residue on the posterior pocket of the β-propeller fold protein was not conserved. The catalytic residues of FoRham1 were instead conserved with ulvan lyases belonging to polysaccharide lyase family 24. Kinetic analysis showed that FoRham1 has the highest catalytic efficiency for the substrate α-l-rhamnose-(1→4)-d-glucuronic acid. The crystal structures of ligand-free and α-l-rhamnose-(1→4)-d-glucuronic acid –bound FoRham1 were determined, and the active site was identified on the anterior side of the β-propeller. The three-dimensional structure of the active site and mutagenesis analysis revealed the detailed catalytic mechanism of FoRham1. Our findings offer a new enzymatic tool for the further analysis of the GA carbohydrate structure and for elucidating its physiological functions in plants. Based on these results, we renamed glycoside hydrolase family 145 as a new polysaccharide lyase family 42, in which FoRham1 is included.     

Recharacterization of the mammalian cytosolic type 2 (R)-β-hydroxybutyrate dehydrogenase as 4-oxo-l-proline reductase (EC 1.1.1.104)

Early studies revealed that chicken embryos incubated with a rare analog of l-proline, 4-oxo-l-proline, showed increased levels of the metabolite 4-hydroxy-l-proline. In 1962, 4-oxo-l-proline reductase, an enzyme responsible for the reduction of 4-oxo-l-proline, was partially purified from rabbit kidneys and characterized biochemically. However, only recently was the molecular identity of this enzyme solved. Here, we report the purification from rat kidneys, identification, and biochemical characterization of 4-oxo-l-proline reductase. Following mass spectrometry analysis of the purified protein preparation, the previously annotated mammalian cytosolic type 2 (R)-β-hydroxybutyrate dehydrogenase (BDH2) emerged as the only candidate for the reductase. We subsequently expressed rat and human BDH2 in Escherichia coli, then purified it, and showed that it catalyzed the reversible reduction of 4-oxo-l-proline to cis-4-hydroxy-l-proline via chromatographic and tandem mass spectrometry analysis. Specificity studies with an array of compounds carried out on both enzymes showed that 4-oxo-l-proline was the best substrate, and the human enzyme acted with 12,500-fold higher catalytic efficiency on 4-oxo-l-proline than on (R)-β-hydroxybutyrate. In addition, human embryonic kidney 293T (HEK293T) cells efficiently metabolized 4-oxo-l-proline to cis-4-hydroxy-l-proline, whereas HEK293T BDH2 KO cells were incapable of producing cis-4-hydroxy-l-proline. Both WT and KO HEK293T cells also produced trans-4-hydroxy-l-proline in the presence of 4-oxo-l-proline, suggesting that the latter compound might interfere with the trans-4-hydroxy-l-proline breakdown in human cells. We conclude that BDH2 is a mammalian 4-oxo-l-proline reductase that converts 4-oxo-l-proline to cis-4-hydroxy-l-proline and not to trans-4-hydroxy-l-proline, as originally thought. We also hypothesize that this enzyme may be a potential source of cis-4-hydroxy-l-proline in mammalian tissues.     

Evidence for a specific glutamate/h cotransport in isolated mesophyll cells

Mechanically isolated Asparagus sprengeri Regel mesophyll cells were suspended in 1 millimolar CaSO₄. Immediate alkalinization of the medium occured on the addition of 1 millimolar concentrations of l-glutamate (Glu) and its analog l-methionine-d,l-sulfoximine (l-MSO). d-Glu and the l isomers of the protein amino acids did not elicit alkalinization. l-Glu dependent alkalinization was transient and acidification resumed after approximately 30 to 45 minutes. At pH 6.0, 5 millimolar l-Glu stimulated initial rates of alkalinization that varied between 1.3 to 4.1 nmol H⁺/10⁶ cells·minute. l-Glu dependent alkalinization was saturable, increased with decreasing pH, was inhibited by carbonyl cyanide-p-trichloromethoxyphenyl hydrazone (CCCP), and was not stimulated by light. Uptake of l-[U-¹⁴C]glutamate increased as the pH decreased from 6.5 to 5.5, and was inhibited by l-MSO. l-Glu had no influence on K⁺ efflux. Although evidence for multiple amino acid/proton cotransport systems has been found in other tissues, the present report indicates that a highly specific l-Glu/proton uptake process is present in Asparagus mesophyll cells.     

Biosynthesis of l-Sorbose and l-Psicose Based on C—C Bond Formation Catalyzed by Aldolases in an Engineered Corynebacterium glutamicum Strain

The property of loose stereochemical control at aldol products from aldolases helped to synthesize multiple polyhydroxylated compounds with nonnatural stereoconfiguration. In this study, we discovered for the first time that some fructose 1,6-diphosphate aldolases (FruA) and tagatose 1,6-diphosphate (TagA) aldolases lost their strict stereoselectivity when using l-glyceraldehyde and synthesized not only l-sorbose but also a high proportion of l-psicose. Among the aldolases tested, TagA from Bacillus licheniformis (BGatY) showed the highest enzyme activity with l-glyceraldehyde. Subsequently, a “one-pot” reaction based on BGatY and fructose-1-phosphatase (YqaB) generated 378 mg/liter l-psicose and 199 mg/liter l-sorbose from dihydroxyacetone-phosphate (DHAP) and l-glyceraldehyde. Because of the high cost and instability of DHAP, a microbial fermentation strategy was used further to produce l-sorbose/l-psicose from glucose and l-glyceraldehyde, in which DHAP was obtained from glucose through the glycolytic pathway, and some recombination pathways based on FruA or TagA and YqaB were constructed in Escherichia coli and Corynebacterium glutamicum strains. After evaluation of different host cells and combinations of FruA or TagA with YqaB and optimization of gene expression, recombinant C. glutamicum strain WT(pXFTY) was selected and produced 2.53 g/liter total ketoses, with a yield of 0.50 g/g l-glyceraldehyde. Moreover, deletion of gene cgl0331, encoding the Zn-dependent alcohol dehydrogenase in C. glutamicum, was confirmed for the first time to significantly decrease conversion of l-glyceraldehyde to glycerol and to increase yield of target products. Finally, fed-batch culture of strain SY14(pXFTY) produced 3.5 g/liter l-sorbose and 2.3 g/liter l-psicose, with a yield of 0.61 g/g l-glyceraldehyde. This microbial fermentation strategy also could be applied to efficiently synthesize other l-sugars.     

Are cyclooxygenase-2 and nitric oxide involved in the dyskinesia of Parkinson's disease induced by l-DOPA?

Inflammatory mechanisms are proposed to play a role in l-DOPA-induced dyskinesia. Cyclooxygenase-2 (COX2) contributes to inflammation pathways in the periphery and is constitutively expressed in the central nervous system. Considering that inhibition of nitric oxide (NO) formation attenuates l-DOPA-induced dyskinesia, this study aimed at investigating if a NO synthase (NOS) inhibitor would change COX2 brain expression in animals with l-DOPA-induced dyskinesia. To this aim, male Wistar rats received unilateral 6-hydroxydopamine microinjection into the medial forebrain bundle were treated daily with l-DOPA (21 days) combined with 7-nitroindazole or vehicle. All hemi-Parkinsonian rats receiving l-DOPA showed dyskinesia. They also presented increased neuronal COX2 immunoreactivity in the dopamine-depleted dorsal striatum that was directly correlated with dyskinesia severity. Striatal COX2 co-localized with choline-acetyltransferase, calbindin and DARPP-32 (dopamine-cAMP-regulated phosphoprotein-32), neuronal markers of GABAergic neurons. NOS inhibition prevented l-DOPA-induced dyskinesia and COX2 increased expression in the dorsal striatum. These results suggest that increased COX2 expression after l-DOPA long-term treatment in Parkinsonian-like rats could contribute to the development of dyskinesia.     

The biological sulphation of l-tyrosyl peptides

1. A rat-liver supernatant preparation can achieve the biological O-sulphation of l-tyrosylglycine and l-tyrosyl-l-alanine at pH7·0. 2. The optimum concentrations of l-tyrosylglycine and l-tyrosyl-l-alanine in this system are 50mm and 60mm respectively. 3. l-Tyrosylglycine yields two sulphated products, whereas l-tyrosyl-l-alanine yields three sulphated products, when used as acceptor for sulphate in the rat-liver system. 4. With both substrates, one of the sulphated products has been identified as the O-sulphate ester of the corresponding parent peptide.     

One-Pot Production of l-threo-3-Hydroxyaspartic Acid Using Asparaginase-Deficient Escherichia coli Expressing Asparagine Hydroxylase of Streptomyces coelicolor A3(2)

We developed a novel process for efficient synthesis of l-threo-3-hydroxyaspartic acid (l-THA) using microbial hydroxylase and hydrolase. A well-characterized mutant of asparagine hydroxylase (AsnO-D241N) and its homologous enzyme (SCO2693-D246N) were adaptable to the direct hydroxylation of l-aspartic acid; however, the yields were strictly low. Therefore, the highly stable and efficient wild-type asparagine hydroxylases AsnO and SCO2693 were employed to synthesize l-THA. By using these recombinant enzymes, l-THA was obtained by l-asparagine hydroxylation by AsnO followed by amide hydrolysis by asparaginase via 3-hydroxyasparagine. Subsequently, the two-step reaction was adapted to one-pot bioconversion in a test tube. l-THA was obtained in a small amount with a molar yield of 0.076% by using intact Escherichia coli expressing the asnO gene, and thus, two asparaginase-deficient mutants of E. coli were investigated. A remarkably increased l-THA yield of 8.2% was obtained with the asparaginase I-deficient mutant. When the expression level of the asnO gene was enhanced by using the T7 promoter in E. coli instead of the lac promoter, the l-THA yield was significantly increased to 92%. By using a combination of the E. coli asparaginase I-deficient mutant and the T7 expression system, a whole-cell reaction in a jar fermentor was conducted, and consequently, l-THA was successfully obtained from l-asparagine with a maximum yield of 96% in less time than with test tube-scale production. These results indicate that asparagine hydroxylation followed by hydrolysis would be applicable to the efficient production of l-THA.     

An Immunomodulating Motif of the HIV-1 Fusion Protein Is Chirality-independent: IMPLICATIONS FOR ITS MODE OF ACTION*

An immunosuppressive motif was recently found within the HIV-1 gp41 fusion protein (termed immunosuppressive loop-associated determinant core motif (ISLAD CM)). Peptides containing the motif interact with the T-cell receptor (TCR) complex; however, the mechanism by which the motif exerts its immunosuppressive activity is yet to be determined. Recent studies showed that interactions between protein domains in the membrane milieu are not always sterically controlled. Therefore, we utilized the unique membrane leniency toward association between d- and l-stereoisomers to investigate the detailed mechanism by which ISLAD CM inhibits T-cell activation. We show that a d-enantiomer of ISLAD CM (termed ISLAD d-CM) inhibited the proliferation of murine myelin oligodendrocyte glycoprotein (MOG)-(35–55)-specific line T-cells to the same extent as the l-motif form. Moreover, the d- and l-forms preferentially bound spleen-derived T-cells over B-cells by 13-fold. Furthermore, both forms of ISLAD CM co-localized with the TCR on activated T-cells and interacted with the transmembrane domain of the TCR. FRET experiments revealed the importance of basic residues for the interaction between ISLAD CM forms and the TCR transmembrane domain. Ex vivo studies demonstrated that ISLAD d-CM administration inhibited the proliferation (72%) and proinflammatory cytokine secretion of pathogenic MOG(35–55)-specific T-cells. This study provides insights into the immunosuppressive mechanism of gp41 and demonstrates that chirality-independent interactions in the membrane can take place in diverse biological systems. Apart from HIV pathogenesis, the d-peptide reported herein may serve as a potential tool for treating T-cell-mediated pathologies.     

Identification of an l-Arabinose Reductase Gene in Aspergillus niger and Its Role in l-Arabinose Catabolism

The first enzyme in the pathway for l-arabinose catabolism in eukaryotic microorganisms is a reductase, reducing l-arabinose to l-arabitol. The enzymes catalyzing this reduction are in general nonspecific and would also reduce d-xylose to xylitol, the first step in eukaryotic d-xylose catabolism. It is not clear whether microorganisms use different enzymes depending on the carbon source. Here we show that Aspergillus niger makes use of two different enzymes. We identified, cloned, and characterized an l-arabinose reductase, larA, that is different from the d-xylose reductase, xyrA. The larA is up-regulated on l-arabinose, while the xyrA is up-regulated on d-xylose. There is however an initial up-regulation of larA also on d-xylose but that fades away after about 4 h. The deletion of the larA gene in A. niger results in a slow growth phenotype on l-arabinose, whereas the growth on d-xylose is unaffected. The l-arabinose reductase can convert l-arabinose and d-xylose to their corresponding sugar alcohols but has a higher affinity for l-arabinose. The K_m for l-arabinose is 54 ± 6 mm and for d-xylose 155 ± 15 mm.     

Novel Enzyme Family Found in Filamentous Fungi Catalyzing trans-4-Hydroxylation of l-Pipecolic Acid

Hydroxypipecolic acids are bioactive compounds widely distributed in nature and are valuable building blocks for the organic synthesis of pharmaceuticals. We have found a novel hydroxylating enzyme with activity toward l-pipecolic acid (l-Pip) in a filamentous fungus, Fusarium oxysporum c8D. The enzyme l-Pip trans-4-hydroxylase (Pip4H) of F. oxysporum (FoPip4H) belongs to the Fe(II)/α-ketoglutarate-dependent dioxygenase superfamily, catalyzes the regio- and stereoselective hydroxylation of l-Pip, and produces optically pure trans-4-hydroxy-l-pipecolic acid (trans-4-l-HyPip). Amino acid sequence analysis revealed several fungal enzymes homologous with FoPip4H, and five of these also had l-Pip trans-4-hydroxylation activity. In particular, the homologous Pip4H enzyme derived from Aspergillus nidulans FGSC A4 (AnPip4H) had a broader substrate specificity spectrum than other homologues and reacted with the l and d forms of various cyclic and aliphatic amino acids. Using FoPip4H as a biocatalyst, a system for the preparative-scale production of chiral trans-4-l-HyPip was successfully developed. Thus, we report a fungal family of l-Pip hydroxylases and the enzymatic preparation of trans-4-l-HyPip, a bioactive compound and a constituent of secondary metabolites with useful physiological activities.     

A Bifunctional Enzyme in a Single Gene Catalyzes the Incorporation of GlcN into the Aeromonas Core Lipopolysaccharide

The core lipopolysaccharide (LPS) of Aeromonas hydrophila AH-3 and Aeromonas salmonicida A450 is characterized by the presence of the pentasaccharide α-d-GlcN-(1→7)-l-α-d-Hep-(1→2)-l-α-d-Hep-(1→3)-l-α-d-Hep-(1→5)-α-Kdo. Previously it has been suggested that the WahA protein is involved in the incorporation of GlcN residue to outer core LPS. The WahA protein contains two domains: a glycosyltransferase and a carbohydrate esterase. In this work we demonstrate that the independent expression of the WahA glycosyltransferase domain catalyzes the incorporation of GlcNAc from UDP-GlcNAc to the outer core LPS. Independent expression of the carbohydrate esterase domain leads to the deacetylation of the GlcNAc residue to GlcN. Thus, the WahA is the first described bifunctional glycosyltransferase enzyme involved in the biosynthesis of core LPS. By contrast in Enterobacteriaceae containing GlcN in their outer core LPS the two reactions are performed by two different enzymes.