L-serine Dehydratase Research Articles

Variations in protein expression associated with oral cancer.

Differential protein expression of the oral microbiome is related to human diseases, including cancer. In order to reveal the potential relationship between oral bacterial protein expression in oral squamous cell carcinoma (OSCC), we designed this study. We obtained samples of the same patient from cancer lesion and anatomically matched normal site. Then, we used the label free quantitative technique based on liquid chromatography tandem mass spectrometry (LC-MS/MS) to analyze the bacteria in the samples of oral squamous cell carcinoma at the protein level, so as to detect the functional proteins. Protein diversity in the cancer samples was significantly greater than in the normal samples. We identified a substantially higher number of the taxa than those detected in previous studies, demonstrating the presence of a remarkable number of proteins in the groups. In particular, proteins involved in energy production and conversion, proton transport, hydrogen transport and hydrogen ion transmembrane transport, ATP-binding cassette (ABC) transporter, PTS system, and L-serine dehydratase were enriched significantly in the experimental group. Moreover, some proteins associated with Actinomyces and Fusobacterium were highly associated with OSCC and provided a good diagnostic outcome. The present study revealed considerable changes in the expression of bacterial proteins in OSCC and enrich our understanding in this point.

Gene sdaB Is Involved in the Nematocidal Activity of Enterobacter ludwigii AA4 Against the Pine Wood Nematode Bursaphelenchus xylophilus.

Bursaphelenchus xylophilus, a plant parasitic nematode, is the causal agent of pine wilt, a devastating forest tree disease. Essentially, no efficient methods for controlling B. xylophilus and pine wilt disease have yet been developed. Enterobacter ludwigii AA4, isolated from the root of maize, has powerful nematocidal activity against B. xylophilus in a new in vitro dye exclusion test. The corrected mortality of the B. xylophilus treated by E. ludwigii AA4 or its cell extract reached 98.3 and 98.6%, respectively. Morphological changes in B. xylophilus treated with a cell extract from strain AA4 suggested that the death of B. xylophilus might be caused by an increased number of vacuoles in non-apoptotic cell death and the damage to tissues of the nematodes. In a greenhouse test, the disease index of the seedlings of Scots pine (Pinus sylvestris) treated with the cells of strain AA4 plus B. xylophilus or those treated by AA4 cell extract plus B. xylophilus was 38.2 and 30.3, respectively, was significantly lower than 92.5 in the control plants treated with distilled water and B. xylophilus. We created a sdaB gene knockout in strain AA4 by deleting the gene that was putatively encoding the beta-subunit of L-serine dehydratase through Red homologous recombination. The nematocidal and disease-suppressing activities of the knockout strain were remarkably impaired. Finally, we revealed a robust colonization of P. sylvestris seedling needles by E. ludwigii AA4, which is supposed to contribute to the disease-controlling efficacy of strain AA4. Therefore, E. ludwigii AA4 has significant potential to serve as an agent for the biological control of pine wilt disease caused by B. xylophilus.

Identification of an l-serine/l-threonine dehydratase with glutamate racemase activity in mammals

Recent investigations have shown that multiple d-amino acids are present in mammals and these compounds have distinctive physiological functions. Free d-glutamate is present in various mammalian tissues and cells and in particular, it is presumably correlated with cardiac function, and much interest is growing in its unique metabolic pathways. Recently, we first identified d-glutamate cyclase as its degradative enzyme in mammals, whereas its biosynthetic pathway in mammals is unclear. Glutamate racemase is a most probable candidate, which catalyzes interconversion between d-glutamate and l-glutamate. Here, we identified the cDNA encoding l-serine dehydratase-like (SDHL) as the first mammalian clone with glutamate racemase activity. This rat SDHL had been deposited in mammalian databases as a protein of unknown function and its amino acid sequence shares ∼60% identity with that of l-serine dehydratase. Rat SDHL was expressed in Escherichia coli, and the enzymatic properties of the recombinant were characterized. The results indicated that rat SDHL is a multifunctional enzyme with glutamate racemase activity in addition to l-serine/l-threonine dehydratase activity. This clone is hence abbreviated as STDHgr. Further experiments using cultured mammalian cells confirmed that d-glutamate was synthesized and l-serine and l-threonine were decomposed. It was also found that SDHL (STDHgr) contributes to the homeostasis of several other amino acids.

Integrated Systems Pharmacology, Urinary Metabonomics, and Quantitative Real-Time PCR Analysis to Uncover Targets and Metabolic Pathways of the You-Gui Pill in Treating Kidney-Yang Deficiency Syndrome.

Kidney-yang deficiency syndrome (KYDS) is a metabolic disease caused by a neuro-endocrine disorder. The You-gui pill (YGP) is a classic traditional Chinese medicine (TCM) formula for the treatment of KYDS and has been widely used to warm and recuperate KYDS clinically for hundreds of years in China. However, it is unknown whetherthe corresponding targets and metabolic pathways can also be found via using metabonomics based on one platform (e.g., 1H NMR) to study different biological samples of KYDS. At the same time, relevant reports on further molecular verification (e.g., RT-qPCR analysis) of these targets associated with biomarkers and metabolic pathways have not yet, to our knowledge, been seen in KYDS’s research. In the present study, a comprehensive strategy integrating systems pharmacology and 1H NMR-based urinary metabonomics analysis was proposed to identify the target proteins and metabolic pathways that YGP acts on KYDS. Thereafter, further validation of target proteins in kidney tissue was performed through quantitative real-time PCR analysis (RT-qPCR). Furthermore, biochemical parameters and histopathological analysis were studied. As a result, seven target proteins (L-serine dehydratase; phosphoenolpyruvate carboxykinase; spermidine synthase; tyrosyl-tRNA synthetase, glutamine synthetase; 3-hydroxyacyl-CoA dehydrogenase; glycine amidinotransferase) in YGP were discovered to play a therapeutic role in KYDS via affecting eight metabolic pathways (glycine, serine and threonine metabolism; butanoate metabolism; TCA cycle, etc.). Importantly, three target proteins (i.e., 3-hydroxyacyl-CoA dehydrogenase; glutamine synthetase; and glycine amidinotransferase) and two metabolic pathways (butanoate metabolism and dicarboxylate metabolism) related to KYDS, to our knowledge, had been newly discovered in our study. The mechanism of action mainly involved energy metabolism, oxidative stress, ammonia metabolism, amino acid metabolism, and fatty acid metabolism. In short, our study demonstrated that targets and metabolic pathways for the treatment of KYDS by YGP can be effectively found via combining with systems pharmacology and urinary metabonomics. In addition to this, common and specific targets and metabolic pathways of KYDS treated by YGP can be found effectively by integration with the analysis of different biological samples (e.g., serum, urine, feces, and tissue). It is; therefore, important that this laid the foundation for deeper mechanism research and drug-targeted therapy of KYDS in future.

Cystathionine β-lyase is involved in d-amino acid metabolism.

Non-canonical d-amino acids play important roles in bacteria including control of peptidoglycan metabolism and biofilm disassembly. Bacteria appear to produce non-canonical d-amino acids to adapt to various environmental changes, and understanding the biosynthetic pathways is important. We identified novel amino acid racemases possessing the ability to produce non-canonical d-amino acids in Escherichia coli and Bacillus subtilis in our previous study, whereas the biosynthetic pathways of these d-amino acids still remain unclear. In the present study, we demonstrated that two cystathionine β-lyases (MetC and MalY) from E. coli produce non-canonical d-amino acids including non-proteinogenic amino acids. Furthermore, MetC displayed d- and l-serine (Ser) dehydratase activity. We characterised amino acid racemase, Ser dehydratase and cysteine lyase activities, and all were higher for MetC. Interestingly, all three activities were at a comparable level for MetC, although optimal conditions for each reaction were distinct. These results indicate that MetC and MalY are multifunctional enzymes involved in l-methionine metabolism and the production of d-amino acids, as well as d- and l-Ser metabolism. To our knowledge, this is the first evidence that cystathionine β-lyase is a multifunctional enzyme with three different activities.

Mutagenic and chemical analyses provide new insight into enzyme activation and mechanism of the type 2 iron-sulfur l-serine dehydratase from Legionella pneumophila

The crystal structure of the Type 2 l-serine dehydratase from Legionella pneumophila (lpLSD), revealed a “tail-in-mouth” configuration where the C-terminal residue acts as an intrinsic competitive inhibitor. This pre-catalytic structure undergoes an activation step prior to catalytic turnover. Mutagenic analysis of residues at or near the active site cleft is consistent with stabilization of substrate binding by many of the same residues that interact with the C-terminal cysteine and highlight the critical role of certain tail residues in activity. pH-rate profiles show that a residue with pK of 5.9 must be deprotonated and a residue with a pK of 8.5 must be protonated for activity. This supports an earlier suggestion that His 61 is the likely catalytic base. An additional residue with a pK of 8.5–9 increases cooperativity when it is deprotonated. This investigation also demonstrates that the Fe-S dehydratases convert the enamine/imine intermediates of the catalytic reaction to products on the enzyme prior to release. This is in contrast to pyridoxyl 5′ phosphate based dehydratases that release an enamine/imine intermediate into solution, which then hydrolyzes to produce the ketoamine product.

Hydrogen-Deuterium Exchange Mass Spectrometry Reveals Unique Conformational and Chemical Transformations Occurring upon [4Fe-4S] Cluster Binding in the Type 2 L-Serine Dehydratase from Legionella pneumophila.

The type 2 L-serine dehydratase from Legionella pneumophila (lpLSD) contains a [4Fe-4S](2+) cluster that acts as a Lewis acid to extract the hydroxyl group of L-serine during the dehydration reaction. Surprisingly, the crystal structure shows that all four of the iron atoms in the cluster are coordinated with protein cysteinyl residues and that the cluster is buried and not exposed to solvent. If the crystal structure of lpLSD accurately reflects the structure in solution, then substantial rearrangement at the active site is necessary for the substrate to enter. Furthermore, repair of the oxidized protein when the cluster has degraded would presumably entail exposure of the buried cysteine ligands. Thus, the conformation required for the substrate to enter may be similar to those required for a new cluster to enter the active site. To address this, hydrogen-deuterium exchange combined with mass spectrometry (HDX MS) was used to probe the conformational changes that occur upon oxidative degradation of the Fe-S cluster. The regions that show the most significant differential HDX are adjacent to the cluster location in the holoenzyme or connect regions that are adjacent to the cluster. The observed decrease in flexibility upon cluster binding provides direct evidence that the "tail-in-mouth" conformation observed in the crystal structure also occurs in solution and that the C-terminal peptide is coordinated to the [4Fe-4S] cluster in a precatalytic conformation. This observation is consistent with the requirement of an activation step prior to catalysis and the unusually high level of resistance to oxygen-induced cluster degradation. Furthermore, peptide mapping of the apo form under nonreducing conditions revealed the formation of disulfide bonds between C396 and C485 and possibly between C343 and C385. These observations provide a picture of how the cluster loci are stabilized and poised to receive the cluster in the apo form and the requirement for a reduction step during cluster formation.

Comparative genomics and proteomics of 13 Porphyromonas gingivalis strains

At the current time, genome sequences of a total of 13 Porphyromonas gingivalis strains are available, including five completed genomes (strains ATCC 33277, HG66, TDC60, JCVISC001, and W83) and eight high-coverage draft sequences (F0185, F0566, F0568, F0569, F0570, SJD2, W4087, and W50) that are assembled into fewer than 300 contigs. This study compared these genomes at both nucleotide and protein sequence levels in order to understand their phylogenetic and functional relatedness. There are four copies of 16S rRNA gene sequences in each of the strains of ATCC 33277, HG66, TDC60, and W83 and one copy in the other nine genomes. These 25 16S rRNA sequences represent only 13 unique sequences. The five copies in W83 and W50 are identical and the three copies in HG66 are identical to the four copies in ATCC 33277, suggesting close evolutionary lineage between W83 and W50, as well as HG66 and ATCC 33277. Genome-wide comparison based on “Rapid Annotation using Subsystem Technology” (RAST) also showed that for the overall biological functions of the genomes, W83 is closer to W50, and HG66 to ATCC33277, than to other genomes. The comparison of the RAST subsystems identified biological functions that are unique to individual, shared by some, or by all genomes. Functions unique to individual genomes include: a tetracycline resistance protein TetQ, DNA metabolism gene YcfH, and DNA repair gene exonuclease SbcC (only in SJD2); very-short-patch mismatch repair endonuclease and a phage packaging terminase similar to Bacteroides phage B124-14 (in W4087); an internalin similar to a Listeria surface virulence protein (W83); a Type I restriction-modification system (F0569); an iron acquisition/heme transport protein (F0566); colicin I receptor and carbamoylputrescine amidase (W50); l-serine dehydratase (TDC60); and spermidine synthase and ribokinase (JCVISC001). The results also identified biological functions that are missing in individual or several genomes. For example, JCVISC001 does not contain the CRISPR (clustered regularly interspaced short palindromic repeats) system – a prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages. Some genomes are enriched with multiple copies of certain genes [e.g., TDC60, W50, and W83 encode 2–4 copies of 4-alpha-glucanotransferase (amylomaltase in glycan metabolism)], while others only have a single copy in the genome. Complete results of this study will be presented and available online for download.

Structure of L-serine dehydratase from Legionella pneumophila: novel use of the C-terminal cysteine as an intrinsic competitive inhibitor.

Here we report the first complete structure of a bacterial Fe–S l-serine dehydratase determined to 2.25 Å resolution. The structure is of the type 2 l-serine dehydratase from Legionella pneumophila that consists of a single polypeptide chain containing a catalytic α domain and a β domain that is structurally homologous to the “allosteric substrate binding” or ASB domain of d-3-phosphoglycerate dehydrogenase from Mycobacterium tuberculosis. The enzyme exists as a dimer of identical subunits, with each subunit exhibiting a bilobal architecture. The [4Fe-4S]2+ cluster is bound by residues from the C-terminal α domain and is situated between this domain and the N-terminal β domain. Remarkably, the model reveals that the C-terminal cysteine residue (Cys 458), which is conserved among the type 2 l-serine dehydratases, functions as a fourth ligand to the iron–sulfur cluster producing a “tail in mouth” configuration. The interaction of the sulfhydryl group of Cys 458 with the fourth iron of the cluster appears to mimic the position that the substrate would adopt prior to catalysis. A number of highly conserved or invariant residues found in the β domain are clustered around the iron–sulfur center. Ser 16, Ser 17, Ser 18, and Thr 290 form hydrogen bonds with the carboxylate group of Cys 458 and the carbonyl oxygen of Glu 457, whereas His 19 and His 61 are poised to potentially act as the catalytic base required for proton extraction. Mutation of His 61 produces an inactive enzyme, whereas the H19A protein variant retains substantial activity, suggesting that His 61 serves as the catalytic base. His 124 and Asn 126, found in an HXN sequence, point toward the Fe–S cluster. Mutational studies are consistent with these residues either binding a serine molecule that serves as an activator or functioning as a potential trap for Cys 458 as it moves out of the active site prior to catalysis.

Construction of an l-serine producing Escherichia coli via metabolic engineering

L-Serine is a nonessential amino acid, but plays a crucial role as a building block for cell growth. Currently, L-serine production is mainly dependent on enzymatic or cellular conversion. In this study, we constructed a recombinant Escherichia coli that can fermentatively produce L-serine from glucose. To accumulate L-serine, sdaA encoding the L-serine dehydratase, iclR encoding the isocitrate lyase regulator, and arcA encoding the aerobic respiration control protein were deleted in turn. In batch fermentation, the engineered E. coli strain YF-5 exhibited obvious L-serine accumulation but poor cell growth. To restore cell growth, aceB encoding the malate synthase was knocked out, and the engineered strain was then transformed with plasmid that overexpressed serA (FR) , serB, and serC genes. The resulting strain YF-7 produced 4.5g/L L-serine in batch cultivation and 8.34g/L L-serine in fed-batch cultivation.

PLP undergoes conformational changes during the course of an enzymatic reaction

Numerous enzymes, such as the pyridoxal 5'-phosphate (PLP)-dependent enzymes, require cofactors for their activities. Using X-ray crystallography, structural snapshots of the L-serine dehydratase catalytic reaction of a bacterial PLP-dependent enzyme were determined. In the structures, the dihedral angle between the pyridine ring and the Schiff-base linkage of PLP varied from 18° to 52°. It is proposed that the organic cofactor PLP directly catalyzes reactions by active conformational changes, and the novel catalytic mechanism involving the PLP cofactor was confirmed by high-level quantum-mechanical calculations. The conformational change was essential for nucleophilic attack of the substrate on PLP, for concerted proton transfer from the substrate to the protein and for directing carbanion formation of the substrate. Over the whole catalytic cycle, the organic cofactor catalyzes a series of reactions, like the enzyme. The conformational change of the PLP cofactor in catalysis serves as a starting point for identifying the previously unknown catalytic roles of organic cofactors.

Identification and characterization of two new types of bacterial l-serine dehydratases and assessment of the function of the ACT domain

Two new types of bacterial Fe–S l-serine dehydratases have been identified. These join two previously recognized enzyme types, for a total of four, that are distinguished on the basis of domain arrangement and amino acid sequence. A Type 3 enzyme from Amphibacillus xylanus (axLSD) and a Type 4 enzyme from Heliscomenobacter hydrossis (hhLSD) were cloned, expressed, purified, and characterized. Like the Type 1 enzyme from Bacillus subtilis (bsLSD), axLSD required a monovalent cation, preferably potassium, for activity. However, the hhLSD was without activity even after reconstitution of the iron–sulfur center by a process that successfully restored activity to oxygen-inactivated axLSD. This and other characteristics suggest that this Type 4 protein may be a pseudoenzyme. The oxygen sensitivity of axLSD was greater than other l-serine dehydratases so far studied and suggested that there may be significant conformational differences among the four types resulting in widely different solvent accessibility of the Fe–S clusters in these enzymes. The role of the ACT domain in these enzymes was explored by deleting it from bsLSD. Although there was an effect on the kinetic parameters, this domain was not responsible for the cation requirement nor did its removal have a significant effect on oxygen sensitivity.

Kinetic Evidence of a Noncatalytic Substrate Binding Site That Regulates Activity in Legionella pneumophilal-Serine Dehydratase

The L-serine dehydratase from Legionella pneumophila (lpLSD) has recently been shown to contain a domain (β domain) that has a high degree of structural homology with the ASB domain of d-3-phosphoglycerate dehydrogenase (PGDH) from Mycobacterium tuberculosis. Furthermore, this domain has been shown by sequence homology to be present in all bacterial L-serine dehydratases that utilize an Fe-S catalytic center. In PGDH, L-serine binds to the ACT domain to inhibit catalytic activity. However, substrate must be bound to the ASB domain for serine to exert its effect. As such, the ASB domain acts as a codomain for the action of L-serine. Pre-steady-state kinetic analysis of L-serine binding to lpLSD demonstrates that L-serine binds to a second noncatalytic site and produces a conformational change in the enzyme. The rate of this conformational change is too slow for its participation in the catalytic cycle but rather occurs prior to catalysis to produce an activated form of the enzyme. That the conformational change must occur prior to catalysis is shown by a lag in the production of product that exhibits essentially the same rate constant as the conformational change. The second, noncatalytic site for L-serine is likely to be the ASB domain (β domain) of lpLSD that functions in a manner similar to that in PGDH. A mechanism whose overall effect is to keep L-serine levels from accumulating to high levels while not completely depleting the L-serine pool in the bacterial cell is proposed.

Allosteric Activation and Contrasting Properties of l-Serine Dehydratase Types 1 and 2

Bacterial L-serine dehydratases differ from mammalian L- and D-serine dehydratases and bacterial D-serine dehydratases by the presence of an iron-sulfur center rather than a pyridoxyl phosphate prosthetic group. They exist in two forms, types 1 and 2, distinguished by their sequence and oligomeric configuration. Both types contain an ASB domain, and the type 1 enzymes also contain an ACT domain in a tandem arrangement with the ASB domain like that in type 1 D-3-phosphoglycerate dehydrogenases (PGDHs). This investigation reveals striking kinetic differences between L-serine dehydratases from Bacillus subtilis (bsLSD, type 1) and Legionella pneumophila (lpLSD, type 2). lpLSD is activated by monovalent cations and inhibited by monovalent anions. bsLSD is strongly activated by cations, particularly potassium, and shows a mixed response to anions. Flouride is a competitive inhibitor for lpLSD but an apparent activator for bsLSD at low concentrations and an inhibitor at high concentrations. The reaction products, pyruvate and ammonia, also act as activators but to different extents for each type. Pyruvate activation is competitive with L-serine, but activation of the enzyme is not compatible with it simply competing for binding at the active site and suggests the presence of a second, allosteric site. Because activation can be eliminated by higher levels of L-serine, it may be that this second site is actually a second serine binding site. This is consistent with type 1 PGDH in which the ASB domain functions as a second site for substrate binding and activation.

Crystal structure of D-serine dehydratase from Escherichia coli

D-Serine dehydratase from Escherichia coli is a member of the β-family (fold-type II) of the pyridoxal 5′-phosphate-dependent enzymes, catalyzing the conversion of D-serine to pyruvate and ammonia. The crystal structure of monomeric D-serine dehydratase has been solved to 1.97Å-resolution for an orthorhombic data set by molecular replacement. In addition, the structure was refined in a monoclinic data set to 1.55Å resolution. The structure of DSD reveals a larger pyridoxal 5′-phosphate-binding domain and a smaller domain. The active site of DSD is very similar to those of the other members of the β-family. Lys118 forms the Schiff base to PLP, the cofactor phosphate group is liganded to a tetraglycine cluster Gly279–Gly283, and the 3-hydroxyl group of PLP is liganded to Asn170 and N1 to Thr424, respectively. In the closed conformation the movement of the small domain blocks the entrance to active site of DSD. The domain movement plays an important role in the formation of the substrate recognition site and the catalysis of the enzyme. Modeling of D-serine into the active site of DSD suggests that the hydroxyl group of D-serine is coordinated to the carboxyl group of Asp238. The carboxyl oxygen of D-serine is coordinated to the hydroxyl group of Ser167 and the amide group of Leu171 (O1), whereas the O2 of the carboxyl group of D-serine is hydrogen-bonded to the hydroxyl group of Ser167 and the amide group of Thr168. A catalytic mechanism very similar to that proposed for L-serine dehydratase is discussed.

Kinetic, mutagenic, and structural homology analysis of l-serine dehydratase from Legionella pneumophila

A structural database search has revealed that the same fold found in the allosteric substrate binding (ASB) domain of Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase (PGDH) is found in l-serine dehydratase from Legionella pneumophila. The M. tuberculosis PGDH ASB domain functions in the control of catalytic activity. Bacterial l-serine dehydratases are 4Fe–4S proteins that convert l-serine to pyruvate and ammonia. Sequence homology reveals two types depending on whether their α and β domains are on the same (Type 2) or separate (Type 1) polypeptides. The α domains contain the catalytic iron–sulfur center while the β domains do not yet have a described function, but the structural homology with PGDH suggests a regulatory role. Type 1 β domains also contain additional sequence homologous to PGDH ACT domains. A continuous assay for l-serine dehydratase is used to demonstrate homotropic cooperativity, a broad pH range, and essential irreversibility. Product inhibition analysis reveals a Uni–Bi ordered mechanism with ammonia dissociating before pyruvate. l-Threonine is a poor substrate and l-cysteine and d-serine are competitive inhibitors with Ki values that differ by almost 10-fold from those reported for Escherichia colil-serine dehydratase. Mutagenesis identifies the three cysteine residues at the active site that anchor the iron–sulfur complex.

The cytotoxic early protein 77 of mycobacteriophage L5 interacts with MSMEG_3532, an L‐serine dehydratase of Mycobacterium smegmatis

Mycobacteriophage L5 is a temperate phage infecting a broad range of mycobacterial species. Upon induction of lytic growth, L5 rapidly switches off host protein synthesis. We have recently identified the mycobacteriophage L5 early protein gp77 as a host shut-off protein that acts growth inhibitory in the mycobacterial host when expressed through the corresponding phage promoter. Here we present data showing that this purified phage protein of unknown function specifically binds to protein MSMEG_3532 when incubated with cell lysates of Mycobacterium smegmatis. This interaction was confirmed by pull-down assays using purified MSMEG_3532 as bait which co-purified with gp77. The amino acid sequence of MSMEG_3532 is nearly identical to that of threonine dehydratases, serine dehydratases and an L-threo-3-hydroxyaspartate dehydratase. An enzymatic assay identified this host protein as a pyridoxal-5'-phosphate-dependent L-serine dehydratase (SdhA) which converts L-serine to pyruvate. This is the first biochemical characterization of a SdhA derived from mycobacteria. Though the addition of purified gp77 to the established in vitro assay had no influence on SdhA activity at a saturating L-serine concentration, the specific interaction of phage protein and dehydratase in vivo may well have a role in altering the amino acid pool or the products of amino acid metabolism in favour of phage maturation.

Characterization of two isotypes of l-threonine dehydratase from Entamoeba histolytica

The genome sequence of the enteric protozoan parasite Entamoeba histolytica suggests that amino acid catabolism plays an important role in energy metabolism. In the present study, we described kinetic and regulatory properties of catabolic l-threonine and l-serine dehydratase (TD) from E. histolytica. TD catalyses the pyridoxal phosphate-dependent dehydrative deamination of l-threonine and l-serine to ammonia and keto acids (2-oxobutyrate and pyruvate, respectively). E. histolytica possesses two TD isotypes (EhTD1-2) showing 38% mutual identity, a calculated molecular mass of 45.0 or 46.5kDa, and an isoelectric point of 6.68 or 5.88, respectively. Only EhTD1 showed l-threonine and l-serine dehydrative deaminating activities whereas EhTD2, in which the amino acid residues involved in the substrate and cofactor binding were not conserved, was devoid of these activities. The kcat/Km value of EhTD1 was >3 fold higher for l-threonine than l-serine. EhTD1 was inhibited by l-cysteine in a competitive manner with the Ki values of 1.1mM and 2.2mM for l-serine and l-threonine, respectively. EhTD1 was insensitive to the allosteric activation by AMP or CMP. Three major substitutions of EhTD1 likely attribute to the insensitivity. EhTD1 was also inhibited about 50% by 20mM 2-oxobutyrate, pyruvate, and glyoxylate; the inhibition was not, however, reversed by AMP. Together, these data showed that EhTD1 possesses unique regulatory properties distinct from other organisms and may play an important role in energy metabolism via amino acid degradation in E. histolytica.

Occurrence of D-serine in rice and characterization of rice serine racemase

Germinated, unpolished rice was found to contain a substantial amount of D-serine, with the ratio of the D-enantiomer to the L-enantiomer being higher for serine than for other amino acids. The relative amount of D-serine (D/(D + L)%) reached approximately 10% six days after germination. A putative serine racemase gene ( serr, clone No. 001-110-B03) was found in chromosome 4 of the genomic DNA of Oryza sativa L. ssp. Japonica cv. Nipponbare. This was expressed as serr in Escherichia coli and its gene product (SerR) was purified to apparent homogeneity. SerR is a homodimer with a subunit molecular mass of 34.5 kDa, and is highly specific for serine. In addition to a serine racemase reaction, SerR catalyzes D- and L-serine dehydratase reactions, for which the specific activities were determined to be 2.73 and 1.42 nkatal/mg, respectively. The optimum temperature and pH were respectively determined for the racemase reaction (35 °C and pH 9.0) and for the dehydratase reaction (35 °C and pH 9.5). SerR was inhibited by PLP-enzyme inhibitors. ATP decreased the serine racemase activity of SerR but increased the serine dehydratase activity. Kinetic analysis showed that Mg 2+ increases the catalytic efficiency of the serine racemase activity of SerR and decreases that of the serine dehydratase activity. Fluorescence-quenching analysis of the tryptophan residues in SerR indicated that the structure of SerR is distorted by the addition of Mg 2+, and this structural change probably regulates the two enzymatic activities.

A Quantum Mechanical/Molecular Mechanical Study on the Catalysis of the Pyridoxal 5′-Phosphate-Dependent Enzyme l-Serine Dehydratase

The catalytic mechanism of a pyridoxal 5'-phosphate-dependent enzyme, l-serine dehydratase, has been investigated using ab initio quantum mechanical/molecular mechanical (QM/MM) methods. New insights into the chemical steps have been obtained, including the chemical role of the substrate carboxyl group in the Schiff base formation step and a proton-relaying mechanism involving the phosphate of the cofactor in the beta-hydroxyl-leaving step. The latter step is of no barrier and follows sequentially after the elimination of the alpha-proton, leading to a single but sequential alpha, beta-elimination step. The rate-limiting transition state is specifically stabilized by the enzyme environment. At this transition state, charges are localized on the substrate carboxyl group, as well as on the amino group of Lys41. Specific interactions of the enzyme environment with these groups are able to lower the activation barrier significantly. One major difficulty associated with studies of complicated enzymatic reactions using ab initio QM/MM models is the appropriate choices of reaction coordinates. In this study, we have made use of efficient semiempirical models and pathway optimization techniques to overcome this difficulty.