Crystal structure of mevalonate 3,5-bisphosphate decarboxylase reveals insight into the evolution of decarboxylases in the mevalonate metabolic pathways.

Mevalonate 3,5-bisphosphate decarboxylase is involved in the recently discovered Thermoplasma-type mevalonate pathway. The enzyme catalyzes the elimination of the 3-phosphate group from mevalonate 3,5-bisphosphate as well as concomitant decarboxylation of the substrate. This entire reaction of the enzyme resembles the latter half-reactions of its homologs, diphosphomevalonate decarboxylase and phosphomevalonate decarboxylase, which also catalyze ATP-dependent phosphorylation of the 3-hydroxyl group of their substrates. However, the crystal structure of mevalonate 3,5-bisphosphate decarboxylase and the structural reasons of the difference between reactions catalyzed by the enzyme and its homologs are unknown. In this study, we determined the X-ray crystal structure of mevalonate 3,5-bisphosphate decarboxylase from Picrophilus torridus, a thermoacidophilic archaeon of the order Thermoplasmatales. Structural and mutational analysis demonstrated the importance of a conserved aspartate residue for enzyme activity. In addition, although crystallization was performed in the absence of substrate or ligands, residual electron density having the shape of a fatty acid was observed at a position overlapping the ATP-binding site of the homologous enzyme, diphosphomevalonate decarboxylase. This finding is in agreement with the expected evolutionary route from phosphomevalonate decarboxylase (ATP-dependent) to mevalonate 3,5-bisphosphate decarboxylase (ATP-independent) through the loss of kinase activity. We found that the binding of geranylgeranyl diphosphate, an intermediate of the archeal isoprenoid biosynthesis pathway, evoked significant activation of mevalonate 3,5-bisphosphate decarboxylase, and several mutations at the putative geranylgeranyl diphosphate-binding site impaired this activation, suggesting the physiological importance of ligand binding as well as a possible novel regulatory system employed by the Thermoplasma-type mevalonate pathway.

Mechanism of Pyridoxine 5'-Phosphate Accumulation in Pyridoxal 5'-Phosphate-Binding Protein Deficiency.

The pyridoxal 5'-phosphate (PLP)-binding protein (PLPBP) plays an important role in vitamin B6 homeostasis. Loss of this protein in organisms such as Escherichia coli and humans disrupts the vitamin B6 pool and induces intracellular accumulation of pyridoxine 5'-phosphate (PNP), which is normally undetectable in wild-type cells. This accumulated PNP could affect diverse metabolic systems through the inhibition of some PLP-dependent enzymes. In this study, we investigated the as-yet-unclear mechanism of intracellular accumulation of PNP due to the loss of PLPBP protein encoded by yggS in E. coli. Genetic studies using several PLPBP-deficient strains of E. coli lacking a known enzyme(s) in the de novo or salvage pathways of vitamin B6, including pyridoxine (amine) 5'-phosphate oxidase (PNPO), PNP synthase, pyridoxal kinase, and pyridoxal reductase, demonstrated that neither the flux from the de novo pathway nor the salvage pathway solely contributed to the PNP accumulation caused by the PLPBP mutation. Studies of the strains lacking both PLPBP and PNPO suggested that PNP shares the same pool with PMP, and showed that PNP levels are impacted by PMP levels and vice versa. Here, we show that disruption of PLPBP perturbs PMP homeostasis, which may result in PNP accumulation in the PLPBP-deficient strains. IMPORTANCE A PLP-binding protein (PLPBP) from the conserved COG0325 family has recently been recognized as a key player in vitamin B6 homeostasis in various organisms. Loss of PLPBP disrupts vitamin B6 homeostasis and perturbs diverse metabolisms, including amino acid and α-keto acid metabolism. Accumulation of PNP is a characteristic phenotype of PLPBP deficiency and is suggested to be a potential cause of the pleiotropic effects, but the mechanism of this accumulation has been poorly understood. In this study, we show that fluxes for PNP synthesis/metabolism are not responsible for the accumulation of PNP. Our results indicate that PLPBP is involved in the homeostasis of pyridoxamine 5'-phosphate, and that its disruption may lead to the accumulation of PNP in PLPBP deficiency.

Role of the conserved pyridoxal 5'-phosphate-binding protein YggS/PLPBP in vitamin B6 and amino acid homeostasis.

The YggS/PLPBP protein (also called COG0325 or PLPHP) is a conserved pyridoxal 5'-phosphate (PLP)-binding protein present in all 3 domains of life. Recent studies have demonstrated that disruption or mutation of this protein has multifaceted effects in various organisms, including vitamin B6-dependent epilepsy in humans. In Escherichia coli, disruption of this protein-encoded by yggS-perturbs Thr-Ile/Val metabolism, one-carbon metabolism, coenzyme A synthesis, and vitamin B6 homeostasis. This protein is critical for maintaining low levels of pyridoxine 5'-phosphate (PNP) in various organisms. In the yggS-deficient E. coli strain, inhibition of PLP-dependent enzymes, such as the glycine cleavage system by PNP, is the root cause of metabolic perturbation. Our data suggest that the YggS/PLPBP protein may be involved in the balancing of B6 vitamers by mediating efficient turnover of protein-bound B6 vitamers. This paper reviews recent findings on the function of the YggS/PLPBP protein.

Isopentenyl diphosphate/dimethylallyl diphosphate-specific Nudix hydrolase from the methanogenic archaeon Methanosarcina mazei.

Nudix hydrolases typically catalyze the hydrolysis of nucleoside diphosphate linked to moiety X and yield nucleoside monophosphate and X-phosphate, while some of them hydrolyze a terminal diphosphate group of non-nucleosidic compounds and convert it into a phosphate group. Although the number of Nudix hydrolases is usually limited in archaea comparing with those in bacteria and eukaryotes, the physiological functions of most archaeal Nudix hydrolases remain unknown. In this study, a Nudix hydrolase family protein, MM_2582, from the methanogenic archaeon Methanosarcina mazei was recombinantly expressed in Escherichia coli, purified, and characterized. This recombinant protein shows higher hydrolase activity toward isopentenyl diphosphate and short-chain prenyl diphosphates than that toward nucleosidic compounds. Kinetic studies demonstrated that the archaeal enzyme prefers isopentenyl diphosphate and dimethylallyl diphosphate, which suggests its role in the biosynthesis of prenylated flavin mononucleotide, a recently discovered coenzyme that is required, for example, in the archaea-specific modified mevalonate pathway.

Inhibition of glycine cleavage system by pyridoxine 5'-phosphate causes synthetic lethality in glyA yggS and serA yggS in Escherichia coli.

The YggS/Ybl036c/PLPBP family includes conserved pyridoxal 5'-phosphate (PLP)-binding proteins that play a critical role in the homeostasis of vitamin B6 and amino acids. Disruption of members of this family causes pleiotropic effects in many organisms by unknown mechanisms. In Escherichia coli, conditional lethality of the yggS and glyA (encoding serine hydroxymethyltransferase) has been described, but the mechanism of lethality was not determined. Strains lacking yggS and serA (3-phosphoglycerate dehydrogenase) were conditionally lethality in the M9-glucose medium supplemented with Gly. Analyses of vitamin B6 pools found the high-levels of pyridoxine 5'-phosphate (PNP) in the two yggS mutants. Growth defects of the double mutants could be eliminated by overexpressing PNP/PMP oxidase (PdxH) to decrease the PNP levels. Further, a serA pdxH strain, which accumulates PNP in the presence of yggS, exhibited similar phenotype to serA yggS mutant. Together these data suggested the inhibition of the glycine cleavage (GCV) system caused the synthetic lethality. Biochemical assays confirmed that PNP disrupts the GCV system by competing with PLP in GcvP protein. Our data are consistent with a model in which PNP-dependent inhibition of the GCV system causes the conditional lethality observed in the glyA yggS or serA yggS mutants.

Modified mevalonate pathway of the archaeon Aeropyrum pernix proceeds via trans-anhydromevalonate 5-phosphate.

The modified mevalonate pathway is believed to be the upstream biosynthetic route for isoprenoids in general archaea. The partially identified pathway has been proposed to explain a mystery surrounding the lack of phosphomevalonate kinase and diphosphomevalonate decarboxylase by the discovery of a conserved enzyme, isopentenyl phosphate kinase. Phosphomevalonate decarboxylase was considered to be the missing link that would fill the vacancy in the pathway between mevalonate 5-phosphate and isopentenyl phosphate. This enzyme was recently discovered from haloarchaea and certain Chroloflexi bacteria, but their enzymes are close homologs of diphosphomevalonate decarboxylase, which are absent in most archaea. In this study, we used comparative genomic analysis to find two enzymes from a hyperthermophilic archaeon, Aeropyrum pernix, that can replace phosphomevalonate decarboxylase. One enzyme, which has been annotated as putative aconitase, catalyzes the dehydration of mevalonate 5-phosphate to form a previously unknown intermediate, trans-anhydromevalonate 5-phosphate. Then, another enzyme belonging to the UbiD-decarboxylase family, which likely requires a UbiX-like partner, converts the intermediate into isopentenyl phosphate. Their activities were confirmed by in vitro assay with recombinant enzymes and were also detected in cell-free extract from A. pernix These data distinguish the modified mevalonate pathway of A. pernix and likely, of the majority of archaea from all known mevalonate pathways, such as the eukaryote-type classical pathway, the haloarchaea-type modified pathway, and another modified pathway recently discovered from Thermoplasma acidophilum.

Pyridoxal Reductase, PdxI, Is Critical for Salvage of Pyridoxal in Escherichia coli.

Pyridoxal 5'-phosphate (PLP) is the biologically active form of vitamin B6 and an essential cofactor in all organisms. In Escherichia coli, PLP is synthesized via the deoxyxylulose 5-phosphate (DXP)-dependent pathway that includes seven enzymatic steps and generates pyridoxine 5'-phosphate as an intermediate. Additionally, E. coli is able to salvage pyridoxal, pyridoxine, and pyridoxamine B6 vitamers to produce PLP using kinases PdxK/PdxY and pyridox(am)ine phosphate oxidase (PdxH). We found that E. coli strains blocked in PLP synthesis prior to the formation of pyridoxine 5'-phosphate (PNP) required significantly less exogenous pyridoxal (PL) than strains lacking pdxH and identified the conversion of PL to pyridoxine (PN) during cultivation to be the cause. Our data showed that PdxI, shown to have PL reductase activity in vitro, was required for the efficient salvage of PL in E. coli The pdxI+E. coli strains converted exogenous PL to PN during growth, while pdxI mutants did not. In total, the data herein demonstrated that PdxI is a critical enzyme in the salvage of PL by E. coliIMPORTANCE The biosynthetic pathway of pyridoxal 5'-phosphate (PLP) has extensively been studied in Escherichia coli, yet limited information is available about the vitamin B6 salvage pathway. We show that the protein PdxI (YdbC) is the primary pyridoxal (PL) reductase in E. coli and is involved in the salvage of PL. The orthologs of PdxI occur in a wide range of bacteria and plants, suggesting that PL reductase in the B6 salvage pathway is more widely distributed than previously expected.

The Role of YggS in Vitamin B6 Homeostasis in Salmonella enterica Is Informed by Heterologous Expression of Yeast SNZ3.

YggS (COG0325) is a pyridoxal 5'-phosphate (PLP)-binding protein proposed to be involved in homeostasis of B6 vitamers. In Salmonella enterica, lack of yggS resulted in phenotypes that were distinct and others that were similar to those of a yggS mutant of Escherichia coli Like other organisms, yggS mutants of S. enterica accumulate endogenous pyridoxine 5'-phosphate (PNP). Data herein show that strains lacking YggS accumulated ∼10-fold more PLP in growth medium than a parental strain. The deoxyxylulose 5-phosphate-dependent biosynthetic pathway for PLP and the PNP/pyridoxamine 5'-phosphate (PMP) oxidase credited with interconverting B6 vitamers were replaced with a single PLP synthase from Saccharomyces cerevisiae The impact of a yggS deletion on the intracellular and extracellular levels of B6 vitamers in this restructured strain supported a role for PdxH in PLP homeostasis and led to a general model for YggS function in PLP-PMP cycling. Our findings uncovered broader consequences of a yggS mutation than previously reported and suggest that the accumulation of PNP is not a direct effect of lacking YggS but rather a downstream consequence.IMPORTANCE Pyridoxal 5'-phosphate (PLP) is an essential cofactor for enzymes in all domains of life. Perturbations in PLP or B6 vitamer content can be detrimental, notably causing B6-dependent epilepsy in humans. YggS homologs are broadly conserved and have been implicated in altered levels of B6 vitamers in multiple organisms. The biochemical activity of YggS, expected to be conserved across domains, is not yet known. Herein, a simplified heterologous pathway minimized metabolic variables and allowed the dissection of this system to generate new metabolic knowledge that will be relevant to understanding YggS.

Conserved Pyridoxal 5'-Phosphate-Binding Protein YggS Impacts Amino Acid Metabolism through Pyridoxine 5'-Phosphate in Escherichia coli.

Escherichia coli YggS (COG0325) is a member of the highly conserved pyridoxal 5'-phosphate (PLP)-binding protein (PLPBP) family. Recent studies suggested a role for this protein family in the homeostasis of vitamin B6 and amino acids. The deletion or mutation of a member of this protein family causes pleiotropic effects in many organisms and is causative of vitamin B6-dependent epilepsy in humans. To date, little has been known about the mechanism by which lack of YggS results in these diverse phenotypes. In this study, we determined that the pyridoxine (PN) sensitivity observed in yggS-deficient E. coli was caused by the pyridoxine 5'-phosphate (PNP)-dependent overproduction of Val, which is toxic to E. coli The data suggest that the yggS mutation impacts Val accumulation by perturbing the biosynthetic of Thr from homoserine (Hse). Exogenous Hse inhibited the growth of the yggS mutant, caused further accumulation of PNP, and increased the levels of some intermediates in the Thr-Ile-Val metabolic pathways. Blocking the Thr biosynthetic pathway or decreasing the intracellular PNP levels abolished the perturbations of amino acid metabolism caused by the exogenous PN and Hse. Our data showed that a high concentration of intracellular PNP is the root cause of at least some of the pleiotropic phenotypes described for a yggS mutant of E. coliIMPORTANCE Recent studies showed that deletion or mutation of members of the YggS protein family causes pleiotropic effects in many organisms. Little is known about the causes, mechanisms, and consequences of these diverse phenotypes. It was previously shown that yggS mutations in E. coli result in the accumulation of PNP and some metabolites in the Ile/Val biosynthetic pathway. This work revealed that some exogenous stresses increase the aberrant accumulation of PNP in the yggS mutant. In addition, the current report provides evidence indicating that some, but not all, of the phenotypes of the yggS mutant in E. coli are due to the elevated PNP level. These results will contribute to continuing efforts to determine the molecular functions of the members of the YggS protein family.

Urinary D-serine level as a predictive biomarker for deterioration of renal function in patients with atherosclerotic risk factors.

BACKGROUND: D-serine, the enantiomer of L-serine, was identified in mammals 20 years ago. Although a close relationship between D-serine and renal dysfunction has been shown, the clinical implications of urinary D- and L-serine in humans are poorly understood. The aim of this study was to evaluate the relationship between urinary D- and L-serine with well-known renal biomarkers, and clarify the prognostic value of D- and L-serine for renal events. METHODS: This cross-sectional, prospective study included 65 patients with atherosclerotic risk factors, who were followed up for a median of 16 months. The primary endpoint was a composite of end-stage renal disease and a decline in estimated glomerular filtration rate (eGFR) ≥ 25% from baseline. RESULTS: Urinary D-serine concentrations showed a better correlation with eGFR than did urinary L-serine, whereas neither urinary D- nor L-serine correlated with tubular markers such as urinary liver-type fatty acid-binding protein and N-acetyl-beta-D-glucosaminidase. A Cox regression analysis revealed that low urinary D-serine levels were significantly associated with the primary endpoint after adjusting for confounding factors (hazard ratio 12.60; 95% confidence interval, 3.49-45.51). CONCLUSIONS: Urinary D-serine is associated with glomerular filtration and can be a prognostic biomarker of renal dysfunction in patients with atherosclerotic risk factors.

Novel imaging detailing the origins of a pneumothorax.

This is a prospective clinical study aimed at introducing a method to visualise the location of an air leak and to identify the bulla responsible on three-dimensional (3-D) cine CT. In 10 patients with spontaneous pneumothorax, dynamic 320-detector row CT was performed with injection of 0.9% saline into the affected pleural cavity via a preplaced chest tube. In eight cases, 3-D cine CT thoracography revealed the location of the air leak and the bulla responsible (7 cases: air stream sign; 1 case: repeated collapse and expansion of a bulla with the patient's breathing).

Production of Ophthalmic Acid Using Engineered Escherichia coli.

Ophthalmic acid (OA; l-γ-glutamyl-l-2-aminobutyryl-glycine) is an analog of glutathione (GSH; l-γ-glutamyl-l-cysteinyl-glycine) in which the cysteine moiety is replaced by l-2-aminobutyrate. OA is a useful peptide for the pharmaceutical and/or food industries. Herein, we report a method for the production of OA using engineered Escherichia coli cells. yggS-deficient E. coli, which lacks the highly conserved pyridoxal 5'-phosphate-binding protein YggS and naturally accumulates OA, was selected as the starting strain. To increase the production of OA, we overexpressed the OA biosynthetic enzymes glutamate-cysteine ligase (GshA) and glutathione synthase (GshB), desensitized the product inhibition of GshA, and eliminated the OA catabolic enzyme γ-glutamyltranspeptidase. The production of OA was further enhanced by the deletion of miaA and ridA with the aim of increasing the availability of ATP and attenuating the unwanted degradation of amino acids, respectively. The final strain developed in this study successfully produced 277 µmol/liter of OA in 24 h without the formation of by-products in a minimal synthetic medium containing 1 mM each glutamate, 2-aminobutyrate, and glycine.IMPORTANCE Ophthalmic acid (OA) is a peptide that has the potential for use in the pharmaceutical and/or food industries. An efficient method for the production of OA would allow us to expand our knowledge about its physiological functions and enable the industrial/pharmaceutical application of this compound. We demonstrated the production of OA using Escherichia coli cells in which OA biosynthetic enzymes and degradation enymes were engineered. We also showed that unique approaches, including the use of a ΔyggS mutant as a starting strain, the establishment of an S495F mutation in GshA, and the deletion of ridA or miaA, facilitated the efficient production of OA in E. coli.

Utilization of an intermediate of the methylerythritol phosphate pathway, (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate, as the prenyl donor substrate for various prenyltransferases.

(E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate (HMBPP) is an intermediate of the methylerythritol phosphate pathway. Utilization of HMBPP by lycopene elongase from Corynebacterium glutamicum, which is a UbiA-family prenyltransferase responsible for C50 carotenoid biosynthesis, was investigated using an Escherichia coli strain that contained the exogenous mevalonate pathway as well as the carotenoid biosynthetic pathway. Inhibition of the endogenous methylerythritol phosphate pathway resulted in loss of the production of C50 carotenoid flavuxanthin, while C40 lycopene formation was retained. Overexpression of E. coli ispH gene, which encodes HMBPP reductase, also decreased the production of flavuxanthin in E. coli cells. These results indicate the preference of lycopene elongase for HMBPP instead of the previously proposed substrate, dimethylallyl diphosphate. Furthermore, several (all-E)-prenyl diphosphate synthases, which are classified in a distinct family of prenyltransferase, were demonstrated to accept HMBPP, which implies that the compound is more widely used as a prenyl donor substrate than was previously expected.

Fascial reinforcement fixing the bronchi to the heart: its anatomy and clinical significance.

PURPOSE: The details of the mediastinal fascia have been scarcely described and the bronchopericardial membrane is the only known structure that is present between the bronchi and the pericardium. However, the anatomical description of this structure is unclear. This study aimed to investigate the fascial structures between the bronchi and the pericardium based on surgical findings. METHODS: The connective tissues in the mid-mediastinum were observed surgically when lung lobectomy, including mediastinal lymph node dissection for lung cancer, was performed at our institute from April 2011 to March 2016. RESULTS: In total, 96 lobectomies were performed in 94 patients. A firm fibrous structure connecting the tracheobronchus and the fibrous pericardium was observed. It fixes the central bronchi to the pericardium and is composed of three parts. The largest part exists in front of the carina, its appearance is membranous, and runs behind the pulmonary artery. The other parts run over the right pulmonary artery and diverge at its superior trunk. The location at which all these structures fuse to the pericardium is the venous part of the hilum cordis (VHC). CONCLUSIONS: The results showed that connections of the dense fibrous tissues existed between the tracheobronchus and VHC. The structure not only works as a ligament that fixes the bronchi to the mid-mediastinum, but also divides the mid-mediastinum into two compartments: the Baréty and subcarinal spaces. The anatomy of the structure observed in this study differs from the previous description of the bronchopericardial membrane.

Role of the aminotransferase domain in Bacillus subtilis GabR, a pyridoxal 5'-phosphate-dependent transcriptional regulator.

MocR/GabR family proteins are widely distributed prokaryotic transcriptional regulators containing pyridoxal 5'-phosphate (PLP), a coenzyme form of vitamin B6. The Bacillus subtilis GabR, probably the most extensively studied MocR/GabR family protein, consists of an N-terminal DNA-binding domain and a PLP-binding C-terminal domain that has a structure homologous to aminotransferases. GabR suppresses transcription of gabR and activates transcription of gabT and gabD, which encode γ-aminobutyrate (GΑΒΑ) aminotransferase and succinate semialdehyde dehydrogenase, respectively, in the presence of PLP and GABA. In this study, we examined the mechanism underlying GabR-mediated gabTD transcription with spectroscopic, crystallographic and thermodynamic studies, focusing on the function of the aminotransferase domain. Spectroscopic studies revealed that GABA forms an external aldimine with the PLP in the aminotransferase domain. Isothermal calorimetry demonstrated that two GabR molecules bind to the 51-bp DNA fragment that contains the GabR-binding region. GABA minimally affected ΔG(binding) upon binding of GabR to the DNA fragment but greatly affected the contributions of ΔH and ΔS to ΔG(binding). GABA forms an external aldimine with PLP and causes a conformational change in the aminotransferase domain, and this change likely rearranges GabR binding to the promoter and thus activates gabTD transcription.

Is D-aspartate produced by glutamic-oxaloacetic transaminase-1 like 1 (Got1l1): a putative aspartate racemase?

D-Aspartate is an endogenous free amino acid in the brain, endocrine tissues, and exocrine tissues in mammals, and it plays several physiological roles. In the testis, D-aspartate is detected in elongate spermatids, Leydig cells, and Sertoli cells, and implicated in the synthesis and release of testosterone. In the hippocampus, D-aspartate strongly enhances N-methyl-D-aspartate receptor-dependent long-term potentiation and is involved in learning and memory. The existence of aspartate racemase, a candidate enzyme for D-aspartate production, has been suggested. Recently, mouse glutamic-oxaloacetic transaminase 1-like 1 (Got1l1) has been reported to synthesize substantially D-aspartate from L-aspartate and to be involved in adult neurogenesis. In this study, we investigated the function of Got1l1 in vivo by generating and analyzing Got1l1 knockout (KO) mice. We also examined the enzymatic activity of recombinant Got1l1 in vitro. We found that Got1l1 mRNA is highly expressed in the testis, but it is not detected in the brain and submandibular gland, where D-aspartate is abundant. The D-aspartate contents of wild-type and Got1l1 KO mice were not significantly different in the testis and hippocampus. The recombinant Got1l1 expressed in mammalian cells showed L-aspartate aminotransferase activity, but lacked aspartate racemase activity. These findings suggest that Got1l1 is not the major aspartate racemase and there might be an as yet unknown D-aspartate-synthesizing enzyme.

Conserved pyridoxal protein that regulates Ile and Val metabolism.

Escherichia coli YggS is a member of the highly conserved uncharacterized protein family that binds pyridoxal 5'-phosphate (PLP). To assist with the functional assignment of the YggS family, in vivo and in vitro analyses were performed using a yggS-deficient E. coli strain (ΔyggS) and a purified form of YggS, respectively. In the stationary phase, the ΔyggS strain exhibited a completely different intracellular pool of amino acids and produced a significant amount of l-Val in the culture medium. The log-phase ΔyggS strain accumulated 2-ketobutyrate, its aminated compound 2-aminobutyrate, and, to a lesser extent, l-Val. It also exhibited a 1.3- to 2.6-fold increase in the levels of Ile and Val metabolic enzymes. The fact that similar phenotypes were induced in wild-type E. coli by the exogenous addition of 2-ketobutyrate and 2-aminobutyrate indicates that the 2 compounds contribute to the ΔyggS phenotypes. We showed that the initial cause of the keto acid imbalance was the reduced availability of coenzyme A (CoA); supplementation with pantothenate, which is a CoA precursor, fully reversed phenotypes conferred by the yggS mutation. The plasmid-borne expression of YggS and orthologs from Bacillus subtilis, Saccharomyces cerevisiae, and humans fully rescued the ΔyggS phenotypes. Expression of a mutant YggS lacking PLP-binding ability, however, did not reverse the ΔyggS phenotypes. These results demonstrate for the first time that YggS controls Ile and Val metabolism by modulating 2-ketobutyrate and CoA availability. Its function depends on PLP, and it is highly conserved in a wide range species, from bacteria to humans.

Catalytic mechanism of serine racemase from Dictyostelium discoideum.

The eukaryotic serine racemase from Dictyostelium discoideum is a fold-type II pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes racemization and dehydration of both isomers of serine. In the present study, the catalytic mechanism and role of the active site residues of the enzyme were examined by site-directed mutagenesis. Mutation of the PLP-binding lysine (K56) to alanine abolished both serine racemase and dehydrase activities. Incubation of D- and L-serine with the resultant mutant enzyme, K56A, resulted in the accumulation of PLP-serine external aldimine, while less amounts of pyruvate, α-aminoacrylate, antipodal serine and quinonoid intermediate were formed. An alanine mutation of Ser81 (S81) located on the opposite side of K56 against the PLP plane converted the enzyme from serine racemase to L-serine dehydrase; S81A showed no racemase activity and had significantly reduced D-serine dehydrase activity, but it completely retained its L-serine dehydrase activity. Water molecule(s) at the active site of the S81A mutant enzyme probably drove D-serine dehydration by abstracting the α-hydrogen in D-serine. Our data suggest that the abstraction and addition of α-hydrogen to L- and D-serine are conducted by K56 and S81 at the si- and re-sides, respectively, of PLP.

d-amino acid auxotrophic Escherichia coli strain for in vivo functional cloning of novel d-amino acid synthetic enzyme.

Various d-amino acids have been found in a wide range of organisms, including mammals. Although the physiological functions of various d-amino acids have been reported or suggested, the molecular basis of these biological functions has been elucidated in only a few cases. The identification of a d-amino acid biosynthetic enzyme is a critical step in understanding the mechanism of the physiological functions of d-amino acids. While in vivo functional screening can be a powerful tool for identifying novel metabolic enzymes, none of the existing organisms exhibit growth dependent on d-amino acid other than d-Ala and d-Glu. Here, we report the first organism that exhibits non-canonical d-amino acid auxotrophy. We found that an Escherichia coli strain lacking the major d-Ala and d-Glu biosynthetic enzymes, alr, dadX, and murI, and expressing the mutated d-amino acid transaminase (DAAT) gene from Bacillus sp. YM-1 (MB3000/mdaat+ ) grew well when supplemented with certain d-amino acid. A multicopy suppression study with plasmids encoding one of the 51 PLP-dependent enzymes of E. coli showed that MB3000/mdaat+ could detect weak and moonlighting racemase activity, such from cystathionine ß-lyase (MetC) and a negative regulator of MalT activity/cystathionine ß-lyase (MalY)-these exhibit only a few tenths to a few thousandths of the racemization activity of canonical amino acid racemases. We believe that this unique platform will contribute to further research in this field by identifying novel d-amino acid-metabolizing enzymes.

A [4Fe-4S] cluster resides at the active center of phosphomevalonate dehydratase, a key enzyme in the archaeal modified mevalonate pathway.

The recent discovery of the archaeal modified mevalonate pathway revealed that the fundamental units for isoprenoid biosynthesis (isopentenyl diphosphate and dimethylallyl diphosphate) are biosynthesized via a specific intermediate, trans-anhydromevalonate phosphate. In this biosynthetic pathway, which is unique to archaea, the formation of trans-anhydromevalonate phosphate from (R)-mevalonate 5-phosphate is catalyzed by a key enzyme, phosphomevalonate dehydratase. This archaea-specific enzyme belongs to the aconitase X family within the aconitase superfamily, along with bacterial homologs involved in hydroxyproline metabolism. Although an iron-sulfur cluster is thought to exist in phosphomevalonate dehydratase and is believed to be responsible for the catalytic mechanism of the enzyme, the structure and role of this cluster have not been well characterized. Here, we reconstructed the iron-sulfur cluster of phosphomevalonate dehydratase from the hyperthermophilic archaeon Aeropyrum pernix to perform biochemical characterization and kinetic analysis of the enzyme. Electron paramagnetic resonance, iron quantification, and mutagenic studies of the enzyme demonstrated that three conserved cysteine residues coordinate a [4Fe-4S] cluster-as is typical in aconitase superfamily hydratases/dehydratases, in contrast to bacterial aconitase X-family enzymes, which have been reported to harbor a [2Fe-2S] cluster.