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.

Molecular basis and functional development of enzymes related to amino acid metabolism.

Enzymology, the study of enzyme structures and reaction mechanisms can be considered a classical discipline. However, enzymes cannot be freely designed to catalyze desired reactions yet, and enzymology is by no means a complete science. I have long studied the reaction mechanisms of enzymes related to amino acid metabolism, such as aminotransferases and racemases, which depend on pyridoxal 5'-phosphate, a coenzyme form of vitamin B6. During these studies, I have often been reminded that enzymatic reactions are extremely sophisticated processes based on chemical principles and enzyme structures, and have often been amazed at the evolutionary mechanisms that bestowed them with such structures. In this review, I described the reaction mechanism of various pyridoxal enzymes especially related to d-amino acids metabolism, whose roles in mammals have recently attracted attention. I hope to convey some of the significance and interest in enzymology through this review.

Identification and characterization of a serine racemase in the silkworm Bombyx mori.

The pupae of lepidopterans contain high concentrations of endogenous d-serine. In the silkworm Bombyx mori, d-serine is negligible during the larval stage but increases markedly during the pupal stage, reaching 50% of the total free serine. However, the physiological function of d-serine and the enzyme responsible for its production is unknown. Herein, we identified a new type of pyridoxal 5'-phosphate (PLP)-dependent serine racemase (SR) that catalyses the racemization of l-serine to d-serine in B. mori. This silkworm SR (BmSR) has an N-terminal PLP-binding domain that is homologous to mammalian SR and a C-terminal putative ligand-binding regulatory-like domain (ACT-like domain) that is absent in mammalian SR. Similar to mammalian SRs, BmSR catalyses the racemization and dehydration of both serine isomers. However, BmSR is different from mammalian SRs as evidenced by its insensitivity to Mg2+/Ca2+ and Mg-ATP-which are required for activation of mammalian SRs-and high d-serine dehydration activity. At the pupal stage, the SR activity was predominantly detected in the fat body, which was consistent with the timing and localization of BmSR expression. The results are an important first step in elucidating the physiological significance of d-serine in lepidopterans.

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.

Evolution and properties of alanine racemase from Synechocystis sp. PCC6803.

Alanine racemase (EC 5.1.1.1) depends on pyridoxal 5'-phosphate and catalyses the interconversion between L- and D-Ala. The enzyme is responsible for the biosynthesis of D-Ala, which is an essential component of the peptidoglycan layer of bacterial cell walls. Phylogenetic analysis of alanine racemases demonstrated that the cyanobacterial enzyme diverged before the separation of gram-positive and gram-negative enzymes. This result is interesting considering that the peptidoglycans observed in cyanobacteria seem to combine the properties of those in both gram-negative and gram-positive bacteria. We cloned the putative alanine racemase gene (slr0823) of Synechocystis sp. PCC6803 in Escherichia coli cells, expressed and purified the enzyme protein and studied its enzymological properties. The enzymatic properties of the Synechocystis enzyme were similar to those of other gram-positive and gram-negative bacterial enzymes. Alignment of the amino acid sequences of alanine racemase enzymes revealed that the conserved tyrosine residue in the active centre of most of the gram-positive and gram-negative bacterial enzymes has been replaced with tryptophan in most of the cyanobacterial enzymes. We carried out the site-directed mutagenesis involving the corresponding residue of Synechocystis enzyme (W385) and revealed that the residue is involved in the substrate recognition by the enzyme.

Enzymatic determination of d-alanine with l-alanine dehydrogenase and alanine racemase.

An enzymatic assay system of d-Ala, which is reported to affect the taste, was constructed using alanine racemase and l-alanine dehydrogenase. d-Ala is converted to l-Ala by alanine racemase and then deaminated by l-alanine dehydrogenase with the reduction of NAD+ to NADH, which is determined with water-soluble tetrazolium. Using the assay system, the d-Ala contents of 7 crustaceans were determined.

Urinary l-erythro-ß-hydroxyasparagine-a novel serine racemase inhibitor and substrate of the Zn2+-dependent d-serine dehydratase.

In the present study, we identified l-erythro-ß-hydroxyasparagine (l-ß-EHAsn) found abundantly in human urine, as a novel substrate of Zn2+-dependent d-serine dehydratase (DSD). l-ß-EHAsn is an atypical amino acid present in large amounts in urine but rarely detected in serum or most organs/tissues examined. Quantitative analyses of urinary l-ß-EHAsn in young healthy volunteers revealed significant correlation between urinary l-ß-EHAsn concentration and creatinine level. Further, for in-depth analyses of l-ß-EHAsn, we developed a simple three-step synthetic method using trans-epoxysuccinic acid as the starting substance. In addition, our research revealed a strong inhibitory effect of l-ß-EHAsn on mammalian serine racemase, responsible for producing d-serine, a co-agonist of the N-methyl-d-aspartate (NMDA) receptor involved in glutamatergic neurotransmission.

Mechanism of eukaryotic serine racemase-catalyzed serine dehydration.

Eukaryotic serine racemase (SR) is a pyridoxal 5'-phosphate enzyme belonging to the Fold-type II group, which catalyzes serine racemization and is responsible for the synthesis of D-Ser, a co-agonist of the N-methyl-d-aspartate receptor. In addition to racemization, SR catalyzes the dehydration of D- and L-Ser to pyruvate and ammonia. The bifuctionality of SR is thought to be important for D-Ser homeostasis. SR catalyzes the racemization of D- and L-Ser with almost the same efficiency. In contrast, the rate of L-Ser dehydration catalyzed by SR is much higher than that of D-Ser dehydration. This has caused the argument that SR does not catalyze the direct D-Ser dehydration and that D-Ser is first converted to L-Ser, then dehydrated. In this study, we investigated the substrate and solvent isotope effect of dehydration of D- and L-Ser catalyzed by SR from Dictyostelium discoideum (DdSR) and demonstrated that the enzyme catalyzes direct D-Ser dehydration. Kinetic studies of dehydration of four Thr isomers catalyzed by D. discoideum and mouse SRs suggest that SR discriminates the substrate configuration at C3 but not at C2. This is probably the reason for the difference in efficiency between L- and D-Ser dehydration catalyzed by SR.

Reconstruction of the "Archaeal" Mevalonate Pathway from the Methanogenic Archaeon Methanosarcina mazei in Escherichia coli Cells.

The mevalonate pathway is a well-known metabolic route that provides biosynthetic precursors for myriad isoprenoids. An unexpected variety of the pathway has been discovered from recent studies on microorganisms, mainly on archaea. The most recently discovered example, called the "archaeal" mevalonate pathway, is a modified version of the canonical eukaryotic mevalonate pathway and was elucidated in our previous study using the hyperthermophilic archaeon Aeropyrum pernix This pathway comprises four known enzymes that can produce mevalonate 5-phosphate from acetyl coenzyme A, two recently discovered enzymes designated phosphomevalonate dehydratase and anhydromevalonate phosphate decarboxylase, and two more known enzymes, i.e., isopentenyl phosphate kinase and isopentenyl pyrophosphate:dimethylallyl pyrophosphate isomerase. To show its wide distribution in archaea and to confirm if its enzyme configuration is identical among species, the putative genes of a lower portion of the pathway-from mevalonate to isopentenyl pyrophosphate-were isolated from the methanogenic archaeon Methanosarcina mazei, which is taxonomically distant from A. pernix, and were introduced into an engineered Escherichia coli strain that produces lycopene, a red carotenoid pigment. Lycopene production, as a measure of isoprenoid productivity, was enhanced when the cells were grown semianaerobically with the supplementation of mevalonolactone, which demonstrates that the archaeal pathway can function in bacterial cells to convert mevalonate into isopentenyl pyrophosphate. Gene deletion and complementation analysis using the carotenogenic E. coli strain suggests that both phosphomevalonate dehydratase and anhydromevalonate phosphate decarboxylase from M. mazei are required for the enhancement of lycopene production.IMPORTANCE Two enzymes that have recently been identified from the hyperthermophilic archaeon A. pernix as components of the archaeal mevalonate pathway do not require ATP for their reactions. This pathway, therefore, might consume less energy than other mevalonate pathways to produce precursors for isoprenoids. Thus, the pathway might be applicable to metabolic engineering and production of valuable isoprenoids that have application as pharmaceuticals. The archaeal mevalonate pathway was successfully reconstructed in E. coli cells by introducing several genes from the methanogenic or hyperthermophilic archaeon, which demonstrated that the pathway requires the same components even in distantly related archaeal species and can function in bacterial cells.

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.

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.

Conversion of Mevalonate 3-Kinase into 5-Phosphomevalonate 3-Kinase by Single Amino Acid Mutations.

Mevalonate 3-kinase plays a key role in a recently discovered modified mevalonate pathway specific to thermophilic archaea of the order Thermoplasmatales The enzyme is homologous to diphosphomevalonate decarboxylase, which is involved in the widely distributed classical mevalonate pathway, and to phosphomevalonate decarboxylase, which is possessed by halophilic archaea and some Chloroflexi bacteria. Mevalonate 3-kinase catalyzes the ATP-dependent 3-phosphorylation of mevalonate but does not catalyze the subsequent decarboxylation as related decarboxylases do. In this study, a substrate-interacting glutamate residue of Thermoplasma acidophilum mevalonate 3-kinase was replaced by smaller amino acids, including its counterparts in diphosphomevalonate decarboxylase and phosphomevalonate decarboxylase, with the aim of altering substrate specificity. These single amino acid mutations resulted in the conversion of mevalonate 3-kinase into 5-phosphomevalonate 3-kinase, which can synthesize 3,5-bisphosphomevalonate from 5-phosphomevalonate. The mutants catalyzing the hitherto undiscovered reaction enabled the construction of an artificial mevalonate pathway in Escherichia coli cells, as was demonstrated by the accumulation of lycopene, a red carotenoid pigment.IMPORTANCE Isoprenoid is the largest family of natural compounds, including important bioactive molecules such as vitamins, hormones, and natural medicines. The mevalonate pathway is a target for metabolic engineering because it supplies precursors for isoprenoid biosynthesis. Mevalonate 3-kinase is an enzyme involved in the modified mevalonate pathway specific to limited species of thermophilic archaea. Replacement of a single amino acid residue in the active site of the enzyme changed its substrate preference and allowed the mutant enzymes to catalyze a previously undiscovered reaction. Using the genes encoding the mutant enzymes and other archaeal enzymes, we constructed an artificial mevalonate pathway, which can produce the precursor of isoprenoid through an unexplored route, in bacterial cells.

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.

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.

D-Serine Metabolism and Its Importance in Development of Dictyostelium discoideum.

In mammals, D-Ser is synthesized by serine racemase (SR) and degraded by D-amino acid oxidase (DAO). D-Ser acts as an endogenous ligand for N-methyl-D-aspartate (NMDA)- and δ2 glutamate receptors, and is involved in brain functions such as learning and memory. Although SR homologs are highly conserved in eukaryotes, little is known about the significance of D-Ser in non-mammals. In contrast to mammals, the slime mold Dictyostelium discoideum genome encodes SR, DAO, and additionally D-Ser specific degradation enzyme D-Ser dehydratase (DSD), but not NMDA- and δ2 glutamate receptors. Here, we studied the significances of D-Ser and DSD in D. discoideum. Enzymatic assays demonstrated that DSD is 460- and 1,700-fold more active than DAO and SR, respectively, in degrading D-Ser. Moreover, in dsd-null cells D-Ser degradation activity is completely abolished. In fact, while in wild-type D. discoideum intracellular D-Ser levels were considerably low, dsd-null cells accumulated D-Ser. These results indicated that DSD but not DAO is the primary enzyme responsible for D-Ser decomposition in D. discoideum. We found that dsd-null cells exhibit delay in development and arrest at the early culmination stage. The efficiency of spore formation was considerably reduced in the mutant cells. These phenotypes were further pronounced by exogenous D-Ser but rescued by plasmid-borne expression of dsd. qRT-PCR analysis demonstrated that mRNA expression of key genes in the cAMP signaling relay is perturbed in the dsd knockout. Our data indicate novel roles for D-Ser and/or DSD in the regulation of cAMP signaling in the development processes of D. discoideum.

Biosynthetic machinery for C25,C25-diether archaeal lipids from the hyperthermophilic archaeon Aeropyrum pernix.

Archaea that thrive in harsh environments usually produce membrane lipids with specific structures such as bipolar tetraether lipids. Only a few genera of archaea, which are hyperthermophiles or halophiles, are known to utilize diether lipids with extended, C25 isoprenoid hydrocarbon chains. In the present study, we identify two prenyltransferases and a prenyl reductase responsible for the biosynthesis of C25,C25-diether lipids in the hyperthermophilic archaeon Aeropyrum pernix. These enzymes are more specific to C25 isoprenoid chains than to C20 chains, which are used for the biosynthesis of ordinary C20,C20-diether archaeal lipids. The recombinant expression of these enzymes with two known archaeal enzymes allows the production of C25,C25-diether archaeal lipids in the cells of Escherichia coli.

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.

Occurrence of the (2R,3S)-Isomer of 2-Amino-3,4-dihydroxybutanoic Acid in the Mushroom Hypsizygus marmoreus.

Here, we report the occurrence of the (2R,3S)-isomer of 2-amino-3,4-dihydroxybutanoic acid (d-ADHB) in the fruiting body of an edible mushroom, Hypsizygus marmoreus. This is an unusual example of the accumulation of a d-amino acid whose enantiomer is not a proteinogenic amino acid. We show that d-ADHB occurs specifically in the mushroom H. marmoreus. Other edible mushrooms examined, including Pholiota microspora, Pleurotus eryngii, Mycena chlorophos, Sparassis crispa, Grifola frondosa, Pleurotus ostreatus, and Flammulina velutipes, do not contain detectable levels of d-ADHB. The concentration of d-ADHB in the fruiting body of H. marmoreus is relatively high (approximately 1.3 mg/g of fruiting body) and is comparable to the concentration of some of the most abundant free proteinogenic amino acids. Quantitative analysis of d-ADHB during fruiting body development demonstrated that the amino acid is synthesized during the fruiting body formation period. The absence of the putative precursors of d-ADHB, the (2S,3S)-isomer of ADHB and 2-oxo-tetronate, and the enzyme activities of d-ADHB racemase (2-epimerase) and transaminase suggested that d-ADHB is synthesized by a unique mechanism in this organism. Our data also suggested that the lack of or low expression of a d-ADHB degradation enzyme is a key determinant of d-ADHB accumulation in H. marmoreus.

Identification of enzymes involved in the mevalonate pathway of Flavobacterium johnsoniae.

The mevalonate pathway is prevalent in eukaryotes, archaea, and a limited number of bacteria. This pathway yields the fundamental precursors for isoprenoid biosynthesis, i.e., isopentenyl diphosphate and dimethylally diphosphate. In the downstream part of the general eukaryote-type mevalonate pathway, mevalonate is converted into isopentenyl diphosphate by the sequential actions of mevalonate kinase, phosphomevalonate kinase, and diphosphomevalonte decarboxylase, while a partial lack of the putative genes of these enzymes is sometimes observed in archaeal and bacterial genomes. The absence of these genes has led to the recent discovery of modified mevalonate pathways. Therefore, we decided to investigate the mevalonate pathway of Flavobacterium johnsoniae, a bacterium of the phylum Bacteroidetes, which is reported to lack the genes of mevalonate kinase and phosphomevalonate kinase. This study provides proof of the existence of the general mevalonate pathway in F. johnsoniae, although the pathway involves the kinases that are distantly related to the known enzymes.