Methylmercury Can Facilitate the Formation of Dehydroalanine in Selenoenzymes: Insight from DFT Molecular Modeling.

Experimental studies have indicated that electrophilic mercury forms (e.g., methylmercury, MeHg+) can accelerate the breakage of selenocysteine in vitro. Particularly, in 2009, Khan et al. (Environ. Toxicol. Chem. 2009, 28, 1567-1577) proposed a mechanism for the degradation of a free methylmercury selenocysteinate complex that was theoretically supported by Asaduzzaman et al. (Inorg. Chem. 2010, 50, 2366-2372). However, little is known about the fate of methylmercury selenocysteinate complexes embedded in an enzyme, especially in conditions of oxidative stress in which methylmercury target enzymes operate. Here, an accurate computational study on molecular models (level of theory: COSMO-ZORA-BLYP-D3(BJ)/TZ2P) was carried out to investigate the formation of dehydroalanine (Dha) in selenoenzymes, which irreversibly impairs their function. Methylselenocysteine as well as methylcysteine and methyltellurocysteine were included to gain insight on the peculiar behavior of selenium. Dha forms in a two-step process, i.e., the oxidation of the chalcogen nucleus followed by a syn-elimination leading to the alkene and the chalcogenic acid. The effect of an excess of hydrogen peroxide, which may lead to the formation of chalcogenones before the elimination, and of MeHg+, a severe toxicant targeting selenoproteins, which leads to the formation of methylmercury selenocysteinate, are also studied with the aim of assessing whether these pathological conditions facilitate the formation of Dha. Indeed, elimination occurs after chalcogen oxidation and MeHg+ facilitates the process. These results indicate a possible mechanism of toxicity of MeHg+ in selenoproteins.

Biochemistry of plants N-heterocyclic non-protein amino acids.

Plants catalyze the biosynthesis of a large number of non-protein amino acids, which are usually toxic for other organisms. In this review, the chemistry and metabolism of N-heterocyclic non-protein amino acids from plants are described. These N-heterocyclic non-protein amino acids are composed of ß-substituted alanines and include mimosine, ß-pyrazol-1-yl-L-alanine, willardiine, isowillardiine, and lathyrine. These ß-substituted alanines consisted of an N-heterocyclic moiety and an alanyl side chain. This review explains how these individual moieties are derived from their precursors and how they are used as the substrate for biosynthesizing the respective N-heterocyclic non-protein amino acids. In addition, known catabolism and possible role of these non-protein amino acids in the actual host is explained.

Covalently immobilize crude d-amino acid transaminase onto UiO-66-NH2 surface for d-Ala biosynthesis.

Enzyme reaction has been accepted widely in numerous applications owing to the high efficiency and stereo-selectivity, as well as simple preparation by gene engineering. However, the fragility and complex purification process of the enzyme are long-standing problems which limit the large-scale application. One possible solution may be the enzyme immobilization. As one type of porous material with high loading capacity and designable functionality, Metal-Organic Frameworks (MOFs) are ideal choices for the immobilization of enzyme with a considerable interest in recent years. In this study, d-amino acid transaminase (DAT), an important enzyme for industrial synthesis of d-Ala, was covalently immobilized on the surface of a star MOFs material, UiO-66-NH2. Interestingly, we found that the nanoscale hybrid enzyme UiO-66-NH2-Gd-DAT not only maintained the high catalytic efficiency but also got rid of the interference of polluting enzymes, which meant that we could obtain efficient and stereo-selective immobilized enzyme without complex purification process. In general, our findings demonstrated that using UiO-66-NH2 might be a promising strategy to immobilize enzyme and produce effective biocatalyst with high activity and stereo-selectivity.

The role of alanine synthesis and nitrate-induced nitric oxide production during hypoxia stress in Cucurbita pepo nectaries.

Floral nectar is a sugary solution produced by nectaries to attract and reward pollinators. Nectar metabolites, such as sugars, are synthesized within the nectary during secretion from both pre-stored and direct phloem-derived precursors. In addition to sugars, nectars contain nitrogenous compounds such as amino acids; however, little is known about the role(s) of nitrogen (N) compounds in nectary function. In this study, we investigated N metabolism in Cucurbita pepo (squash) floral nectaries in order to understand how various N-containing compounds are produced and determine the role of N metabolism in nectar secretion. The expression and activity of key enzymes involved in primary N assimilation, including nitrate reductase (NR) and alanine aminotransferase (AlaAT), were induced during secretion in C. pepo nectaries. Alanine (Ala) accumulated to about 35% of total amino acids in nectaries and nectar during peak secretion; however, alteration of vascular nitrate supply had no impact on Ala accumulation during secretion, suggesting that nectar(y) amino acids are produced by precursors other than nitrate. In addition, nitric oxide (NO) is produced from nitrate and nitrite, at least partially by NR, in nectaries and nectar. Hypoxia-related processes are induced in nectaries during secretion, including lactic acid and ethanolic fermentation. Finally, treatments that alter nitrate supply affect levels of hypoxic metabolites, nectar volume and nectar sugar composition. The induction of N metabolism in C. pepo nectaries thus plays an important role in the synthesis and secretion of nectar sugar.

Serine restriction alters sphingolipid diversity to constrain tumour growth.

Serine, glycine and other nonessential amino acids are critical for tumour progression, and strategies to limit their availability are emerging as potential therapies for cancer1-3. However, the molecular mechanisms driving this response remain unclear and the effects on lipid metabolism are relatively unexplored. Serine palmitoyltransferase (SPT) catalyses the de novo biosynthesis of sphingolipids but also produces noncanonical 1-deoxysphingolipids when using alanine as a substrate4,5. Deoxysphingolipids accumulate in the context of mutations in SPTLC1 or SPTLC26,7-or in conditions of low serine availability8,9-to drive neuropathy, and deoxysphinganine has previously been investigated as an anti-cancer agent10. Here we exploit amino acid metabolism and the promiscuity of SPT to modulate the endogenous synthesis of toxic deoxysphingolipids and slow tumour progression. Anchorage-independent growth reprogrammes a metabolic network involving serine, alanine and pyruvate that drives the endogenous synthesis and accumulation of deoxysphingolipids. Targeting the mitochondrial pyruvate carrier promotes alanine oxidation to mitigate deoxysphingolipid synthesis and improve spheroid growth, similar to phenotypes observed with the direct inhibition of SPT or ceramide synthesis. Restriction of dietary serine and glycine potently induces the accumulation of deoxysphingolipids while decreasing tumour growth in xenograft models in mice. Pharmacological inhibition of SPT rescues xenograft growth in mice fed diets restricted in serine and glycine, and the reduction of circulating serine by inhibition of phosphoglycerate dehydrogenase (PHGDH) leads to the accumulation of deoxysphingolipids and mitigates tumour growth. The promiscuity of SPT therefore links serine and mitochondrial alanine metabolism to membrane lipid diversity, which further sensitizes tumours to metabolic stress.

Rational modification of the carbon metabolism of Corynebacterium glutamicum to enhance L-leucine production.

L-Leucine is an essential amino acid that has wide and expanding applications in the industry. It is currently fast-growing market demand that provides a powerful impetus to further increase its bioconversion productivity and production stability. In this study, we rationally engineered the metabolic flux from pyruvate to L-leucine synthesis in Corynebacterium glutamicum to enhance both pyruvate availability and L-leucine synthesis. First, the pyc (encoding pyruvate carboxylase) and avtA (encoding alanine-valine aminotransferase) genes were deleted to weaken the metabolic flux of the tricarboxylic acid cycle and reduce the competitive consumption of pyruvate. Next, the transcriptional level of the alaT gene (encoding alanine aminotransferase) was down regulated by inserting a terminator to balance L-leucine production and cell growth. Subsequently, the genes involved in L-leucine biosynthesis were overexpressed by replacing the native promoters PleuA and PilvBNC of the leuA gene and ilvBNC operon, respectively, with the promoter Ptuf of eftu (encoding elongation factor Tu) and using a shuttle expression vector. The resulting strain WL-14 produced 28.47 ± 0.36 g/L L-leucine in shake flask fermentation.

Whole genome mining reveals a diverse repertoire of lanthionine synthetases and lanthipeptides among the genus Paenibacillus.

AIM: Lanthionine or methyllanthionine-containing lanthipeptides belongs to ribosomally synthesized and post-translationally modified peptides (RiPPs) family. Recent revolution in sequencing has made available huge genome sequence dataset of micro-organisms. In this study, we performed genome mining of the complete and partial genome sequences of 479 bacteria of the genus Paenibacillus to determine the diversity and distribution of lanthipeptide gene clusters. METHODS AND RESULTS: All genome sequences were annotated by RAST and subsequently analysed by BAGEL and antiSMASH. A total of 221 lanthipeptide gene clusters were identified in 127 strains of the genus Paenibacillus. One hundred and fifty gene clusters were found associated with the production of class I lanthipeptides while 58 and 13 gene clusters were related to class II and class IV lanthipeptide production respectively. Frequency of strains whose genomes encode putative lanthipeptide precursors was 26·5%. CONCLUSIONS: The results of lanthionine synthetases analysis suggested that diversity of lanthipeptides is much more than anticipated, while lanthionine synthetases must have been co-evolved among various species of the genus Paenibacillus. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report showing diversity and distribution of different classes of lanthipeptides among various species of the genus Paenibacillus. This study also reveals the novel lanthipeptide sequences which may be further developed as potential antimicrobials for therapeutic applications.

Roseocin, a novel two-component lantibiotic from an actinomycete.

Lantibiotics are lanthionine ring containing natural products that belong to the class of ribosomally synthesized and posttranslationally modified peptides (RiPPs). Recent expansion in the availability of microbial genome data and in silico analysis tools have accelerated the discovery of these promising alternatives to antibiotics. Following the genome-mining approach, a biosynthetic gene cluster for a putative two-component lantibiotic, roseocin, was identified in the genome of an Actinomycete, Streptomyces roseosporus NRRL 11379. Posttranslationally modified lanthipeptides of this cluster were obtained by heterologous expression of the genes in Escherichia coli, and were in vitro reconstituted to their bioactive form by exploiting commercial proteases like endoproteinase GluC, and proteinase K. The two peptides displayed synergistic antimicrobial activity against Gram-positive bacteria including the WHO high-priority pathogens, MRSA and VRE. Structural characterization confirmed the installation of four (methyl)lanthionine rings with an indispensable disulfide bond in the α-peptide, and six (methyl)lanthionine rings in the ß-peptide, by a single promiscuous lanthionine synthetase, RosM. Roseocin is the first two-component lantibiotic from a non-Firmicute, with extensive lanthionine bridging.

Biosynthesis of the N-N-Bond-Containing Compound l-Alanosine.

The formation of a N-N bond is a unique biochemical transformation, and nature employs diverse biosynthetic strategies to activate nitrogen for bond formation. Among molecules that contain a N-N bond, biosynthetic routes to diazeniumdiolates remain enigmatic. We here report the biosynthetic pathway for the diazeniumdiolate-containing amino acid l-alanosine. Our work reveals that the two nitrogen atoms in the diazeniumdiolate of l-alanosine arise from glutamic acid and aspartic acid, and we clarify the early steps of the biosynthetic pathway by using both in vitro and in vivo approaches. Our work demonstrates a peptidyl-carrier-protein-based mechanism for activation of the precursor l-diaminopropionate, and we also show that nitric oxide can participate in non-enzymatic diazeniumdiolate formation. Furthermore, we demonstrate that the gene alnA, which encodes a fusion protein with an N-terminal cupin domain and a C-terminal AraC-like DNA-binding domain, is required for alanosine biosynthesis.

Catalytic Production of Alanine from Waste Glycerol.

Chemical synthesis of amino acids directly from biomass feedstock is rare. Reported here is a one-step protocol to convert crude glycerol, from the biodiesel industry, into 43 % alanine over a Ru1 Ni7 /MgO catalyst. The multifunctional catalytic system promotes glycerol conversion into lactic acid, and then into alanine. X-ray absorption spectroscopy and scanning transmission electron microscopy revealed the existence of bimetallic RuNi species, whereas density-functional theory calculations suggested Ni-doped Ru substantially decreased the Ea of C-H bond dissociation of lactate alkoxide to form pyruvate, which is the rate-determining step. The catalytic route established in this work creates new opportunities for glycerol utilization and enriches the substrate scope of renewable feedstock to access value-added amino acids.

Structural and Kinetic Insight into the Biosynthesis of H2S and l-Lanthionine from l-Cysteine by a Pyridoxal l-Phosphate-Dependent Enzyme from Fusobacterium nucleatum.

Fusobacterium nucleatum is a common oral bacterium and a major producer of H2S, a toxic gas linked to the pathogenesis of periodontal disease. The bacterium encodes a fold type II pyridoxal l-phosphate (PLP)-dependent enzyme, Fn1220 or lanthionine synthase (LS), that generates H2S and l-lanthionine (a component of the peptidoglycan layer) through ß-replacement of l-cysteine by a second molecule of l-cysteine. Herein, we show through detailed kinetic analysis that LS elicits catalytic promiscuity as demonstrated for other fold type II PLP-dependent homologues, namely, O-acetylserine sulfhydrylase (OASS) and cystathionine ß-synthase (CBS). Like OASS, LS can assimilate H2S by catalyzing the ß-replacement of O-acetyl-l-serine by sulfide to form l-cysteine. However, the turnover for this reaction in LS is slower than that of other studied OASS enzymes due to slower conversion to the α-aminoacrylate intermediate. Similar to yeast and human CBS, LS can generate H2S and l-cystathionine through ß-replacement of l-cysteine by a second molecule of l-homocysteine; however, whereas this is the main H2S-forming reaction in CBS, it is not for LS. LS shows a marked preference for forming H2S and l-lanthionine through the condensation of 2 equiv of l-cysteine. Sequence alignment of LS with other CBS and OASS enzymes and inspection of the LS crystal structure in the external aldimine state with l-lanthionine reveal that LS possesses a unique loop that engages in hydrogen-bond contact with the product, providing a structural rationale for the enzyme's catalytic preference for H2S and l-lanthionine biosynthesis.

Optimization of a reduced enzymatic reaction cascade for the production of L-alanine.

Cell-free enzymatic reaction cascades combine the advantages of well-established in vitro biocatalysis with the power of multi-step in vivo pathways. The absence of a regulatory cell environment enables direct process control including methods for facile bottleneck identification and process optimization. Within this work, we developed a reduced, enzymatic reaction cascade for the direct production of L-alanine from D-glucose and ammonium sulfate. An efficient, activity based enzyme selection is demonstrated for the two branches of the cascade. The resulting redox neutral cascade is composed of a glucose dehydrogenase, two dihydroxyacid dehydratases, a keto-deoxy-aldolase, an aldehyde dehydrogenase and an L-alanine dehydrogenase. This artificial combination of purified biocatalysts eliminates the need for phosphorylation and only requires NAD as cofactor. We provide insight into in detail optimization of the process parameters applying a fluorescamine based L-alanine quantification assay. An optimized enzyme ratio and the necessary enzyme load were identified and together with the optimal concentrations of cofactor (NAD), ammonium and buffer yields of >95% for the main branch and of 8% for the side branch were achieved.

Use of the dehydrophos biosynthetic enzymes to prepare antimicrobial analogs of alaphosphin.

The C-terminal domain of the dehydrophos biosynthetic enzyme DhpH (DhpH-C) catalyzes the condensation of Leu-tRNALeu with (R)-1-aminoethylphosphonate, the aminophosphonate analog of alanine called Ala(P). The product of this reaction, Leu-Ala(P), is a phosphonodipeptide, a class of compounds that have previously been investigated for use as clinical antibiotics. In this study, we show that DhpH-C is highly substrate tolerant and can condense various aminophosphonates (Gly(P), Ser(P), Val(P), 1-amino-propylphosphonate, and phenylglycine(P)) to Leu. Moreover, the enzyme is also tolerant with respect to the amino acid attached to tRNALeu. Using a mutant of leucyl tRNA synthetase that is deficient in its proofreading ability allowed the preparation of a series of aminoacyl-tRNALeu derivatives (Ile, Ala, Val, Met, norvaline, and norleucine). DhpH-C accepted these aminoacyl-tRNA derivatives and condensed the amino acid with l-Ala(P) to form the corresponding phosphonodipeptides. A subset of these peptides displayed antimicrobial activities demonstrating that the enzyme is a versatile biocatalyst for the preparation of antimicrobial peptides. We also investigated another enzyme from the dehydrophos biosynthetic pathway, the 2-oxoglutarate dependent enzyme DhpA. This enzyme oxidizes 2-hydroxyethylphosphonate to 1,2-dihydroxyethylphosphonate en route to l-Ala(P), but longer incubation results in overoxidation to 1-oxo-2-hydroxyethylphosphonate. This α-ketophosphonate was converted by the pyridoxal phosphate dependent enzyme DhpD into l-Ser(P). Thus, the dehydrophos biosynthetic enzymes can generate not only l-Ala(P) but also l-Ser(P).

Peptidoglycan precursor synthesis along the sidewall of pole-growing mycobacteria.

Rod-shaped mycobacteria expand from their poles, yet d-amino acid probes label cell wall peptidoglycan in this genus at both the poles and sidewall. We sought to clarify the metabolic fates of these probes. Monopeptide incorporation was decreased by antibiotics that block peptidoglycan synthesis or l,d-transpeptidation and in an l,d-transpeptidase mutant. Dipeptides complemented defects in d-alanine synthesis or ligation and were present in lipid-linked peptidoglycan precursors. Characterizing probe uptake pathways allowed us to localize peptidoglycan metabolism with precision: monopeptide-marked l,d-transpeptidase remodeling and dipeptide-marked synthesis were coincident with mycomembrane metabolism at the poles, septum and sidewall. Fluorescent pencillin-marked d,d-transpeptidation around the cell perimeter further suggested that the mycobacterial sidewall is a site of cell wall assembly. While polar peptidoglycan synthesis was associated with cell elongation, sidewall synthesis responded to cell wall damage. Peptidoglycan editing along the sidewall may support cell wall robustness in pole-growing mycobacteria.

Activity-Based Probes Developed by Applying a Sequential Dehydroalanine Formation Strategy to Expressed Proteins Reveal a Potential α-Globin-Modulating Deubiquitinase.

We report a general and novel semisynthetic strategy for the preparation of ubiquitinated protein-activity-based probes on the basis of sequential dehydroalanine formation on expressed proteins. We applied this approach to construct a physiologically and therapeutically relevant ubiquitinated α-globin probe, which was used for the enrichment and proteomic identification of α-globin-modulating deubiquitinases. We found USP15 as a potential deubiquitinase for the modulation of α-globin, an excess of which aggravates ß-thalassemia symptoms. This development opens new opportunities for activity-based-probe design to shed light on the important aspects underlying ubiquitination and deubiquitination in health and disease.

Self-Directed in Cell Production of Methionine Analogue Azidohomoalanine by Synthetic Metabolism and Its Incorporation into Model Proteins.

Common protocols for the incorporation of noncanonical amino acids (ncAAs) into proteins require addition of the desired ncAA to the growth medium, its cellular uptake, and subsequent intracellular accumulation. This feeding scheme is generally suitable for small-scale proof-of-concept incorporation experiments. However, it is no general solution for orthogonal translation of ncAAs, as their chemical synthesis is generally tedious and expensive. Here, we describe a simple protocol that efficiently couples in situ semi-synthetic biosynthesis of L-azidohomoalanine and its incorporation into proteins at L-methionine (Met) positions. In our metabolically engineered Met-auxotrophic Escherichia coli strain, Aha is biosynthesized from externally added sodium azide and O-acetyl-L-homoserine as inexpensive precursors. This represents an efficient platform for expression of azide-containing proteins suitable for site-selective bioorthogonal strategies aimed at noninvasive protein modifications (Tornøe et al., J Org Chem 67:3057-3064, 2002; Kiick et al., Angew Chem Int Ed 39:2148-2152, 2000; Budisa, Angew Chem Int Ed Engl 47:6426-6463, 2004; van Hest, J Am Chem Soc 122:1282-1288, 2000).

Diversity of Polyketide Chains Achieved by Deleting the Tailoring Genes in the Biosynthesis of Ansatrienins.

The ast gene cluster (GenBank accession numbers KF813023.1 and KP284551) was characterized to be responsible for the biosynthesis of ansatrienins in Streptomyces sp. XZQH13, which contains astC, astF1, and astF2 genes involved in the assembly of the N-cyclohexanoyl d-alanyl side chain and the hydroxylation of C-19, respectively. Further to investigating the biosynthetic mechanism of ansatrienins, herein we constructed the mutant strains XZQH13OEΔastF2 and XZQH13OEΔastCΔastF2. Three new ansatrienin analogues, namely, ansatrienols I-K (1-3), along with trienomycinol (4) and 3-O-demethyltrienomycinol (5), were isolated from the XZQH13OEΔastCΔastF2 strain, and trienomycin A (6) and trienomycin G (7) were isolated from the XZQH13OEΔastF2 strain. Their structures were determined by a combination of high-resolution MS (ESI) and 1D and 2D NMR spectroscopy. Accordingly, a pathway for the biosynthesis of these new ansatrienins was proposed.

LanCL proteins are not Involved in Lanthionine Synthesis in Mammals.

LanC-like (LanCL) proteins are mammalian homologs of bacterial LanC enzymes, which catalyze the addition of the thiol of Cys to dehydrated Ser residues during the biosynthesis of lanthipeptides, a class of natural products formed by post-translational modification of precursor peptides. The functions of LanCL proteins are currently unclear. A recent proposal suggested that LanCL1 catalyzes the addition of the Cys of glutathione to protein- or peptide-bound dehydroalanine (Dha) to form lanthionine, analogous to the reaction catalyzed by LanC in bacteria. Lanthionine has been detected in human brain as the downstream metabolite lanthionine ketimine (LK), which has been shown to have neuroprotective effects. In this study, we tested the proposal that LanCL1 is involved in lanthionine biosynthesis by constructing LanCL1 knock-out mice and measuring LK concentrations in their brains using a mass spectrometric detection method developed for this purpose. To investigate whether other LanCL proteins (LanCL2/3) may confer a compensatory effect, triple knock-out (TKO) mice were also generated and tested. Very similar concentrations of LK (0.5-2.5 nmol/g tissue) were found in LanCL1 knock-out, TKO and wild type (WT) mouse brains, suggesting that LanCL proteins are not involved in lanthionine biosynthesis.

Assessment of Metabolic Changes in Mycobacterium smegmatis Wild-Type and alr Mutant Strains: Evidence of a New Pathway of d-Alanine Biosynthesis.

In mycobacteria, d-alanine is an essential precursor for peptidoglycan biosynthesis. The only confirmed enzymatic pathway to form d-alanine is through the racemization of l-alanine by alanine racemase (Alr, EC 5.1.1.1). Nevertheless, the essentiality of Alr in Mycobacterium tuberculosis and Mycobacterium smegmatis for cell survivability in the absence of d-alanine has been a point of controversy with contradictory results reported in the literature. To address this issue, we examined the effects of alr inactivation on the cellular metabolism of M. smegmatis. The M. smegmatis alr insertion mutant TAM23 exhibited essentially identical growth to wild-type mc2155 in the absence of d-alanine. NMR metabolomics revealed drastically distinct phenotypes between mc2155 and TAM23. A metabolic switch was observed for TAM23 as a function of supplemented d-alanine. In the absence of d-alanine, the metabolic response directed carbon through an unidentified transaminase to provide the essential d-alanine required for survival. The process is reversed when d-alanine is available, in which the d-alanine is directed to peptidoglycan biosynthesis. Our results provide further support for the hypothesis that Alr is not an essential function of M. smegmatis and that specific Alr inhibitors will have no bactericidal action.

Nitrile-synthesizing enzyme: Screening, purification and characterization.

Cyanide is known as a toxic compound for almost all living organisms. We have searched for cyanide-resistant bacteria from the soil and stock culture collection of our laboratory, and have found the existence of a lot of microorganisms grown on culture media containing 10 mM potassium cyanide. Almost all of these cyanide-resistant bacteria were found to show ß-cyano-L-alanine (ß-CNAla) synthetic activity. ß-CNAla synthase is known to catalyze nitrile synthesis: the formation of ß-CNAla from potassium cyanide and O-acetyl-L-serine or L-cysteine. We found that some microorganisms were able to detoxify cyanide using O-methyl-DL-serine, O-phospho-L-serine and ß-chloro-DL-alanine. In addition, we purified ß-CNAla synthase from Pseudomonas ovalis No. 111 in nine steps, and characterized the purified enzyme. This enzyme has a molecular mass of 60,000 and appears to consist of two identical subunits. The purified enzyme exhibits a maximum activity at pH 8.5-9.0 at an optimal temperature of 40-50°C. The enzyme is specific for O-acetyl-L-serine and ß-chloro-DL-alanine. The Km value for O-acetyl-L-serine is 10.0 mM and Vmax value is 3.57 µmol/min/mg.