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.

Characterization and application of l-methionine γ-lyase Q349S mutant enzyme with an enhanced activity toward l-homocysteine.

l-Methionine γ-lyse (MGL), a pyridoxal 5'-phosphate-dependent enzyme, catalyzes the α,γ-elimination of l-methionine (l-Met) and l-homocysteine (l-Hcy) to produce α-keto acids, thiols, and ammonia. Previously, various mutant enzymes of Pseudomonas putida MGL (PpMGL) were prepared to identify a homocysteine (Hcy)-specific enzyme that would assist the diagnosis of homocystinuria. Among the mutat enzymes the Q349S mutant exhibited high degradation activity toward l-Hcy. In the present study, PpMGL Q349S was characterized; the results suggested that it could be applied to determine the amount of l-Hcy. Compared to the wild-type PpMGL, specific activities of the Q349S mutant with l-Hcy and l-Met were 1.5 and 0.7 times, respectively. Additionally, we confirmed that l-Hcy in plasma samples could be accurately detected using the Q349S mutant by preincubating it with cysteine desulfurase (CsdA). Furthermore, we determined the X-ray crystal structure of PpMGL Q349S l-Met or l-Hcy complexes Michaelis complex, germinal diamine, and external aldimine at 2.25-2.40 Å. These 3D structures showed that the interaction partner of the ß-hydroxyl group of Thr355 in the wild-type PpMGL was changed to the carboxyl group of the Hcy-PLP external aldimine in the Q349S mutant. The interaction of Ser349 and Arg375 was different between l-Met and l-Hcy recognition, indicating that it was important for the recognition of the carboxyl group of the substrate.

Multiple mutations in RNA polymerase ß-subunit gene (rpoB) in Streptomyces incarnatus NRRL8089 enhance production of antiviral antibiotic sinefungin: modeling rif cluster region by density functional theory.

Streptomyces incarnatus NRRL8089 produces the antiviral, antifungal, antiprotozoal nucleoside antibiotic sinefungin. To enhance sinefungin production, multiple mutations were introduced to the rpoB gene encoding RNA polymerase (RNAP) ß-subunit at the target residues, D447, S453, H457, and R460. Sparse regression analysis using elastic-net lasso-ridge penalties on previously reported H457X mutations identified a numeric parameter set, which suggested that H457R/Y/F may cause production enhancement. H457R/R460C mutation successfully enhanced the sinefungin production by 3-fold, while other groups of mutations, such as D447G/R460C or D447G/H457Y, made moderate or even negative effects. To identify why the rif cluster residues have diverse effects on sinefungin production, an RNAP/DNA/mRNA complex model was constructed by homology modeling and molecular dynamics simulation. The 4 residues were located near the mRNA strand. Density functional theory-based calculation suggested that D447, H457, and R460 are in direct contact with ribonucleotide, and partially positive charges are induced by negatively charged chain of mRNA.

A new l-arginine oxidase engineered from l-glutamate oxidase.

The alternation of substrate specificity expands the application range of enzymes in industrial, medical, and pharmaceutical fields. l-Glutamate oxidase (LGOX) from Streptomyces sp. X-119-6 catalyzes the oxidative deamination of l-glutamate to produce 2-ketoglutarate with ammonia and hydrogen peroxide. LGOX shows strict substrate specificity for l-glutamate. Previous studies on LGOX revealed that Arg305 in its active site recognizes the side chain of l-glutamate, and replacement of Arg305 by other amino acids drastically changes the substrate specificity of LGOX. Here we demonstrate that the R305E mutant variant of LGOX exhibits strict specificity for l-arginine. The oxidative deamination activity of LGOX to l-arginine is higher than that of l-arginine oxidase form from Pseudomonas sp. TPU 7192. X-ray crystal structure analysis revealed that the guanidino group of l-arginine is recognized not only by Glu305 but also Asp433, Trp564, and Glu617, which interact with Arg305 in wild-type LGOX. Multiple interactions by these residues provide strict specificity and high activity of LGOX R305E toward l-arginine. LGOX R305E is a thermostable and pH stable enzyme. The amount of hydrogen peroxide, which is a byproduct of oxidative deamination of l-arginine by LGOX R305E, is proportional to the concentration of l-arginine in a range from 0 to 100 µM. The linear relationship is maintained around 1 µM of l-arginine. Thus, LGOX R305E is suitable for the determination of l-arginine.

Structural basis of enzyme activity regulation by the propeptide of l-lysine α-oxidase precursor from Trichoderma viride.

Harmuful proteins are usually synthesized as inactive precursors and are activated by proteolytic processing. l-Amino acid oxidase (LAAO) is a flavoenzyme that catalyzes the oxidative deamination of l-amino acid to produce a 2-oxo acid with ammonia and highly toxic hydrogen peroxide and, therefore, is expressed as a precursor. The LAAO precursor shows significant variation in size and the cleavage pattern for activation. However, the molecular mechanism of how the propeptide suppresses the enzyme activity remains unclear except for deaminating/decarboxylating Pseudomonasl-phenylalanine oxidase (PAO), which has a short N-terminal propeptide composed of 14 residues. Here we show the inactivation mechanism of the l-lysine oxidase (LysOX) precursor (prLysOX), which has a long N-terminal propeptide composed of 77 residues, based on the crystal structure at 1.97 Šresolution. The propeptide of prLysOX indirectly changes the active site structure to inhibit the enzyme activity. prLysOX retains weak enzymatic activity with strict specificity for l-lysine and shows raised activity in acidic conditions. The structures of prLysOX crystals that soaked in a solution with various concentrations of l-lysine have revealed that prLysOX can adopt two conformations; one is the inhibitory form, and the other is very similar to mature LysOX. The propeptide region of the latter form is disordered, and l-lysine is bound to the latter form. These results indicate that prLysOX uses a different strategy from PAO to suppress the enzyme activity and suggest that prLysOX can be activated quickly in response to the environmental change without proteolytic processing.

Structural basis for substrate specificity of l-methionine decarboxylase.

l -Methionine decarboxylase (MetDC) from Streptomyces sp. 590 is a vitamin B6 -dependent enzyme and catalyzes the non-oxidative decarboxylation of l -methionine to produce 3-methylthiopropylamine and carbon dioxide. We present here the crystal structures of the ligand-free form of MetDC and of several enzymatic reaction intermediates. Group II amino acid decarboxylases have many residues in common around the active site but the residues surrounding the side chain of the substrate differ. Based on information obtained from the crystal structure, and mutational and biochemical experiments, we propose a key role for Gln64 in determining the substrate specificity of MetDC, and for Tyr421 as the acid catalyst that participates in protonation after the decarboxylation reaction.

Structural basis of strict substrate recognition of l-lysine α-oxidase from Trichoderma viride.

l-Lysine oxidase (LysOX) is a FAD-dependent homodimeric enzyme that catalyzes the oxidative deamination of l-lysine to produce α-keto-ε-aminocaproate with ammonia and hydrogen peroxide. LysOX shows strict substrate specificity for l-lysine, whereas most l-amino acid oxidases (LAAOs) exhibit broad substrate specificity for l-amino acids. Previous studies of LysOX showed that overall structural similarity to the well-studied snake venom LAAOs. However, the molecular mechanism of strict specificity for l-lysine was still unclear. We here determined the structure of LysOX in complex with l-lysine at 1.7 Å resolution. The structure revealed that the hydrogen bonding network formed by D212, D315, and A440 with two water molecules is responsible for the recognition of the side chain amino group. In addition, a narrow hole formed by five hydrophobic residues in the active site contributes to strict substrate specificity. Mutation studies demonstrated that D212 and D315 are essential for l-lysine recognition, and the D212A/D315A double mutant LysOX showed different substrate specificity from LysOX. Moreover, the structural basis of the substrate specificity change has also been revealed by the structural analysis of the mutant variant and its substrate complexes. These results clearly explain the molecular mechanism of the strict specificity of LysOX and suggest that LysOX is a potential candidate for a template to design LAAOs specific to other l-amino acids.

Comparative Gene Analysis Focused on Silica Cell Wall Formation: Identification of Diatom-Specific SET Domain Protein Methyltransferases.

Silica cell walls of diatoms have attracted attention as a source of nanostructured functional materials and have immense potential for a variety of applications. Previous studies of silica cell wall formation have identified numerous involved proteins, but most of these proteins are species-specific and are not conserved among diatoms. However, because the basic process of diatom cell wall formation is common to all diatom species, ubiquitous proteins and molecules will reveal the mechanisms of cell wall formation. In this study, we assembled de novo transcriptomes of three diatom species, Nitzschia palea, Achnanthes kuwaitensis, and Pseudoleyanella lunata, and compared protein-coding genes of five genome-sequenced diatom species. These analyses revealed a number of diatom-specific genes that encode putative endoplasmic reticulum-targeting proteins. Significant numbers of these proteins showed homology to silicanin-1, which is a conserved diatom protein that reportedly contributes to cell wall formation. These proteins also included a previously unrecognized SET domain protein methyltransferase family that may regulate functions of cell wall formation-related proteins and long-chain polyamines. Proteomic analysis of cell wall-associated proteins in N. palea identified a protein that is also encoded by one of the diatom-specific genes. Expression analysis showed that candidate genes were upregulated in response to silicon, suggesting that these genes play roles in silica cell wall formation. These candidate genes can facilitate further investigations of silica cell wall formation in diatoms.

Suppressed Methionine γ-Lyase Expression Causes Hyperaccumulation of S-Methylmethionine in Soybean Seeds.

Several soybean (Glycine max) germplasms, such as Nishiyamahitashi 98-5 (NH), have an intense seaweed-like flavor after cooking because of their high seed S-methylmethionine (SMM) content. In this study, we compared the amounts of amino acids in the phloem sap, leaves, pods, and seeds between NH and the common soybean cultivar Fukuyutaka. This revealed a comparably higher SMM content alongside a higher free Met content in NH seeds, suggesting that the SMM-hyperaccumulation phenotype of NH soybean was related to Met metabolism in seeds. To investigate the molecular mechanism behind SMM hyperaccumulation, we examined the phenotype-associated gene locus in NH plants. Analyses of the quantitative trait loci in segregated offspring of the cross between NH and the common soybean cultivar Williams 82 indicated that one locus on chromosome 10 explains 71.4% of SMM hyperaccumulation. Subsequent fine-mapping revealed that a transposon insertion into the intron of a gene, Glyma.10g172700, is associated with the SMM-hyperaccumulation phenotype. The Glyma.10g172700-encoded recombinant protein showed Met-γ-lyase (MGL) activity in vitro, and the transposon-insertion mutation in NH efficiently suppressed Glyma.10g172700 expression in developing seeds. Exogenous administration of Met to sections of developing soybean seeds resulted in transient increases in Met levels, followed by continuous increases in SMM concentrations, which was likely caused by Met methyltransferase activity in the seeds. Accordingly, we propose that the SMM-hyperaccumulation phenotype is caused by suppressed MGL expression in developing soybean seeds, resulting in transient accumulation of Met, which is converted into SMM to avoid the harmful effects caused by excess free Met.

Novel method for l-methionine determination using l-methionine decarboxylase and application of the enzyme for l-homocysteine determination.

For many years, clinical studies have suggested that blood levels of l-methionine and L-homocysteine correlate with health status or homocystinuria/hypermethioninemia. l-Methionine in a solution containing 0%, 10%, or 20% human serum was detected in 10-200 µM using l-methionine decarboxylase (MetDC). Spike and recovery tests showed that the enzymatic assay could accurately and reproducibly determine the increases in l-methionine in serum samples. These results suggest that our enzymatic method using MetDC is useful for primary screening of hypermethioninemia or homocystinuria based on serum l-methionine concentration. Additionally, we confirmed that l-methionine (100 nmol) in solution was degraded to less than the detection limit by incubation at 37ºC for 10 min using 2 U of MetDC. Therefore, l-homocysteine in serum samples can be detected with equivalent sensitivity using l-methionine γ-lyase (MGL), in solutions that either did not contain l-methionine or contained l-methionine preincubated with MetDC.Abbreviations: DTT: dithiothreitol; IPTG: isopropyl-ß-d-thiogalactopyranoside; KPB: potassium phosphate buffer; MBTH: 3-methyl-2-benzothiazolinonehydrazone; mdc: the gene coding l-methionine decarboxylase; MetDC: l-methionine decarboxylase; mgl: the gene coding l-methionine γ-lyase; MGL: l-methionine γ-lyase; PLP: pyridoxal 5'-phosphate.

Antiviral effect of sinefungin on in vitro growth of feline herpesvirus type 1.

Feline herpesvirus type 1 (FHV-1) causes a potentially fatal disease in cats. Through the use of virus inhibition and cytotoxicity assays, sinefungin, a nucleoside antibiotic, was assessed for its potential to inhibit the growth of FHV-1. Sinefungin inhibited in vitro growth of FHV-1 most significantly over other animal viruses, such as feline infectious peritonitis virus, equine herpesvirus, pseudorabies virus and feline calicivirus. Our results revealed that sinefungin specifically suppressed the replication of FHV-1 after its adsorption to the host feline kidney cells in a dose-dependent manner without obvious cytotoxicity to the host cells. This antibiotic can potentially offer a highly effective treatment for animals infected with FHV-1, providing alternative medication to currently available antiviral therapies.

Application of l-methionine γ-lyase in chiral amino acid analysis.

Here, a conventional chiral amino acid analysis method using high-performance liquid chromatography was coupled with a sample pretreatment using l-methionine γ-lyase from Pseudomonas putida ICR 3460 for the selective analysis of l-methionine and l-tryptophan. The sample was analyzed after the degradation of l-methionine with l-methionine γ-lyase, as l-methionine coelutes with l-tryptophan under the standard chiral amino acid analytical conditions used for precolumn derivatization with o-phthalaldehyde and N-acetyl-l-cysteine. The l-tryptophan in the sample was then eluted as a clearly separated peak and analyzed further. Since the l-methionine γ-lyase did not act on l-tryptophan, we were able to calculate the l-methionine or l-tryptophan concentration based on the data obtained from 2 individual runs: the sample with and without l-methionine γ-lyase pretreatment. The concentration of l-tryptophan was calculated from the data obtained from the sample with l-methionine γ-lyase pretreatment, while the concentration of l-methionine was calculated using the following equation: l-methionine concentration = {the data from the sample without l-methionine γ-lyase pretreatment}-{the data from the sample with l-methionine γ-lyase pretreatment}. Model samples containing authentic amino acids and a fermented food sample were analyzed by our method, and the calculated concentrations of l-methionine and l-tryptophan were consistently in agreement with the theoretical values.

Development of Recombinant Methioninase for Cancer Treatment.

The elevated requirement for methionine (MET) of cancer cells is termed MET dependence. To selectively target the MET dependence of tumors for treatment on a large-scale preclinical and clinical basis, the L-methionine α-deamino-γ-mercaptomethane-lyase (EC 4.4.1.11) (methioninase, [METase]) gene from Pseudomonas putida has been cloned in Escherichia coli using the polymerase chain reaction (PCR). Purification using two DEAE Sepharose FF ion-exchange column and one ActiClean Etox endotoxin-affinity chromatography column has been established. Plasmid pMGLTrc03, which has a trc promoter and a spacing of 12 nucleotides between the Shine-Dalgarno sequence and the ATG translation initiation codon, was selected as the most suitable plasmid. The recombinant bacteria produced rMETase at 43% of the total proteins in soluble fraction by simple batch fermentation using a 500 L fermentor. Crystals were directly obtained from crude enzyme with 87% yield by a crystallization in the presence of 9.0% polyethylene glycol 6000, 3.6% ammonium sulfate, and 0.18 M sodium chloride using a 100 L crystallizer. After recrystallization, the enzyme was purified by anion-exchange column chromatography to remove endotoxins and by gel filtration for polishing. Purified rMETase is stable to lyophilization. In order to prevent immunological reactions which might be produced by multiple dosing of rMETase and to prolong the serum half-life of rMETase, the N-hydroxysuccinimidyl ester of methoxypolyethylene glycol propionic acid (M-SPA-PEG 5000) has been coupled to rMETase. The PEGylated molecules (PEG-rMETase) were purified from unreacted PEG with Amicon 30 K centriprep concentrators or by Sephacryl S-300 HR gel-filtration chromatography. Unreacted rMETase was removed by DEAE Sepharose FF anion-exchange chromatography. The resulting PEG-rMETase subunit, produced from a PEG/rMETase ratio of 30/1 in the synthetic reaction, had a molecular mass of approximately 53 kda determined by matrix-assisted laser desorption/ionization mass spectrometry, indicating the conjugation of two PEG molecules per subunit of rMETase and eight per tetramer. PEG-rMETase molecules obtained from reacting ratios of PEG/rMETase of 30/1 had an enzyme activity of 70% of unmodified rMETase. PEGylation of rMETase increased the serum half-life of the enzyme in rats to approximately 160 min compared to 80 min for unmodified rMETase. PEG-rMETase could deplete serum MET levels to less than 0.1 µM for approximately 8 h compared to 2 h for rMETase in rats. A significant prolongation of in vivo activity and effective MET depletion by the PEG-rMETase were achieved by the simultaneous administration of pyridoxal 5'-phosphate. rMETase was also conjugated with methoxypolyethylene glycol succinimidyl glutarate 5000 (MEGC-PEG). Miniosmotic pumps containing various concentrations of PLP were implanted in BALB-C mice. PLP-infused mice were then injected with a single dose of 4000 or 8000 units/kg PEG-rMETase. Mice infused with 5, 50, 100, 200, and 500 mg/mL PLP-containing miniosmotic pumps increased plasma PLP to 7, 24, 34, 60, and 95 µM, respectively, from the PLP baseline of 0.3 µM. PLP increased the half-life of MEGC-PEG-rMETase holoenzyme in a dose-dependent manner. The extended time of MET depletion by MEGC-PEG-rMETase was due to the maintenance of active MEGC-PEG-rMETase holoenzyme by infused PLP.

Integrated transcriptomic and proteomic analyses of a molecular mechanism of radular teeth biomineralization in Cryptochiton stelleri.

Many species of chiton are known to deposit magnetite (Fe3O4) within the cusps of their heavily mineralized and ultrahard radular teeth. Recently, much attention has been paid to the ultrastructural design and superior mechanical properties of these radular teeth, providing a promising model for the development of novel abrasion resistant materials. Here, we constructed de novo assembled transcripts from the radular tissue of C. stelleri that were used for transcriptome and proteome analysis. Transcriptomic analysis revealed that the top 20 most highly expressed transcripts in the non-mineralized teeth region include the transcripts encoding ferritin, while those in the mineralized teeth region contain a high proportion of mitochondrial respiratory chain proteins. Proteomic analysis identified 22 proteins that were specifically expressed in the mineralized cusp. These specific proteins include a novel protein that we term radular teeth matrix protein1 (RTMP1), globins, peroxidasins, antioxidant enzymes and a ferroxidase protein. This study reports the first de novo transcriptome assembly from C. stelleri, providing a broad overview of radular teeth mineralization. This new transcriptomic resource and the proteomic profiles of mineralized cusp are valuable for further investigation of the molecular mechanisms of radular teeth mineralization in chitons.

Redox-tuning of oxidizing disulfide oxidoreductase generates a potent disulfide isomerase.

Oxidative folding of extracellular proteins is pivotal for the biogenesis of bacterial virulence factors. Escherichia coli DsbA catalyzes disulfide bond formation in extracellular proteins and in multicomponent architectures on the cell surface. The present study assessed the significance of the redox properties of DsbA by exploiting the plaque-forming ability of bacteriophage M13, which specifically recognizes F-pili during infection of the host cell. A library of mutant dsbA genes was constructed by randomizing the dipeptide XX sequence in the active-site redox motif CXXC and then screened for mutants that altered plaque yield and appearance. In total, 24 dsbA mutant alleles produced substantially different degrees of complementation, and one mutant dsbA gene that encodes a CDIC sequence produced over 40-fold more clear plaques than wild type dsbA. The redox potential of purified DsbA [CDIC] was -172 mV, representing a less-oxidizing catalysis than the wild type DsbA (-122 mV), but one that is closer to yeast protein disulfide isomerase (-175 mV). DsbA [CDIC] exhibited a greater ability to refold fully denatured glutathionylated ribonuclease A than the wild type enzyme and a DsbA [CRIC] mutant, which has the same redox potential of -172 mV. Homology modeling and molecular dynamics simulation suggest that the CDIC mutant may have an enlarged substrate-binding cleft near the redox center, which confers kinetic advantages when acting on protein substrates.

Structural and mechanistic insights into homocysteine degradation by a mutant of methionine γ-lyase based on substrate-assisted catalysis.

Methionine γ-lyse (MGL) catalyzes the α, γ-elimination of l-methionine and its derivatives as well as the α, ß-elimination of l-cysteine and its derivatives to produce α-keto acids, volatile thiols, and ammonia. The reaction mechanism of MGL has been characterized by enzymological studies using several site-directed mutants. The Pseudomonas putida MGL C116H mutant showed drastically reduced degradation activity toward methionine while retaining activity toward homocysteine. To understand the underlying mechanism and to discern the subtle differences between these substrates, we analyzed the crystal structures of the reaction intermediates. The complex formed between the C116H mutant and methionine demonstrated that a loop structure (Ala51-Asn64) in the adjacent subunit of the catalytic dimer cannot approach the cofactor pyridoxal 5'-phosphate (PLP) because His116 disrupts the interaction of Asp241 with Lys240, and the liberated side chain of Lys240 causes steric hindrance with this loop. Conversely, in the complex formed between C116H mutant and homocysteine, the thiol moiety of the substrate conjugated with PLP offsets the imidazole ring of His116 via a water molecule, disrupting the interaction of His116 and Asp241 and restoring the interaction of Asp241 with Lys240. These structural data suggest that the Cys116 to His mutation renders the enzyme inactive toward the original substrate, but activity is restored when the substrate is homocysteine due to substrate-assisted catalysis.

The hyperthermophilic cystathionine γ-synthase from the aerobic crenarchaeon Sulfolobus tokodaii: expression, purification, crystallization and structural insights.

Cystathionine γ-synthase (CGS; EC 2.5.1.48), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, catalyzes the formation of cystathionine from an L-homoserine derivative and L-cysteine in the first step of the transsulfuration pathway. Recombinant CGS from the thermoacidophilic archaeon Sulfolobus tokodaii (StCGS) was overexpressed in Escherichia coli and purified to homogeneity by heat treatment followed by hydroxyapatite and gel-filtration column chromatography. The purified enzyme shows higher enzymatic activity at 353 K under basic pH conditions compared with that at 293 K. Crystallization trials yielded three crystal forms from different temperature and pH conditions. Form I crystals (space group P21; unit-cell parameters a = 58.4, b = 149.3, c = 90.2 Å, ß = 108.9°) were obtained at 293 K under acidic pH conditions using 2-methyl-2,4-pentanediol as a precipitant, whereas under basic pH conditions the enzyme crystallized in form II at 293 K (space group C2221; unit-cell parameters a = 117.7, b = 117.8, c = 251.3 Å) and in form II' at 313 K (space group C2221; unit-cell parameters a = 107.5, b = 127.7, c = 251.1 Å) using polyethylene glycol 3350 as a precipitant. X-ray diffraction data were collected to 2.2, 2.9 and 2.7 Šresolution for forms I, II and II', respectively. Structural analysis of these crystal forms shows that the orientation of the bound PLP in form II is significantly different from that in form II', suggesting that the change in orientation of PLP with temperature plays a role in the thermophilic enzymatic activity of StCGS.

Gene cloning, recombinant expression, purification and characterization of l-methionine decarboxylase from Streptomyces sp. 590.

l-Methionine decarboxylase (MetDC) from Streptomyces sp. 590 depends on pyridoxal 5'-phosphate and catalyzes the non-oxidative decarboxylation of l-methionine to produce 3-methylthiopropylamine and carbon dioxide. MetDC gene (mdc) was determined to consist of 1,674 bp encoding 557 amino acids, and the amino acid sequence is similar to that of l-histidine decarboxylases and l-valine decarboxylases from Streptomyces sp. strains. The mdc gene was cloned and recombinant MetDC was heterologously expressed by Escherichia coli. The purification of recombinant MetDC was carried out by DEAE-Toyopearl and Ni-NTA agarose column chromatography. The recombinant enzyme was homodimeric with a molecular mass of 61,000 Da and showed optimal activity between 45 to 55 °C and at pH 6.6, and the stability below 30 °C and between pH 4.6 to 7.0. l-Methionine and l-norleucine were good substrates for MetDC. The Michaelis constants for l-methionine and l-norleucine were 30 and 73 mM, respectively. The recombinant MetDC (0.50 U/ml) severely inhibited growth of human tumour cells A431 (epidermoid ovarian carcinoma cell line) and MDA-MB-231 (breast cancer cell line), however showed relatively low cytotoxicity for human normal cell NHDF-Neo (dermal fibroblast cell line from neonatal foreskin). This study revealed the properties of the gene and the protein sequence of MetDC for the first time.

A non-radioactive assay for selenophosphate synthetase activity using recombinant pyruvate pyrophosphate dikinase from Thermus thermophilus HB8.

Biosynthesis of selenocysteine-containing proteins requires monoselenophosphate, a selenium-donor intermediate generated by selenophosphate synthetase (Sephs). A non-radioactive assay was developed as an alternative to the standard [8-(14)C] AMP-quantifying assay. The product, AMP, was measured using a recombinant pyruvate pyrophosphate dikinase from Thermus thermophilus HB8. The KM and kcat for Sephs2-Sec60Cys were determined to be 26 µM and 0.352 min(-1), respectively.