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• W2012108328 abstract "The cyanobacterium Microcystis aeruginosa is widely known for its production of the potent hepatotoxin microcystin. This cyclic heptapeptide is synthesized non-ribosomally by the thio-template function of a large modular enzyme complex encoded within the 55-kb microcystin synthetase gene (mcy) cluster. The mcy gene cluster also encodes several stand-alone enzymes, putatively involved in the tailoring and export of microcystin. This study describes the characterization of the 2-hydroxy-acid dehydrogenase McyI, putatively involved in the production of d-methyl aspartate at position 3 within the microcystin cyclic structure. A combination of bioinformatics, molecular, and biochemical techniques was used to elucidate the structure, function, regulation, and evolution of this unique enzyme. The recombinant McyI enzyme was overexpressed in Escherichia coli and enzymatically characterized. The hypothesized native activity of McyI, the interconversion of 3-methyl malate to 3-methyl oxalacetate, was demonstrated using an in vitro spectrophotometric assay. The enzyme was also able to reduce α-ketoglutarate to 2-hydroxyglutarate and to catalyze the interconversion of malate and oxalacetate. Although NADP(H) was the preferred cofactor of the McyI-catalyzed reactions, NAD(H) could also be utilized, although rates of catalysis were significantly lower. The combined results of this study suggest that hepatotoxic cyanobacteria such as M. aeruginosa PCC7806 are capable of producing methyl aspartate via a novel glutamate mutase-independent pathway, in which McyI plays a pivotal role. The cyanobacterium Microcystis aeruginosa is widely known for its production of the potent hepatotoxin microcystin. This cyclic heptapeptide is synthesized non-ribosomally by the thio-template function of a large modular enzyme complex encoded within the 55-kb microcystin synthetase gene (mcy) cluster. The mcy gene cluster also encodes several stand-alone enzymes, putatively involved in the tailoring and export of microcystin. This study describes the characterization of the 2-hydroxy-acid dehydrogenase McyI, putatively involved in the production of d-methyl aspartate at position 3 within the microcystin cyclic structure. A combination of bioinformatics, molecular, and biochemical techniques was used to elucidate the structure, function, regulation, and evolution of this unique enzyme. The recombinant McyI enzyme was overexpressed in Escherichia coli and enzymatically characterized. The hypothesized native activity of McyI, the interconversion of 3-methyl malate to 3-methyl oxalacetate, was demonstrated using an in vitro spectrophotometric assay. The enzyme was also able to reduce α-ketoglutarate to 2-hydroxyglutarate and to catalyze the interconversion of malate and oxalacetate. Although NADP(H) was the preferred cofactor of the McyI-catalyzed reactions, NAD(H) could also be utilized, although rates of catalysis were significantly lower. The combined results of this study suggest that hepatotoxic cyanobacteria such as M. aeruginosa PCC7806 are capable of producing methyl aspartate via a novel glutamate mutase-independent pathway, in which McyI plays a pivotal role. The hepatotoxic microcystins compose the largest and most structurally diverse group of cyanobacterial toxins. Over 65 isoforms of microcystin varying by degree of methylation, hydroxylation, epimerization, peptide sequence, and toxicity have been identified (1.Sivonen K. Jones G. Chorus I. Bartram J. Toxic Cyanobacteria in Water: a Guide to Their Public Health Consequences, Monitoring and Management. E&FN Spon, London1999: 42-49Google Scholar). Underlying the extraordinary heterogeneity present among the microcystins is their common cyclic structure and possession of several rare nonproteinogenic amino acid moieties. Collectively, the microcystins may be described as monocyclic heptapeptides containing both d- and l-amino acids plus N-methyldehydroalanine and a unique β-amino acid side group, 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (Fig. 1) (2.Botes D. Wessels P. Kruger H. Runnegar M. Santikarn S. Smith R. Barna J. Williams D. J. Chem. Soc. 1985; 1: 2747-2748Google Scholar). Although various amino acid substitutions can occur within the microcystin cyclic structure, the most common toxin isoform, microcystin LR, contains lysine and arginine at positions 2 and 4, respectively (Fig. 1). The microcystin biosynthesis (mcy) gene cluster was the first complex metabolite gene cluster to be fully sequenced from a cyanobacterium. In Microcystis aeruginosa PCC7806, the mcy gene cluster spans 55 kb and comprises 10 genes arranged in two divergently transcribed operons, mcyA–C and mcyD–J. The larger of the two operons, mcyD–J, encodes a modular polyketide synthase (McyD), two hybrid enzymes comprising non-ribosomal peptide synthetase (McyE) and polyketide synthase (McyG) modules, and enzymes putatively involved in the tailoring (McyI, McyJ, and McyF) and transport (McyH) of the toxin. The smaller operon, mcyA–C, encodes three non-ribosomal peptide synthetases (McyA–C) (3.Tillett D. Dittmann E. Erhard M. von Dohren H. Borner T. Neilan B.A. Chem. Biol. 2000; 7: 753-764Abstract Full Text Full Text PDF PubMed Scopus (750) Google Scholar). Although most open reading frames within the mcy gene cluster have been assigned a function based on homologous entries in the data bases, no obvious function could be assigned to the 1014-bp gene mcyI. Preliminary sequence analysis of the inferred primary peptide sequence of mcyI by Tillett et al. (3.Tillett D. Dittmann E. Erhard M. von Dohren H. Borner T. Neilan B.A. Chem. Biol. 2000; 7: 753-764Abstract Full Text Full Text PDF PubMed Scopus (750) Google Scholar) revealed a 41% identity to the catalytic region of the serA-encoded d-3-phosphoglycerate dehydrogenase (PGDH 2The abbreviations used are: PGDH, d-3-phosphoglycerate dehydrogenase; 3-PGA, 3-phosphoglycerate; α-KG, α-ketoglutarate; 2-HGA, 2-hydroxyglutarate; LDH, d-lactate dehydrogenase; MDH, malate dehydrogenase; Mal, malate; OAA, oxalacetate; MeAsp, methyl aspartate; 3-MeMal, 3-methyl malate; MES, 4-morpholineethanesulfonic acid; 3-MeOAA, 3-methyl oxalacetate; ACT, aspartokinase chorismate mutase and TyrA (prephenate dehydrogenase).; EC 1.1.1.95) from Methanobacterium thermoautotrophicum (4.Smith D.R. Doucette-Stamm L.A. Deloughery C. Lee H. Dubois J. Aldredge T. Bashirzadeh R. Blakely D. Cook R. Gilbert K. Harrison D. Hoang L. Keagle P. Lumm W. Pothier B. Qiu D. Spadafora R. Vicaire R. Wang Y. Wierzbowski J. Gibson R. Jiwani N. Caruso A. Bush D. Safer H. Patwell D. Prabhakar S. McDougall S.N. Shimer G. Goyal A. Pietrokovski S. Church G. Daniels C. Mao J. Rice P. Nolling J. Reeve J.N. J. Bacteriol. 1997; 179: 7135-7155Crossref PubMed Scopus (1040) Google Scholar). Although this archaeal PGDH homolog has not yet been characterized, extensive research has been carried out on PGDH homologs from other organisms, including mammals, plants, and bacteria. PGDH belongs to the 2-hydroxy acid family of dehydrogenases and catalyzes the first committed step in the phosphorylated serine biosynthesis pathway: the oxidation of 3-phosphoglycerate (3-PGA) to 3-phosphohydroxypyruvate, with the concomitant reduction of NAD to NADH. Interestingly, PGDH is also capable of catalyzing the interconversion of α-ketoglutarate (α-KG) and 2-hydroxyglutarate (2-HGA) (5.Zhao G. Winkler M.E. J. Bacteriol. 1996; 178: 232-239Crossref PubMed Scopus (117) Google Scholar). The d-2-hydroxy-acid dehydrogenase family also includes d-lactate dehydrogenase (LDH; EC 1.1.1.28) and malate dehydrogenase (MDH; EC 1.1.1.37), which catalyze the conversion of lactate to pyruvate and malate (Mal) to oxalacetate (OAA), respectively. The family members share an overall sequence identity of ∼22% and a similarity of ∼50% (6.Bell J.K. Pease P.J. Bell J.E. Grant G.A. Banaszak L.J. Eur. J. Biochem. 2002; 269: 4176-4184Crossref PubMed Scopus (15) Google Scholar). These enzymes also possess a conserved nucleotide-binding motif that preferentially binds to either NAD(H) or NADP(H). Despite its similarity to PGDH and other 2-hydroxy-acid dehydrogenases, McyI was originally predicted to function as a dehydratase enzyme, playing a role in microcystin biosynthesis by catalyzing the dehydration of serine to dehydroalanine (3.Tillett D. Dittmann E. Erhard M. von Dohren H. Borner T. Neilan B.A. Chem. Biol. 2000; 7: 753-764Abstract Full Text Full Text PDF PubMed Scopus (750) Google Scholar). The recently sequenced nodularin gene cluster of Nodularia spumigena NSOR10 also encodes a 2-hydroxy-acid dehydrogenase homolog, NdaH. Like McyI, this enzyme was originally predicted to catalyze a dehydration reaction: the conversion of threonine to dehydrobutyrine (7.Moffitt M.C. Neilan B.A. Appl. Environ. Microbiol. 2004; 70: 6353-6362Crossref PubMed Scopus (183) Google Scholar). Although the microcystin and nodularin biosynthesis pathways may involve serine/threonine dehydration reactions, it is unlikely that these reactions are catalyzed by McyI and NdaH, as originally predicted. McyI and NdaH are homologous to 2-hydroxy-acid dehydrogenases, and this family of enzymes is neither structurally nor functionally related to the family of dehydratase enzymes known to convert serine to dehydroalanine in other secondary metabolite pathways (8.Karakas S. Narbad A. Horn N. Dodd H. Par A. Colquhoun I. Gasson M. Eur. J. Biochem. 1999; 261: 524-532Crossref PubMed Scopus (93) Google Scholar). Whereas dehydrogenases such as PGDH catalyze redox reactions (i.e. the transfer of electrons involving pyridinium nucleotides), the amino-acid dehydratases catalyze dehydration and rehydration reactions (i.e. the removal or addition of H2O). The chemical nature of these reactions and their respective substrates are very different. Therefore, alternative putative functions for McyI and NdaH in hepatotoxin production needed to be investigated. As McyI shares sequence homology with PGDH, the first committed step in the phosphorylated serine biosynthesis pathway, it is conceivable that this enzyme may also play a role in serine metabolism. However, as no other serine biosynthesis enzymes (e.g. phosphoserine transaminase and phosphoserine phosphatase) are encoded within the mcy gene cluster, it is unlikely that McyI is involved in the production of this nonpolar amino acid. An alternative hypothesis is that McyI is involved in the production of the methyl aspartate unit (MeAsp3) of the microcystin cyclic structure. A feeding study with [1,2-13C]acetate suggested that the MeAsp residues in microcystin and nodularin are synthesized via a condensation reaction between acetyl-CoA and pyruvic acid. 2-Hydroxy-2-methylsuccinic acid is then converted to 2-hydroxy-3-methylsuccinic acid, oxidized to 2-oxo-3-methylsuccinic acid, and finally transaminated to MeAsp (9.Moore R. Chen J. Moore B. Patterson G. J. Am. Chem. Soc. 1991; 113: 5083-5084Crossref Scopus (69) Google Scholar). As McyI is a 2-hydroxy-acid dehydrogenase homolog, we predict that this enzyme catalyzes the interconversion of 2-hydroxy-3-methylsuccinic acid (3-methyl malate) to 2-oxo-3-methylsuccinic acid (3-methyl oxalacetate), the penultimate step in methyl aspartate biosynthesis (Fig. 2). This study describes the bioinformatics, genetic, and enzymatic characterization of McyI. The results of these experiments are discussed with respect to the role of this unique methyl-malate dehydrogenase in microcystin biosynthesis. Cyanobacterial Strains and Cultures−The microcystin-producing strain M. aeruginosa PCC7806 was kindly provided by E. Dittmann (Institute for Biology, Humboldt University, Berlin). Other cyanobacteria were obtained from the Institute for Biology, the Microbiology Division of the Biocenter at the University of Helsinki (Helsinki, Finland), and the cyanobacterial culture collection (University of New South Wales). Cyanobacteria were grown as batch cultures in BG11 medium (Fluka) under 16 μmol photons m-2 s-1 white light. Light intensities were measured using a LI-COR LI-250 light meter (LI-COR Biosciences). The absorbance (A650) of the cultures was measured using an Ultrospec II spectrophotometer (Biochrom Ltd.). Cyanobacterial DNA Extractions−DNA was isolated from cyanobacteria according to the xanthogenate-SDS method of Tillett and Neilan (10.Tillett D. Neilan B.A. J. Phycol. 2000; 36: 251-258Crossref Scopus (267) Google Scholar). Screening Various Strains of Cyanobacteria for mcyI Orthologs−The degenerate oligonucleotide primers mcyIdegenF (5′-TGTGCGTTATCCTAMTAA-3′) and mcyIdegenR (5′-GGCTTCTCDCCCTGAAGC-3′) were designed to amplify, via PCR, an ∼790-bp sequence within the mcyI locus of M. aeruginosa PCC7806 and Anabaena sp. 90. These primers were used to screen chromosomal DNA samples from several toxic and nontoxic strains of cyanobacteria for mcyI orthologs (see Table 1).TABLE 1Distribution of mcyI orthologs among toxic and nontoxic cyanobacteriaStrainRef./sourceMicrocystinaProduction of toxins was detected by mouse bioassay, high pressure liquid chromatography, or enzyme-linked immunosorbent assaymcyIdegenF/RbmcyI orthologs were detected by PCR using mcyIdegenF/RAnabaena cylindrica PCC7122Ref. 38.Herdman M. Castenholz R.W. Iteman I. Waterbury J.B. Rippka R. Boone D.R. Castenholz R.W. Garrity G.M. Bergey's Manual of Systematic Bacteriology. 2nd Ed. Vol. 1. Springer-Verlag, New York2001: 776Google Scholar––Anabaena flosaquaeUTEXcUTEX, University of Texas; NIES, National Institute for Environmental Studies; PCC, Pasteur Culture Collection; N, nodularin-producing strain––Anabaena PCC7108Ref. 39.Rippka R. Deruelles J. Waterbury J.B. Herdman M. Stanier R.Y. J. Gen. Microbiol. 1979; 111: 1-61Crossref Google Scholar–+Anabaena sp. 90Ref. 40.Sivonen K. Namikoshi M. Evans W.R. Carmichael W.W. Sun F. Rouhiainen L. Luukkainen R. Rinehart K.L. Appl. Environ. Microbiol. 1992; 58: 2495-2500Crossref PubMed Google Scholar++M. aeruginosa HUB 5.3Ref. 41.Henning M. Hertel H. Wall H. Kohl J.G. Int. Rev. Ges. Hydrobiol. 1991; 76: 37-45Crossref Scopus (34) Google Scholar–+M. aeruginosa NIES298NIEScUTEX, University of Texas; NIES, National Institute for Environmental Studies; PCC, Pasteur Culture Collection; N, nodularin-producing strain++M. aeruginosa PCC7005PCCcUTEX, University of Texas; NIES, National Institute for Environmental Studies; PCC, Pasteur Culture Collection; N, nodularin-producing strain++M. aeruginosa PCC7804PCC++M. aeruginosa PCC7806Ref. 42.Rippka R. Herdman M. Pasteur Culture Collection of Cyanobacterial Strains in Axenic Culture. Catalogue and Taxonomic Handbook, Vol. 1: Catalogue of Strains. Institute Pasteur, Paris1992Google Scholar++M. aeruginosa PCC7820Ref. 42.Rippka R. Herdman M. Pasteur Culture Collection of Cyanobacterial Strains in Axenic Culture. Catalogue and Taxonomic Handbook, Vol. 1: Catalogue of Strains. Institute Pasteur, Paris1992Google Scholar++M. aeruginosa UWOCC MR-ARef. 43.Jackson A.R. McInnes A. Falconer I.R. Runnegar M.T. Vet. Pathol. 1984; 21: 102-113Crossref PubMed Scopus (88) Google Scholar++M. aeruginosa UWOCC MR-CRef. 43.Jackson A.R. McInnes A. Falconer I.R. Runnegar M.T. Vet. Pathol. 1984; 21: 102-113Crossref PubMed Scopus (88) Google Scholar–+M. aeruginosa UWOCC MR-DRef. 43.Jackson A.R. McInnes A. Falconer I.R. Runnegar M.T. Vet. Pathol. 1984; 21: 102-113Crossref PubMed Scopus (88) Google Scholar++M. aeruginosa UWOC CBSRef. 44.Starr R.C. Zeikus J.A. J. Phycol. 1993; 29: 1-106Crossref Scopus (607) Google Scholar–+M. aeruginosa UWOCE7.GCRef. 44.Starr R.C. Zeikus J.A. J. Phycol. 1993; 29: 1-106Crossref Scopus (607) Google Scholar++Microcystis viridis NIES102Ref. 45.Neilan B.A. Jacobs D. Del Dot T. Blackall L.L. Hawkins P.R. Cox P.T. Goodman A.E. Int. J. Syst. Bacteriol. 1997; 47: 693-697Crossref PubMed Scopus (442) Google Scholar++Microcystis wesenbergii NIES107NIES++Microcystis PCC7840PCC++Microcystis sp. UTEX2664UTEX++Microcystis ssp. UTEX2667UTEX++Nodularia harveyana PCC73104PCCNcUTEX, University of Texas; NIES, National Institute for Environmental Studies; PCC, Pasteur Culture Collection; N, nodularin-producing strain–N. spumigena NSOR10Ref. 46.Moffitt M.C. Neilan B.A. FEMS Microbiol. Lett. 2001; 196: 207-214Crossref PubMed Google ScholarN–P. agardhii CYA126Ref. 22.Christiansen G. Fastner J. Erhard M. Borner T. Dittmann E. J. Bacteriol. 2003; 185: 564-572Crossref PubMed Scopus (296) Google Scholar+–Synechocystis PCC6803PCC––a Production of toxins was detected by mouse bioassay, high pressure liquid chromatography, or enzyme-linked immunosorbent assayb mcyI orthologs were detected by PCR using mcyIdegenF/Rc UTEX, University of Texas; NIES, National Institute for Environmental Studies; PCC, Pasteur Culture Collection; N, nodularin-producing strain Open table in a new tab Sequence Analysis−The 1011-bp DNA sequence of mcyI (complement of nucleotides 2004–3017; GenBank™ accession number AF183408) was analyzed using several different computer programs accessed via the ExPASy proteomics server. Codon usage within the mcyI sequence was assessed with the Countcodon program (Kazusa DNA Research Institute). The percentage similarity and identity scores of McyI and other peptide sequences were determined using the PSI-BLAST program (NCBI). Conserved domains within McyI were detected using CD-Search (NCBI) and ScanProsite (ExPASy). Phylogenetic Analysis−A PSI-BLAST search with the McyI sequence returned 26 sequences with BLAST scores >200 (see Table 2). These sequences were subsequently used to generate a multiple sequence alignment and corresponding phylogenetic tree (ClustalX). Four reference sequences from characterized enzymes (PGDH, LDH, MDH, and formate dehydrogenase) were also included, as was Escherichia coli NAD-independent d-lactate dehydrogenase. The latter was designated as an artificial outgroup because it is a membrane-bound FAD flavoenzyme and does not belong to the d-isomer-specific 2-hydroxy-acid dehydrogenase family (11.Campbell H.D. Rogers B.L. Young I.G. Eur. J. Biochem. 1984; 144: 367-373Crossref PubMed Scopus (19) Google Scholar). Phylogenetic trees were generated using the neighbor-joining method with gaps removed. Trees were displayed graphically using NJplot and AppleWorks Version 6 (Apple Computer, Inc.).TABLE 2Sequences used to create the phylogenetic tree in Fig. 4OrganismProteinPrimary functionaPrimary enzymatic function is given for experimentally characterized proteins onlyLengthbThe values indicate the lengths of the amino acid sequencesAccession no.cThese are the NCBI accession numbers. References can be found in the corresponding data base filesAnabaena sp.McyI337AA062580A. thaliana602NP_195146A. thalianaPGDHd-3-Phosphoglycerate dehydrogenase624T52296A. thalianaPGDHd-3-Phosphoglycerate dehydrogenase603AAM60833Archaeoglobus fulgidus527NP_069647Crocosphaera watsonii525ZP_00179809E. coliLDH (LdhA)Fermentative d-lactate dehydrogenase329AAB51772E. coliPGDH (SerA)d-3-Phosphoglycerate dehydrogenase410P08328E. coliDLDd-Lactate dehydrogenase (NADH-independent)571AAC75194Flaveria trinerviaMDHd-Malate dehydrogenase385AAA87008Methanocaldococcus jannaschii524NP_248012M. kandleri522NP_613584Methanothermobacter thermoautotrophicus525NP_276105M. aeruginosaMcyI337AAF00955M. aeruginosaMcyI337AB032549N. spumigenaNdaH341AA064409Nostoc punctiforme526ZP_00112058Nostoc sp.526NP_485930O. iheyensis319NP_693766Pseudomonas sp.FDHNAD-dependent formate dehydrogenase401P33160Pyrococcus abyssi307NP_126444Pyrococcus furiosus306NP_579123R. xylanophilus527ZP_00199880Synechococcus elongatus529ZP_00164567Synechocystis sp.554NP_441198Synechocystis sp.528NP_896628T. tengcongensis533NP_624129Thermosynechococcus elongatus527NP_681115Trichodesmium erythraeum527ZP_00327083a Primary enzymatic function is given for experimentally characterized proteins onlyb The values indicate the lengths of the amino acid sequencesc These are the NCBI accession numbers. References can be found in the corresponding data base files Open table in a new tab Overexpression of Histidine-tagged McyI−Expression cultures were inoculated with 1% overnight starter culture (Rosetta(DE3)pLysS plus pET30(mcyI)) and grown in Tryptone phosphate medium supplemented with kanamycin (50 μg/ml) and chloramphenicol (34 μg/ml). Cultures were grown with vigorous shaking (160 rpm) at 37 °C to A650 = 0.6 and either retained at 37 °C or transferred to 30 or 25 °C and incubated for an additional 20 min (or until A650 ∼ 1.0). Expression of McyI was subsequently induced with 1 mm isopropyl β-d-thiogalactopyranoside for 0–18 h at 25–37 °C. Cell pellets were harvested by centrifugation 5000 × g for 10 min at 4 °C, washed with phosphate buffer (0.5 m NaCl and 20 mm sodium phosphate (pH 7.4)), and stored at -80 °C until required. Purification of Histidine-tagged McyI−Cell pellets were thawed on ice, resuspended in 2% of the original culture volume of cold phosphate buffer containing 1 mm phenylmethylsulfonyl fluoride, passed several times through an 18-gauge needle, and then briefly sonicated (Branson Sonifier model 250; amplitude of 25, 50% duty cycle, pulsed). The lysate was cleared by centrifugation at 20,000 × g for 30 min at 4 °C and precipitated on ice using 40% ammonium sulfate. The final pellet was resuspended in 0.4% of the original culture volume of cold phosphate buffer containing 100 mm imidazole, filtered through a 22-μm membrane, and applied to a 5-ml HiTrap column (Amersham Biosciences) charged with Ni2+. The column was washed with 10 column volumes of phosphate buffer containing 100 mm imidazole and eluted with 2 column volumes of phosphate buffer containing 300 mm imidazole. The wash and eluate were collected in 1-ml volumes and analyzed by SDS-PAGE (12% polyacrylamide gels) and Western blotting using a nickel-nitrilotriacetic acid-alkaline phosphatase conjugate (Qiagen Inc.) with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium staining (Sigma). Purified protein samples intended for enzyme assays were desalted by size exclusion filtration using Amicon Ultra 10-kDa cutoff columns and 50 mm HEPES exchange buffer (pH 7). Determining the Subunit Organization of Native McyI−The molecular masses of the individual subunits of McyI were estimated by size exclusion chromatography using an ÄKTA Basic 900 series fast protein liquid chromatography apparatus fitted with a Frac-920 fraction collector, a UV detector, and a Superdex 200 10/300 GL column (Amersham Biosciences). The column was equilibrated with wash buffer (50 mm sodium phosphate (pH 7) and 150 mm NaCl) and calibrated with ∼1 mg each of seven molecular mass standards (Sigma) (see Fig. 6). One milligram of purified McyI was subsequently loaded. All samples were run at a flow rate of 0.5 ml/min. Eluted proteins were detected spectrophotometrically (220–280 nm) and collected in 250-μl fractions. Standard curves (log of subunit molecular mass versus volume eluted) were based on the molecular masses of the protein standards. The eluted McyI fractions were diluted to 5 μg/ml and checked for activity by performing an OAA reductase assay (see below). Chemical Synthesis of 2-Hydroxy-3-methylsuccinic Acid− 2-Hydroxy-3-methylsuccinic acid (3-methyl malate (3-MeMal)) was synthesized by reduction of diethyl oxalpropionate (10 g, 0.05 mol; Aldrich) in 95% ethanol (100 ml) by the addition of an 8-fold excess of sodium borohydride (3.8 g, 0.1 mol). After the mixture was stirred at room temperature for 24 h, 4 m HCl (20 ml) was slowly added, and the most of the ethanol evaporated under reduced pressure. Saturated sodium chloride solution (100 ml) was added, and the mixture was extracted five times with ethyl acetate (100 ml). The combined extracts were filtered, dried (MgSO4), and evaporated, yielding a viscous oil (4.5 g, ∼45%). The 1H NMR spectrum (CDCl3) showed signals for the threo-isomer (δ 1.07 (d, J = 7.2 Hz, 3-Me) and 2.9 (m, H-3)) and the erythro-isomer (δ 3.0 (m, H-3)). The ratio of the threo-to erythro-isomers was 1:6. The other signals of the two isomers (erythro and threo H-2 and erythro 3-Me signals) are underneath the CH2 resonances (δ 4.1–4.3) and the CH3 resonances (δ 1.1–1.2) of the ethyl ester groups (data not shown). Hydrolysis of the crude esters (2.0 g) was carried out by refluxing with 4 m HCl (50 ml) for 4 h. The cooled mixture was diluted with water (100 ml) and extracted with ethyl acetate (2 × 50 ml) to remove any unhydrolyzed esters and some unsaturated acids formed. The aqueous phase was evaporated to dryness, giving an oil (1.2 g). This was left to stand at -20 °C for 72 h after approximately half had crystallized. The oily crystals were washed briefly with cold chloroform, in which hydroxymethylsuccinic acids are insoluble, leaving a colorless crystalline solid (0.54 g). The 600-MHz 1H NMR (2H2O) spectrum now showed only the clearly resolved resonances for the two isomers (threo/erythro ratio of 1:6): threo-isomer, δ 4.54 (d, J = 4.05 Hz, H-2), 2.91 (m, H-3), and 1.0 (d, J = 7.14 Hz, 3-Me); and erythro-isomer, δ 4.28 (d, J = 4.17 Hz, H-2), 2.97 (m, H-3), and 1.05 (d, J = 7.20 Hz, 3-Me). The latter assignments are identical to those reported for the erythro-isomer (12.Herter S. Fuchs G. Bacher A. Eisenreich W. J. Biol. Chem. 2002; 277: 20277-20283Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). The resonances are also consistent with earlier NMR data recorded at lower fields and thus lacking some of the dispersion reported here for both the free acids and some derivatives (13.Baht K.S. Dixit K.N. Rao A.S. Indian J. Chem. 1985; 24: 509-512Google Scholar, 14.Renaud P. Hurzler M. Seebach D. Helv. Chim. Acta. 1987; 70: 292-298Crossref Scopus (18) Google Scholar, 15.Kakinuma K. Teresawa H. Li H.-Y. Miyazaki K. Oshima T. Biosci. Biotechnol. Biochem. 1993; 57: 1916-1923Crossref Scopus (3) Google Scholar). No attempts were made to separate these two isomers, and they were used as inhibitors in the ratio indicated above. Oxidoreductase Assays−The dehydrogenase and reductase activities of recombinant McyI were analyzed in triplicate in 1-ml cuvettes. The production or disappearance of NAD(P)/H was monitored spectrophotometrically at 340 nm using a Cary 100 UV spectrophotometer (Varian, Inc.). Reactions were initiated by the addition of substrate or enzyme. The dehydrogenase activities of McyI were measured by monitoring the conversion of NADP to NADPH. Each reaction contained 1 mm NADP, 0.1–10 mm substrate (3-MeMal, d-Mal, l-Mal, d-2-HGA, d-3-PGA), and 50 μg of purified McyI in 1 ml of assay buffer. The production of NADPH was monitored spectrophotometrically at 340 nm for 0.5–1 min at 37 °C. The reductase activities of McyI were measured by monitoring the conversion of NADPH to NADP. Each reaction contained 0.25 mm NADPH, 0.1–5 mm substrate (α-KG or OAA), and 2–5 μg of purified McyI in 1 ml of assay buffer. The disappearance of NADPH was monitored spectrophotometrically at 340 nm for 0.5–1 min at 37 °C. End Product Analysis−The end products of the Mal/OAA oxidoreductase reactions were analyzed by NMR spectroscopy. NMR spectroscopy was carried out on a Bruker Advance DMX-600 spectrometer using a triple broadband inverse probe. 1H NMR spectra were acquired at 600.13 MHz with a 90 ° pulse of 9 μs, a spectral width of 8992 Hz, 33,000 data points and a 2-s delay between pulses. Samples were run in 0.5 ml of 95% potassium phosphate (pH 7) and 5% 2H2O. The standard Bruker water saturation pulse program was used. The free induction decay was zero-filled to 64,000 data points and processed with a line-broadening factor of 1 Hz before Fourier transformation. Determining the Optimal pH of the McyI OAA Reductase Assay−The pH optimum for the McyI OAA reductase assay was determined by monitoring the disappearance of NADPH at 340 nm over a pH range of 5.5–9 at 25 °C. Each 1-ml reaction contained 0.25 mm NADPH, 0.8 mm OAA, and 5 μg of purified McyI in 1 ml of assay buffer (1.0 mm dithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride, and 100 mm buffer (MES at pH 5.5–6.5, HEPES at pH 7–8, or Tris-Cl at pH 8.5–9)). Determining the Optimal Temperature and Thermostability of the McyI OAA Reductase Assay−The temperature optimum for the McyI OAA reductase assay was determined by monitoring the disappearance of NADPH at 340 nm over a temperature range of 25–60 °C. Each 1-ml reaction contained 0.25 mm NADPH, 0.8 mm OAA, and 5 μg of purified McyI in 1 ml of assay buffer (pH 7). The stability of McyI at different temperatures was determined by incubating the enzyme for 3 min at 25–60 °C and then chilling it immediately on ice prior to performing the OAA reductase assay at 37 °C as described above. McyI Specificity−Various structurally related compounds (lactate, pyruvate, d-Ser, l-Ser, l-Phe, oxalic acid, cinnamate, phenyl acetate, phenyl lactate, phenylalanine, and diethyl oxalpropionate; 0.1–10 mm) were tested as substrates of McyI in the forward (dehydrogenase) or reverse (reductase) direction at 37 °C for 1 min at pH 7 as described above. To test whether McyI is able to use non-phosphorylated cofactors, the 3-MeMal, Mal, OAA, and α-KG oxidoreductase assays were repeated using NAD/H in place of NADP/H. McyI Kinetic Analysis−Kinetic analysis of the α-KG and OAA reductase activities of McyI was carried out in 1-ml reactions each containing 0.25 mm NADPH, 5 μg of purified enzyme, and various amounts of substrate in reaction buffer (pH 7). Initial velocities were measured by monitoring the disappearance of NADPH at 340 nm for 0.5 min at 37 °C. Kinetic analysis of the NADPH oxidase activity of McyI was performed in 1-ml reactions each containing 5 μg of purified enzyme, 0.8 mm OAA, and various amounts of NADPH in reaction buffer (pH 7). Initial velocities were measured as described above. Inhibition Assays−The inhibitory effects of various substances on the OA" @default.
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• W2012108328 date "2007-02-01" @default.
• W2012108328 modified "2023-10-01" @default.
• W2012108328 title "Characterization of the 2-Hydroxy-acid Dehydrogenase McyI, Encoded within the Microcystin Biosynthesis Gene Cluster of Microcystis aeruginosa PCC7806" @default.
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• W2012108328 doi "https://doi.org/10.1074/jbc.m606986200" @default.
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