Abstract
The peptidoglycan of the hyperthermophile Thermotoga maritima contains an unusual component, d-lysine (d-Lys), in addition to the typical d-alanine (d-Ala) and d-glutamate (d-Glu). In a previous study, we identified a Lys racemase that is presumably associated with d-Lys biosynthesis. However, our understanding of d-amino acid metabolism in T. maritima and other bacteria remains limited, although d-amino acids in the peptidoglycan are crucial for preserving bacterial cell structure and resistance to environmental threats. Herein, we characterized enzymatic and structural properties of TM0356 that shares a high amino acid sequence identity with serine (Ser) racemase. The results revealed that TM0356 forms a tetramer with each subunit containing a pyridoxal 5′-phosphate as a cofactor. The enzyme did not exhibit racemase activity toward various amino acids including Ser, and dehydratase activity was highest toward l-threonine (l-Thr). It also acted on l-Ser and l-allo-Thr, but not on the corresponding d-amino acids. The catalytic mechanism did not follow typical Michaelis–Menten kinetics; it displayed a sigmoidal dependence on substrate concentration, with highest catalytic efficiency (kcat/K0.5) toward l-Thr. Interestingly, dehydratase activity was insensitive to allosteric regulators l-valine and l-isoleucine (l-Ile) at low concentrations, while these l-amino acids are inhibitors at high concentrations. Thus, TM0356 is a biosynthetic Thr dehydratase responsible for the conversion of l-Thr to α-ketobutyrate and ammonia, which is presumably involved in the first step of the biosynthesis of l-Ile.
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Abbreviations
- Dpm:
-
Diaminopimelate
- GlcNAc:
-
N-Acetylglucosamine
- MurNAc:
-
N-Acetylmuramic acid
- PLP:
-
Pyridoxal 5′-phosphate
- OPA:
-
o-Phthalaldehyde
- NAC:
-
N-Acetyl-l-cysteine
- Boc-l-Cys:
-
N-tert-Butoxycarbonyl-l-cysteine
- Aba:
-
Aminobutyric acid
- Hse:
-
Homoserine
- Cit:
-
Citrulline
- Orn:
-
Ornithine
- SDS:
-
Sodium dodecyl sulfate
- AOAA:
-
Aminoxyacetic acid
- CHES:
-
N-Cyclohexyl-2-aminoethanesulfonic acid
- CAPS:
-
N-Cyclohexyl-3-aminopropanesulfonic acid
- BN-PAGE:
-
Blue native polyacrylamide gel electrophoresis
- CBB:
-
Coomassie Brilliant Blue
References
Adachi M, Shimizu R, Kato S, Oikawa T (2019) The first identification and characterization of a histidine-specific amino acid racemase, histidine racemase from a lactic acid bacterium, Leuconostoc mesenteroides subsp. sake NBRC 102480. Amino Acids 51:331–343. https://doi.org/10.1007/s00726-018-2671-y
Amorim Franco TM, Blanchard JS (2017) Bacterial branched-chain amino acid biosynthesis: structures, mechanisms, and drugability. Biochemistry 56:5849–5865. https://doi.org/10.1021/acs.biochem.7b00849
Arias CA, Weisner J, Blackburn JM, Reynolds PE (2000) Serine and alanine racemase activities of VanT: a protein necessary for vancomycin resistance in Enterococcus gallinarum BM4174. Microbiology 146:1727–1734. https://doi.org/10.1099/00221287-146-7-1727
Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22:195–201
Bellais S, Arthur M, Dubost L, Hugonnet JE, Gutmann L, van Heijenoort J, Legrand R, Brouard JP, Rice L, Mainardi JL (2006) Aslfm, the d-aspartate ligase responsible for the addition of d-aspartic acid onto the peptidoglycan precursor of Enterococcus faecium. J Biol Chem 281:11586–11594. https://doi.org/10.1074/jbc.M600114200
Boniface A, Bouhss A, Mengin-Lecreulx D, Blanot D (2006) The MurE synthetase from Thermotoga maritima is endowed with an unusual d-lysine adding activity. J Biol Chem 281:15680–15686. https://doi.org/10.1074/jbc.M506311200
Boniface A, Parquet C, Arthur M, Mengin-Lecreulx D, Blanot D (2009) The elucidation of the structure of Thermotoga maritima peptidoglycan reveals two novel types of cross-link. J Biol Chem 284:21856–21862. https://doi.org/10.1074/jbc.M109.034363
Borchert AJ, Downs DM (2018) Analyses of variants of the Ser/Thr dehydratase IlvA provide insight into 2-aminoacrylate metabolism in Salmonella enterica. J Biol Chem 293:19240–19249. https://doi.org/10.1074/jbc.RA118.005626
Cava F, de Pedro MA, Lam H, Davis BM, Waldor MK (2011) Distinct pathways for modification of the bacterial cell wall by non-canonical d-amino acids. EMBO J 30:3442–3453. https://doi.org/10.1038/emboj.2011.246
Chen IC, Lin WD, Hsu SK, Thiruvengadam V, Hsu WH (2009) Isolation and characterization of a novel lysine racemase from a soil metagenomic library. Appl Environ Microbiol 75:5161–5166. https://doi.org/10.1128/AEM.00074-09
de Miranda J, Panizzutti R, Foltyn VN, Wolosker H (2002) Cofactors of serine racemase that physiologically stimulate the synthesis of the N-methyl-d-aspartate (NMDA) receptor coagonist d-serine. Proc Natl Acad Sci USA 99:14542–14547. https://doi.org/10.1073/pnas.222421299
Eisenstein E (1991) Cloning, expression, purification, and characterization of biosynthetic threonine deaminase from Escherichia coli. J Biol Chem 266:5801–5807
Eisenstein E (1995) Allosteric regulation of biosynthetic threonine deaminase from Escherichia coli: effects of isoleucine and valine on active-site ligand binding and catalysis. Arch Biochem Biophys 316:311–318. https://doi.org/10.1006/abbi.1995.1042
Espaillat A, Carrasco-López C, Bernardo-García N, Pietrosemoli N, Otero LH, Álvarez L, de Pedro MA, Pazos F, Davis BM, Waldor MK, Hermoso JA, Cava F (2014) Structural basis for the broad specificity of a new family of amino-acid racemases. Acta Crystallogr D Biol Crystallogr 70:79–90. https://doi.org/10.1107/S1399004713024838
Favrot L, Amorim Franco TM, Blanchard JS (2018) Biochemical characterization of the Mycobacterium smegmatis threonine deaminase. Biochemistry 57:6003–6012. https://doi.org/10.1021/acs.biochem.8b00871
Gallagher DT, Gilliland GL, Xiao G, Zondlo J, Fisher KE, Chinchilla D, Eisenstein E (1998) Structure and control of pyridoxal phosphate dependent allosteric threonine deaminase. Structure 6:465–475. https://doi.org/10.1016/s0969-2126(98)00048-3
Goytia M, Chamond N, Cosson A, Coatnoan N, Hermant D, Berneman A, Minoprio P (2007) Molecular and structural discrimination of proline racemase and hydroxyproline-2-epimerase from nosocomial and bacterial pathogens. PLoS ONE 2:e885. https://doi.org/10.1371/journal.pone.0000885
Grohs P, Gutmann L, Legrand R, Schoot B, Mainardi JL (2000) Vancomycin resistance is associated with serine-containing peptidoglycan in Enterococcus gallinarum. J Bacteriol 182:6228–6232. https://doi.org/10.1128/JB.182.21.6228-6232.2000
Guinand M, Ghuysen JM, Schleifer KH, Kandler O (1969) The peptidoglycan in walls of Butyribacterium rettgeri. Biochemistry 8:200–207. https://doi.org/10.1021/bi00829a029
Hernández SB, Cava F (2016) Environmental roles of microbial amino acid racemases. Environ Microbiol 18:1673–1685. https://doi.org/10.1111/1462-2920.13072
Hochbaum AI, Kolodkin-Gal I, Foulston L, Kolter R, Aizenberg J, Losick R (2011) Inhibitory effects of d-amino acids on Staphylococcus aureus biofilm development. J Bacteriol 193:5616–5622. https://doi.org/10.1128/JB.05534-11
Huber R, Langworthy TA, König H, Thomm M, Woese CR, Sleytr UB, Stetter KO (1986) Thermotoga maritima sp. nov. represents a new genus of unique extremely thermophilic eubacteria growing up to 90 °C. Arch Microbiol 144:324–333. https://doi.org/10.1007/BF00409880
Jia R, Yang D, Xu D, Gu T (2017a) Mitigation of a nitrate reducing Pseudomonas aeruginosa biofilm and anaerobic biocorrosion using ciprofloxacin enhanced by d-tyrosine. Sci Rep 7:6946. https://doi.org/10.1038/s41598-017-07312-7
Jia R, Li Y, Al-Mahamedh HH, Gu T (2017b) Enhanced biocide treatments with d-amino acid mixtures against a biofilm consortium from a water cooling tower. Front Microbiol 8:1538. https://doi.org/10.3389/fmicb.2017.01538
Kato S, Hemmi H, Yoshimura T (2012) Lysine racemase from a lactic acid bacterium, Oenococcus oeni: structural basis of substrate specificity. J Biochem 152:505–508. https://doi.org/10.1093/jb/mvs120
Kolodkin-Gal I, Romero D, Cao S, Clardy J, Kolter R, Losick R (2010) d-Amino acids trigger biofilm disassembly. Science 328:627–629. https://doi.org/10.1126/science.1188628
Kuan YC, Kao CH, Chen CH, Chen CC, Hu HY, Hsu WH (2011) Biochemical characterization of a novel lysine racemase from Proteus mirabilis BCRC10725. Process Biochem 46:1914–1920. https://doi.org/10.1016/j.procbio.2011.06.019
Kubota T, Shimamura S, Kobayashi T, Nunoura T, Deguchi S (2016) Distribution of eukaryotic serine racemases in the bacterial domain and characterization of a representative protein in Roseobacter litoralis Och 149. Microbiology 162:53–61. https://doi.org/10.1099/mic.0.000200
Lam H, Oh DC, Cava F, Takacs CN, Clardy J, de Pedro MA, Waldor MK (2009) d-Amino acids govern stationary phase cell wall remodeling in bacteria. Science 325:1552–1555. https://doi.org/10.1126/science.1178123
Leiman SA, May JM, Lebar MD, Kahne D, Kolter R, Losick R (2013) d-Amino acids indirectly inhibit biofilm formation in Bacillus subtilis by interfering with protein synthesis. J Bacteriol 195:5391–5395. https://doi.org/10.1128/JB.00975-13
Li E, Wang P, Zhang D (2016) d-Phenylalanine inhibits biofilm development of a marine microbe, Pseudoalteromonas sp. SC2014. FEMS Microbiol Lett 363:fnw198. https://doi.org/10.1093/femsle/fnw198
Luginbuhl GH, Hofler JG, Decedue CJ, Burns RO (1974) Biodegradative l-threonine deaminase of Salmonella typhimurium. J Bacteriol 120:559–561. https://doi.org/10.1128/JB.120.1.559-561.1974
Matsui D, Oikawa T, Arakawa N, Osumi S, Lausberg F, Stäbler N, Freudl R, Eggeling L (2009) A periplasmic, pyridoxal-5’-phosphate-dependent amino acid racemase in Pseudomonas taetrolens. Appl Microbiol Biotechnol 83:1045–1054. https://doi.org/10.1007/s00253-009-1942-7
Miyamoto T, Katane M, Saitoh Y, Sekine M, Homma H (2017) Identification and characterization of novel broad-spectrum amino acid racemases from Escherichia coli and Bacillus subtilis. Amino Acids 49:1885–1894. https://doi.org/10.1007/s00726-017-2486-2
Miyamoto T, Katane M, Saitoh Y, Sekine M, Homma H (2018) Cystathionine β-lyase is involved in d-amino acid metabolism. Biochem J 475:1397–1410. https://doi.org/10.1042/BCJ20180039
Miyamoto T, Katane M, Saitoh Y, Sekine M, Homma H (2019) Elucidation of the d-lysine biosynthetic pathway in the hyperthermophile Thermotoga maritima. FEBS J 286:601–614. https://doi.org/10.1111/febs.14720
Miyamoto T, Katane M, Saitoh Y, Sekine M, Homma H (2020a) Involvement of penicillin-binding proteins in the metabolism of a bacterial peptidoglycan containing a non-canonical d-amino acid. Amino Acids 52:487–497. https://doi.org/10.1007/s00726-020-02830-7
Miyamoto T, Moriya T, Homma H, Oshima T (2020b) Enzymatic properties and physiological function of glutamate racemase from Thermus thermophilus. Biochim Biophys Acta Proteins Proteom 1868:140461. https://doi.org/10.1016/j.bbapap.2020.140461
Mutaguchi Y, Ohmori T, Wakamatsu T, Doi K, Ohshima T (2013) Identification, purification, and characterization of a novel amino acid racemase, isoleucine 2-epimerase, from Lactobacillus species. J Bacteriol 195:5207–5215. https://doi.org/10.1128/JB.00709-13
Nakazawa A, Tokushige M, Hayaishi O, Ikehara M, Mizuno Y (1967) Studies on the interaction between regulatory enzymes and effectors. I. Effect of adenosine 5’-monophosphate analogues on threonine deaminase. J Biol Chem 242:3868–3872
Radkov AD, Moe LA (2013) Amino acid racemization in Pseudomonas putida KT2440. J Bacteriol 195:5016–5024. https://doi.org/10.1128/JB.00761-13
Radkov AD, Moe LA (2014) Bacterial synthesis of d-amino acids. Appl Microbiol Biotechnol 98:5363–5374. https://doi.org/10.1007/s00253-014-5726-3
Ramón-Peréz ML, Diaz-Cedillo F, Ibarra JA, Torales-Cardeña A, Rodríguez-Martínez S, Jan-Roblero J, Cancino-Diaz ME, Cancino-Diaz JC (2014) d-Amino acids inhibit biofilm formation in Staphylococcus epidermidis strains from ocular infections. J Med Microbiol 63:1369–1376. https://doi.org/10.1099/jmm.0.075796-0
Rosen E, Tsesis I, Elbahary S, Storzi N, Kolodkin-Gal I (2016) Eradication of Enterococcus faecalis biofilms on human dentin. Front Microbiol 7:2055. https://doi.org/10.3389/fmicb.2016.02055
Sanchez Z, Tani A, Kimbara K (2013) Extensive reduction of cell viability and enhanced matrix production in Pseudomonas aeruginosa PAO1 flow biofilms treated with a d-amino acid mixture. Appl Environ Microbiol 79:1396–1399. https://doi.org/10.1128/AEM.02911-12
Sharma R, Keshari D, Singh KS, Singh SK (2017) Biochemical and functional characterization of MRA_1571 of Mycobacterium tuberculosis H37Ra and effect of its down-regulation on survival in macrophages. Biochem Biophys Res Commun 487:892–897. https://doi.org/10.1016/j.bbrc.2017.04.149
Shulman A, Zalyapin E, Vyazmensky M, Yifrach O, Barak Z, Chipman DM (2008) Allosteric regulation of Bacillus subtilis threonine deaminase, a biosynthetic threonine deaminase with a single regulatory domain. Biochemistry 47:11783–11792. https://doi.org/10.1021/bi800901n
Simanshu DK, Savithri HS, Murthy MR (2006) Crystal structures of Salmonella typhimurium biodegradative threonine deaminase and its complex with CMP provide structural insights into ligand-induced oligomerization and enzyme activation. J Biol Chem 281:39630–39641. https://doi.org/10.1074/jbc.M605721200
Simanshu DK, Chittori S, Savithri HS, Murthy MR (2007) Structure and function of enzymes involved in the anaerobic degradation of l-threonine to propionate. J Biosci 56:1195–1206. https://doi.org/10.1007/s12038-007-0121-1
Stadtman TC, Elliott P (1957) Studies on the enzymic reduction of amino acids. II. Purification and properties of d-proline reductase and a proline racemase from Clostridium sticklandii. J Biol Chem 228:983–997
Umbarger HE, Brown B (1957) Threonine deamination in Escherichia coli. II. Evidence for two l-threonine deaminases. J Bacteriol 73:105–112. https://doi.org/10.1128/JB.73.1.105-112.1957
Wittig I, Braun HP, Schägger H (2006) Blue native PAGE. Nat Protoc 1:418–428. https://doi.org/10.1038/nprot.2006.62
Yamashita T, Ashiuchi M, Ohnishi K, Kato S, Nagata S, Misono H (2004) Molecular identification of monomeric aspartate racemase from Bifidobacterium bifidum. Eur J Biochem 271:4798–4803. https://doi.org/10.1111/j.1432-1033.2004.04445.x
Yamauchi T, Choi SY, Okada H, Yohda M, Kumagai H, Esaki N, Soda K (1992) Properties of aspartate racemase, a pyridoxal 5’-phosphate-independent amino acid racemase. J Biol Chem 267:18361–18364
Yorifuji T, Ogata K (1971) Arginine racemase of Pseudomonas graveolens. I. Purification, crystallization, and properties. J Biol Chem 246:5085–5092
Yu X, Li Y, Wang X (2012) Molecular evolution of threonine dehydratase in bacteria. PLoS ONE 8:e80750. https://doi.org/10.1371/journal.pone.0080750
Yu C, Li X, Zhang N, Wen D, Liu C, Li Q (2016) Inhibition of biofilm formation by d-tyrosine: effect of bacterial type and d-tyrosine concentration. Water Res 92:173–179. https://doi.org/10.1016/j.watres.2016.01.037
Zilm PS, Butnejski V, Rossi-Fedele G, Kidd SP, Edwards S, Vasilev K (2017) d-Amino acids reduce Enterococcus faecalis biofilms in vitro and in the presence of antimicrobials used for root canal treatment. PLoS ONE 12:e0170670. https://doi.org/10.1371/journal.pone.0170670
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TM designed the study, performed most of the experiments, analyzed the data, and wrote the manuscript. HH and KS-K supervised and wrote the manuscript. All authors contributed to data interpretation throughout the study.
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Miyamoto, T., Katane, M., Saitoh, Y. et al. Identification and biochemical characterization of threonine dehydratase from the hyperthermophile Thermotoga maritima. Amino Acids 53, 903–915 (2021). https://doi.org/10.1007/s00726-021-02993-x
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DOI: https://doi.org/10.1007/s00726-021-02993-x