Cloning, expression and characterization of thermostable YdaP from Bacillus licheniformis 9A

• Authors: Joseph Wani Lako , Jada Yengkopiong , William Stafford , Marla Tuffin , Don Cowan
• Languages of publication: EN

Abstracts

EN

The Bacillus licheniformis ydaP gene encodes for a pyruvate oxidase that catalyses the oxidative decarboxylation of pyruvate to acetate and CO2. The YdaP form of this enzyme was purified about 48.6-folds to homogeneity in three steps. The enzyme was recovered in a soluble form and demonstrated significant activity on pyruvate using 2, 6-dichlorophenolindophenol (DCPIP) as an artificial electron acceptor. HPLC analysis of the YdaP-enzyme catalysed conversion of pyruvate showed acetate as the sole product, confirming the putative identity of pyruvate oxidase. Analysis of the substrate specificity showed that the YdaP enzyme demonstrated preference for short chain oxo acids; however, it was activated by 1% Triton X-100. The YdaP substrate-binding pocket from the YdaP protein differed substantially from the equivalent site in all of the so far characterized pyruvate oxidases, suggesting that the B. licheniformis YdaP might accept different substrates. This could allow more accessibility of large substrates into the active site of this enzyme. The thermostability and pH activity of the YdaP enzyme were determined, with optimums at 50ºC and pH 5.8, respectively. The amino acid residues forming the catalytic cavity were identified as Gln460 to Ala480.

Keywords

EN

Bacillus licheniformis; pyruvate oxidase; YdaP; thiamine diphosphate enzyme

• Publisher: Polish Biochemical Society
• Journal: Acta Biochimica Polonica
• Year: 2018
• Volume: 65
• Issue: 1
• Pages: 59-66

Dates

published

2018

received

2017-01-06

revised

2017-04-09

accepted

2017-04-11

(unknown)

2018-03-15

Contributors

author

Joseph Wani Lako

• John Garang Memorial University of Science and Technology, College of Science and Technology, Bor, Jonglei State, Republic of South Sudan

author

Jada Yengkopiong

• John Garang Memorial University of Science and Technology, College of Science and Technology, Bor, Jonglei State, Republic of South Sudan

author

William Stafford

• CSIR Bioscience, Stellenbosch, Cape Town, South Africa

author

Marla Tuffin

• Institute for Microbial Biotechnology and Metagenomics (IMBM), Department of Biotechnology, University of the Western Cape, Bellville 7535, Cape Town, South Africa

author

Don Cowan

• Center of Genomics, University of Pretoria, Pretoria, South Africa

References

• Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 3389-3402. doi: 10.1093/nar/25.17.3389.
• Bertagnolli BL, Hager LP (1991) Activation of Escherichia coli pyruvate oxidase enhances the oxidation of hydroxyethylthiamin pyrophosphate. J Biol Chem 266: 10168-10173.
• Blake R, O'Brien TA, Gennis RB, Hager LP (1982) Role of the divalent metal cation in the pyruvate oxidase reaction. J Biol Chem 257: 9605-9611.
• Blake R, Hager LP (1978) Activation of pyruvate oxidase by monomeric and micellar amphiphiles. J Biol Chem 253: 1963-1971.
• Chang YY, Cronan JE, Jr (1986) Molecular cloning, DNA sequencing, and enzymatic analyses of two Escherichia coli pyruvate oxidase mutants defective in activation by lipids. J Bacteriol 167: 312-318.
• Chang YY, Cronan JE, Jr (1988) Common ancestry of Escherichia coli pyruvate oxidase and the acetohydroxy acid synthases of the branched-chain amino acid biosynthetic pathway. J Bacteriol 170: 3937-3945.
• Chang YY, Cronan JE, Jr (1995) Detection by site-specific disulfide cross-linking of a conformational change in binding of Escherschia coli pyruvate oxidase to lipid bilayers. J Biol Chem 270: 7896-7901.
• Claus SC, Berkeley RCW (1986) Genus Bacillus cohn 1872. In Sneath PHA et al., eds. pp 1105-1139. Baltimore, MD: Williams and Wilkins Co
• Cunningham CC, Hager LP (1971) Crystalline pyruvate oxidase from Escherichia coli phospholipids as an allosteric effector for the enzyme. J Biol Chem 246: 1583-1589.
• Duggleby RG (2006) Domain relationships in thiamine diphosphate-dependent enzymes. Acc Chem Res 39: 550-557. doi: 10.1021/ar068022z.
• Diallo MD, Martens M, Vloemans N, Cousin A, Vandekerckhov TT, Neyra M, de Lajudie P, Willems A, Gillis M, Vyverman W, Van der Gucht K (2004) Phylogenetic analysis of partial bacterial 16S rDNA sequence of tropical grass pasture soil under Acacia tortilis subsp raddiana in Senegal. Syst Appl Microbiol 27: 238-252. doi: 10.1078/072320204322881862.
• Erickson RJ (1976) Industrial application of the Bacilli: A review and prospectus. In Schlensiger D ed, pp 406-419. Microbiology. Washington, DC: Amer Soc Microbiol
• Eveleigh DE (1981) The microbial production of industrial chemicals. Scientific American 245: 155-178
• Goffin P, Muscariello L, Lorquet F, Stukkens A, Prozzi D, Sacco M, Kleerebezem M, Hols P (2006) Involvement of pyruvate oxidase activity and acetate production in the survival of Lactobacillus plantarum during the stationary phase of aerobic growth. Appl Environ Microbiol 72: 7933-7940. doi: 10.1128/AEM.00659-06.
• Grabski A, Mehler M, Drott D (2005) The overnight expression autoinduction system: High-density cell growth and protein expression while you sleep. Nature Meths 2: 233-235. doi: 10.1038/nmeth0305-233
• Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/NT. Nucleic Acids Symp Ser 95-98
• Hamilton SE (1986) Identification of the high-affinity lipid binding site in Escherichia coli pyruvate oxidase. Biochemistry 25: 8178-8183. PMID: 3545288.
• Hawkins CF, Borges A, Perham RN (1989) A common structural motif in thiamin pyrophosphate-binding enzymes. FEBS Lett 255: 77-82.
• Juan EC, Hoque MM, Hossain MT, Yamamoto T, Imamura S, Suzuki K, Sekiguchi T, Takénaka A (2007) The structures of pyruvate oxidase from Aerococcus viridans with cofactors and with a reaction intermediate reveal the flexibility of the active-site tunnel for catalysis. Acta Crystallogr Sect F Struct Biol Cryst Commun 63: 900-907. doi: 10.1107/s1744309107041012.
• Lorquet F, Goffin P, Muscariello L, Baudry JB, Ladero V, Sacco M, Kleerebezem M, Hols P (2004) Characterization and functional analysis of the poxB gene, which encodes pyruvate oxidase in Lactobacillus plantarum. J Bacteriol 186: 3749-3759. doi: 10.1128/JB.186.12.3749-3759.2004.
• Lovell SC, Davis IW, Arendall WB 3rd, de Bakker PI, Word JM, Prisant MG, Richardson JS, Richardson DC (2001) Structure validation by Calpha geometry phi, psi and C beta deviation. Proteins 50: 437-450.
• Mather M, Schopfer LM, Massey V, Gennis RB (1982) Studies of the flavin adenine dinucleotide binding region in Escherichia coli pyruvate oxidase. J Biol Chem 257: 12887-12892.
• Miller DN, Bryant JE, Madsen EL, Ghiorse WC (1999) Evaluation and optimization of DNA extraction and purification procedures for soil sediment samples. Appl Environ Microbiol 65: 4715-4724. PMID: 10543776. PMCID: PMC 91634.
• Muller YA, Schulz GE (1993) Structure of the thiamine- and flavin-dependent enzyme pyruvate oxidase. Science 259: 965-967.
• Muller YA, Schumacher G, Rudolph R, Schulz GE (1994) The refined structures of a stabilized mutant and of wild-type pyruvate oxidase from Lactobacillus plantarum. J Mol Biol 237: 315-335.
• Neale AD, Scopes RK, Wettenhall RE, Hoogenraad NJ (1987) Pyruvate decarboxylase of Zymomonas mobilis: isolation, properties and genetic expression in Escherichia coli. J Bacteriol 169: 1024-1028. PMCID: PMC211896.
• Neumann P, Weidner A, Pech A, Stubbs MT, Tittmann K (2008) Structural basis for membrane binding and catalytic activation of the peripheral membrane enzyme pyruvate oxidase from Escherichia coli. Proc Nat Acad Sci 105: 17390-17395. doi: 10.1073/pnas.0805027105.
• O'Brien TA, Blake R, Gennis RB (1977) Regulation by lipids of cofactor binding to a peripheral membrane enzyme: Binding of thiamine pyrophosphate to pyruvate oxidase. Biochemistry 16: 3105-3109.
• Okuyama T, Takata M, Takahashi K (1989) High-performance liquid chromatographic analysis of naturally occurring glycosides and saponins. J Chromatogr 466: 390-398. doi: 10.1016/s0021-9673(01)84636-7
• Patton TG, Rice KC, Foster MK, Bayles KW (2005) The Staphylococcus aureus cidC gene encodes a pyruvate oxidase that affects acetate metabolism and cell death in stationary phase. Mol Microbiol 56: 1664-1674. doi: 10.1111/j.1365-2958.2005.04653.x.
• Priest FG, Goodfellow M, Shute LA, Berkeley RCW (1987) Bacillus amyloliquefaciens sp. nov., no. rev. Int J Syst Bacteriol 37: 69-71. doi: 10.1099/00207713-37-1-69
• Priest FG, Goodfellow M, Todd C (1988) A numerical classification of the genus Bacillus. J Gen Microbiol 134: 1847-1882. doi: 10.1099/00221287-134-7-1847.
• Radnedge L, Agron PG, Hill KH, Jackson PJ, Ticknor LO, Keim P, Andersen GL (2003) Genome differences that distinguish Bacillus anthracis from Bacillus cereus and Bacillus thuringiensis. Appl Environ Microbiol 69: 2755-2764. doi: 10.1128/AEM.69.5.2755-2764.2003.
• Rey MW, Ramaiya P, Nelson BA, Brody-Karpin SD, Zaretsky EJ, Tang M, Lopez de Leon A, Xiang H, Gusti V, Clausen IG, Olsen PB, Rasmussen MD, Andersen JT, Jørgensen PL, Larsen TS, Sorokin A, Bolotin A, Lapidus A, Galleron N, Ehrlich SD, Berka RM (2004) Complete genome sequence of the industrial bacterium Bacillus licheniformis and comparison with closely related Bacillus species. Genome Biol 5: R77. doi: 10.1186/gb-2004-5-10-r77.
• Šali A (1995) Modeling mutations and homologous proteins. Curr Opin Biotech 6: 437-451. doi: 10.1016/0958-1669(95)80074-3.
• San KY, Bennet GN, Aristidou AA, Chou CH (1994) Strategies in high-level expression of recombinant protein in Escherichia coli. Ann N Y Acad Sci 721: 257-267. doi: 10.1111/j.1749-6632.1994.tb47399.x.
• Schreiner ME, Eikmanns BJ (2005) Pyruvate:quinone oxidoreductase from Corynebacterium glutamicum: purification and biochemical characterization. J Bacteriol 187: 862-871. doi: 10.1128/JB.187.3.862-871.2005.
• Sedewitz B, Schleifer KH, Gotz F (1984) Purification and biochemical characterization of pyruvate oxidase from Lactobacillus plantarum. J Bacteriol 160: 273-278. PMCID: PMC214712.
• Shi J, Blundell TL, Mizuguchi K (2001) FUGUE: sequence-structure homology recognition using environment-specific substitution tables and structure-dependent gap penalties. J Mol Biol 310: 243-257. doi: 10.1006/jmbi.2001.4762.
• Steele HL, Jaeger KE, Daniel R, Streit WR (2008) Advances in recovery of novel biocatalysts from metagenomes. J Mol Microbiol Biotechnol 16: 25-37. doi: 10.1159/000142892.
• Tomar A, Eiteman MA, Altman E (2003) The effect of acetate pathway mutations on the production of pyruvate in Escherichia coli. Appl Microbiol Biotechnol 62: 76-82. doi: 10.1007/s00253-003-1234-6.
• Veith B, Herzberg C, Steckel S, Feesche J, Maurer KH, Ehrenreich P, Bäumer S, Henne A, Liesegang H, Merkl R, Ehrenreich A, Gottschalk G (2004) The complete genome sequence of Bacillus licheniformis DSM13, an organism with great industrial potential. J Mol Microbiol Biotechnol 7: 204-211. doi: 10.1159/000079829.
• Wang A. Y, Chang YY, Cronan, JE (1991) Role of the tetrameric structure of Escherichia coli pyruvate oxidase in enzyme activation and lipid binding. J Biol Chem 266: 10959-10966.
• Wille G, Meyer D, Steinmetz A, Hinze E., Golbik R, Tittmann K (2006) The catalytic cycle of a thiamin diphosphate enzyme examined by cryocrystallography. Nat Chem Biol 2: 324-328. doi: 10.1038/nchembio.788.
• Yilmaztekin M, Erten H, Cabaroglu T (2008) Production of isoamyl acetate from sugar beet molasses by Williopsis saturnus var. saturnus. J Inst Brewing 114: 34-38. doi: 10.1016/j.foodchem.2008.05.079
• Yoneda N, Kusano S, Yasui M, Pujado P, Wilcher S (2001) Recent advances in processes and catalysts for the production of acetic acid. Appl Catalysis 221: 253-265. doi: 10.1016/s0926-860x(01)00800-6
• Zhang TF, Hager LP (1987) A single-step large-scale purification of pyruvate oxidase. Arch Biochem Biophys 257: 485-487.

Identifiers

DOI

10.18388/abp.2017_1499