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. 2000 Dec 15;352(Pt 3):717–724.

Conversion of Escherichia coli pyruvate oxidase to an 'alpha-ketobutyrate oxidase'.

Y Y Chang 1, J E Cronan Jr 1
PMCID: PMC1221509  PMID: 11104678

Abstract

Escherichia coli pyruvate oxidase (PoxB), a lipid-activated homotetrameric enzyme, is active on both pyruvate and 2-oxobutanoate ('alpha-ketobutyrate'), although pyruvate is the favoured substrate. By localized random mutagenesis of residues chosen on the basis of a modelled active site, we obtained several PoxB enzymes that had a markedly decreased activity with the natural substrate, pyruvate, but retained full activity with 2-oxobutanoate. In each of these mutant proteins Val-380 had been replaced with a smaller residue, namely alanine, glycine or serine. One of these, PoxB V380A/L253F, was shown to lack detectable pyruvate oxidase activity in vivo; this protein was purified, studied and found to have a 6-fold increase in K(m) for pyruvate and a 10-fold lower V(max) with this substrate. In contrast, the mutant had essentially normal kinetic constants with 2-oxobutanoate. The altered substrate specificity was reflected in a decreased rate of pyruvate binding to the latent conformer of the mutant protein owing to the V380A mutation. The L253F mutation alone had no effect on PoxB activity, although it increased the activity of proteins carrying substitutions at residue 380, as it did that of the wild-type protein. The properties of the V380A/L253F protein provide new insights into the mode of substrate binding and the unusual activation properties of this enzyme.

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Selected References

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  1. Bertagnolli B. L., Hager L. P. Activation of Escherichia coli pyruvate oxidase enhances the oxidation of hydroxyethylthiamin pyrophosphate. J Biol Chem. 1991 Jun 5;266(16):10168–10173. [PubMed] [Google Scholar]
  2. Bertagnolli B. L., Hager L. P. Minimum requirements for protease activation of flavin pyruvate oxidase. Biochemistry. 1991 Aug 20;30(33):8131–8137. doi: 10.1021/bi00247a006. [DOI] [PubMed] [Google Scholar]
  3. Blake R., Hager L. P. Activation of pyruvate oxidase by monomeric and micellar amphiphiles. J Biol Chem. 1978 Mar 25;253(6):1963–1971. [PubMed] [Google Scholar]
  4. Carter K., Gennis R. B. Reconstitution of the Ubiquinone-dependent pyruvate oxidase system of Escherichia coli with the cytochrome o terminal oxidase complex. J Biol Chem. 1985 Sep 15;260(20):10986–10990. [PubMed] [Google Scholar]
  5. Chang Y. Y., Cronan J. E. An Escherichia coli mutant deficient in pyruvate oxidase activity due to altered phospholipid activation of the enzyme. Proc Natl Acad Sci U S A. 1984 Jul;81(14):4348–4352. doi: 10.1073/pnas.81.14.4348. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chang Y. Y., Cronan J. E., Jr Common ancestry of Escherichia coli pyruvate oxidase and the acetohydroxy acid synthases of the branched-chain amino acid biosynthetic pathway. J Bacteriol. 1988 Sep;170(9):3937–3945. doi: 10.1128/jb.170.9.3937-3945.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chang Y. Y., Cronan J. E., Jr Detection by site-specific disulfide cross-linking of a conformational change in binding of Escherichia coli pyruvate oxidase to lipid bilayers. J Biol Chem. 1995 Apr 7;270(14):7896–7901. doi: 10.1074/jbc.270.14.7896. [DOI] [PubMed] [Google Scholar]
  8. Chang Y. Y., Cronan J. E., Jr Mapping nonselectable genes of Escherichia coli by using transposon Tn10: location of a gene affecting pyruvate oxidase. J Bacteriol. 1982 Sep;151(3):1279–1289. doi: 10.1128/jb.151.3.1279-1289.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chang Y. Y., Cronan J. E., Jr Molecular cloning, DNA sequencing, and enzymatic analyses of two Escherichia coli pyruvate oxidase mutants defective in activation by lipids. J Bacteriol. 1986 Jul;167(1):312–318. doi: 10.1128/jb.167.1.312-318.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chang Y. Y., Cronan J. E., Jr Sulfhydryl chemistry detects three conformations of the lipid binding region of Escherichia coli pyruvate oxidase. Biochemistry. 1997 Sep 30;36(39):11564–11573. doi: 10.1021/bi9709102. [DOI] [PubMed] [Google Scholar]
  11. Chang Y. Y., Wang A. Y., Cronan J. E., Jr Expression of Escherichia coli pyruvate oxidase (PoxB) depends on the sigma factor encoded by the rpoS(katF) gene. Mol Microbiol. 1994 Mar;11(6):1019–1028. doi: 10.1111/j.1365-2958.1994.tb00380.x. [DOI] [PubMed] [Google Scholar]
  12. Chang Y. Y., Wang A. Y., Cronan J. E., Jr Molecular cloning, DNA sequencing, and biochemical analyses of Escherichia coli glyoxylate carboligase. An enzyme of the acetohydroxy acid synthase-pyruvate oxidase family. J Biol Chem. 1993 Feb 25;268(6):3911–3919. [PubMed] [Google Scholar]
  13. Chipman D., Barak Z., Schloss J. V. Biosynthesis of 2-aceto-2-hydroxy acids: acetolactate synthases and acetohydroxyacid synthases. Biochim Biophys Acta. 1998 Jun 29;1385(2):401–419. doi: 10.1016/s0167-4838(98)00083-1. [DOI] [PubMed] [Google Scholar]
  14. Dailey F. E., Cronan J. E., Jr Acetohydroxy acid synthase I, a required enzyme for isoleucine and valine biosynthesis in Escherichia coli K-12 during growth on acetate as the sole carbon source. J Bacteriol. 1986 Feb;165(2):453–460. doi: 10.1128/jb.165.2.453-460.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dailey F. E., Cronan J. E., Jr, Maloy S. R. Acetohydroxy acid synthase I is required for isoleucine and valine biosynthesis by Salmonella typhimurium LT2 during growth on acetate or long-chain fatty acids. J Bacteriol. 1987 Feb;169(2):917–919. doi: 10.1128/jb.169.2.917-919.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Dietrich J., Henning U. Regulation of pyruvate dehydrogenase complex synthesis in Escherichia coli K 12. Identification of the inducing metabolite. Eur J Biochem. 1970 Jun;14(2):258–269. doi: 10.1111/j.1432-1033.1970.tb00285.x. [DOI] [PubMed] [Google Scholar]
  17. Gollop N., Damri B., Chipman D. M., Barak Z. Physiological implications of the substrate specificities of acetohydroxy acid synthases from varied organisms. J Bacteriol. 1990 Jun;172(6):3444–3449. doi: 10.1128/jb.172.6.3444-3449.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Grabau C., Cronan J. E., Jr In vivo function of Escherichia coli pyruvate oxidase specifically requires a functional lipid binding site. Biochemistry. 1986 Jul 1;25(13):3748–3751. doi: 10.1021/bi00361a003. [DOI] [PubMed] [Google Scholar]
  19. Grabau C., Cronan J. E., Jr Molecular cloning of the gene (poxB) encoding the pyruvate oxidase of Escherichia coli, a lipid-activated enzyme. J Bacteriol. 1984 Dec;160(3):1088–1092. doi: 10.1128/jb.160.3.1088-1092.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Grabau C., Cronan J. E., Jr Nucleotide sequence and deduced amino acid sequence of Escherichia coli pyruvate oxidase, a lipid-activated flavoprotein. Nucleic Acids Res. 1986 Jul 11;14(13):5449–5460. doi: 10.1093/nar/14.13.5449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ibdah M., Bar-Ilan A., Livnah O., Schloss J. V., Barak Z., Chipman D. M. Homology modeling of the structure of bacterial acetohydroxy acid synthase and examination of the active site by site-directed mutagenesis. Biochemistry. 1996 Dec 17;35(50):16282–16291. doi: 10.1021/bi961588i. [DOI] [PubMed] [Google Scholar]
  22. Koland J. G., Miller M. J., Gennis R. B. Reconstitution of the membrane-bound, ubiquinone-dependent pyruvate oxidase respiratory chain of Escherichia coli with the cytochrome d terminal oxidase. Biochemistry. 1984 Jan 31;23(3):445–453. doi: 10.1021/bi00298a008. [DOI] [PubMed] [Google Scholar]
  23. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  24. LaRossa R. A., Van Dyk T. K. Metabolic mayhem caused by 2-ketoacid imbalances. Bioessays. 1987 Sep;7(3):125–130. doi: 10.1002/bies.950070308. [DOI] [PubMed] [Google Scholar]
  25. LaRossa R. A., Van Dyk T. K., Smulski D. R. Toxic accumulation of alpha-ketobutyrate caused by inhibition of the branched-chain amino acid biosynthetic enzyme acetolactate synthase in Salmonella typhimurium. J Bacteriol. 1987 Apr;169(4):1372–1378. doi: 10.1128/jb.169.4.1372-1378.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lang V. J., Leystra-Lantz C., Cook R. A. Characterization of the specific pyruvate transport system in Escherichia coli K-12. J Bacteriol. 1987 Jan;169(1):380–385. doi: 10.1128/jb.169.1.380-385.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Maloy S. R., Nunn W. D. Selection for loss of tetracycline resistance by Escherichia coli. J Bacteriol. 1981 Feb;145(2):1110–1111. doi: 10.1128/jb.145.2.1110-1111.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mather M., Schopfer L. M., Massey V., Gennis R. B. Studies of the flavin adenine dinucleotide binding region in Escherichia coli pyruvate oxidase. J Biol Chem. 1982 Nov 10;257(21):12887–12892. [PubMed] [Google Scholar]
  29. Muller Y. A., Schulz G. E. Structure of the thiamine- and flavin-dependent enzyme pyruvate oxidase. Science. 1993 Feb 12;259(5097):965–967. doi: 10.1126/science.8438155. [DOI] [PubMed] [Google Scholar]
  30. Muller Y. A., Schumacher G., Rudolph R., Schulz G. E. The refined structures of a stabilized mutant and of wild-type pyruvate oxidase from Lactobacillus plantarum. J Mol Biol. 1994 Apr 1;237(3):315–335. doi: 10.1006/jmbi.1994.1233. [DOI] [PubMed] [Google Scholar]
  31. Ott K. H., Kwagh J. G., Stockton G. W., Sidorov V., Kakefuda G. Rational molecular design and genetic engineering of herbicide resistant crops by structure modeling and site-directed mutagenesis of acetohydroxyacid synthase. J Mol Biol. 1996 Oct 25;263(2):359–368. doi: 10.1006/jmbi.1996.0580. [DOI] [PubMed] [Google Scholar]
  32. Recny M. A., Hager L. P. Reconstitution of native Escherichia coli pyruvate oxidase from apoenzyme monomers and FAD. J Biol Chem. 1982 Nov 10;257(21):12878–12886. [PubMed] [Google Scholar]
  33. Sedewitz B., Schleifer K. H., Götz F. Purification and biochemical characterization of pyruvate oxidase from Lactobacillus plantarum. J Bacteriol. 1984 Oct;160(1):273–278. doi: 10.1128/jb.160.1.273-278.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wang A. Y., Chang Y. Y., Cronan J. E., Jr Role of the tetrameric structure of Escherichia coli pyruvate oxidase in enzyme activation and lipid binding. J Biol Chem. 1991 Jun 15;266(17):10959–10966. [PubMed] [Google Scholar]

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