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. 1986 Jul;167(1):312–318. doi: 10.1128/jb.167.1.312-318.1986

Molecular cloning, DNA sequencing, and enzymatic analyses of two Escherichia coli pyruvate oxidase mutants defective in activation by lipids.

Y Y Chang, J E Cronan Jr
PMCID: PMC212877  PMID: 3522547

Abstract

Two Escherichia coli pyruvate oxidase (EC 1.2.2.2) mutant genes, poxB3 and poxB4, were cloned on plasmid pBR322. The poxB3 mutant oxidase which was described previously (Y. Y. Chang and J. E. Cronan, Jr., Proc. Natl. Acad. Sci. USA 81:4348-4352, 1984) was deficient in lipid activation but retained full catalytic activity. The poxB3 mutation was located in the C-terminal half of the gene, and the nucleotide alteration has been determined by DNA sequencing of this part of the gene and by comparing the sequence with that of the wild-type strain (C. Grabau and J. E. Cronan, Jr., submitted for publication). The poxB3 oxidase mutation is the substitution of a serine residue for Pro-536. poxB4, another pyruvate oxidase mutant gene, was also deficient in lipid activation. The major difference between the poxB3 and poxB4 oxidase was in the binding of Triton detergents. The poxB4 mutation was also located in the C-terminal half of the gene, and sequence analysis has shown that only one nucleotide base was altered, which resulted in Ala-467 being converted to a threonine residue. The results of the amino acid substitutions in the mutant proteins, leading to the functional alteration of the enzyme, are discussed.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. 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]
  2. Bordier C. Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem. 1981 Feb 25;256(4):1604–1607. [PubMed] [Google Scholar]
  3. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Chang Y. Y., Cronan J. E., Jr Genetic and biochemical analyses of Escherichia coli strains having a mutation in the structural gene (poxB) for pyruvate oxidase. J Bacteriol. 1983 May;154(2):756–762. doi: 10.1128/jb.154.2.756-762.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Chang Y. Y., Cronan J. E., Jr Selection for the transfer of phenotypically nonselectable chromosomal mutations to recombinant plasmids through introduction of an altered restriction site. Gene. 1985;40(2-3):353–357. doi: 10.1016/0378-1119(85)90061-7. [DOI] [PubMed] [Google Scholar]
  8. Chattoraj D. K., Cordes K., Berman M. L., Das A. Mutagenesis and mutation transfer induced by ultraviolet light in plasmid-cloned DNA. Gene. 1984 Feb;27(2):213–222. doi: 10.1016/0378-1119(84)90142-2. [DOI] [PubMed] [Google Scholar]
  9. Chothia C. Principles that determine the structure of proteins. Annu Rev Biochem. 1984;53:537–572. doi: 10.1146/annurev.bi.53.070184.002541. [DOI] [PubMed] [Google Scholar]
  10. Chou P. Y., Fasman G. D. Empirical predictions of protein conformation. Annu Rev Biochem. 1978;47:251–276. doi: 10.1146/annurev.bi.47.070178.001343. [DOI] [PubMed] [Google Scholar]
  11. Cohen S. N., Chang A. C., Hsu L. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A. 1972 Aug;69(8):2110–2114. doi: 10.1073/pnas.69.8.2110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cunningham C. C., Hager L. P. Crystalline pyruvate oxidase from Escherichia coli. 3. Phospholipid as an allosteric effector for the enzyme. J Biol Chem. 1971 Mar 25;246(6):1583–1589. [PubMed] [Google Scholar]
  13. Dretzen G., Bellard M., Sassone-Corsi P., Chambon P. A reliable method for the recovery of DNA fragments from agarose and acrylamide gels. Anal Biochem. 1981 Apr;112(2):295–298. doi: 10.1016/0003-2697(81)90296-7. [DOI] [PubMed] [Google Scholar]
  14. Eisenberg D. Three-dimensional structure of membrane and surface proteins. Annu Rev Biochem. 1984;53:595–623. doi: 10.1146/annurev.bi.53.070184.003115. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Levitt M. Effect of proline residues on protein folding. J Mol Biol. 1981 Jan 5;145(1):251–263. doi: 10.1016/0022-2836(81)90342-9. [DOI] [PubMed] [Google Scholar]
  18. Lo K. M., Jones S. S., Hackett N. R., Khorana H. G. Specific amino acid substitutions in bacterioopsin: Replacement of a restriction fragment in the structural gene by synthetic DNA fragments containing altered codons. Proc Natl Acad Sci U S A. 1984 Apr;81(8):2285–2289. doi: 10.1073/pnas.81.8.2285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Maquat L. E., Reznikoff W. S. In vitro analysis of the Escherichia coli RNA polymerase interaction with wild-type and mutant lactose promoters. J Mol Biol. 1978 Nov 15;125(4):467–490. doi: 10.1016/0022-2836(78)90311-x. [DOI] [PubMed] [Google Scholar]
  20. Mather M., Blake R., Koland J., Schrock H., Russell P., O'Brien T., Hager L. P., Gennis R. B., O'Leary M. Escherichia coli pyruvate oxidase: interaction of a peripheral membrane protein with lipids. Biophys J. 1982 Jan;37(1):87–88. doi: 10.1016/S0006-3495(82)84613-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  22. O'Brien T. A., Blake R., 2nd, Gennis R. B. Regulation by lipids of cofactor binding to a peripheral membrane enzyme: binding of thiamin pyrophosphate to pyruvate oxidase. Biochemistry. 1977 Jul 12;16(14):3105–3109. doi: 10.1021/bi00633a010. [DOI] [PubMed] [Google Scholar]
  23. Recny M. A., Hager L. P. Isolation and characterization of the protease-activated form of pyruvate oxidase. Evidence for a conformational change in the environment of the flavin prosthetic group. J Biol Chem. 1983 Apr 25;258(8):5189–5195. [PubMed] [Google Scholar]
  24. Russell P., Hager L. P., Gennis R. B. Characterization of the proteolytic activation of pyruvate oxidase. Control by specific ligands and by the flavin oxidation-reduction state. J Biol Chem. 1977 Nov 10;252(21):7877–7882. [PubMed] [Google Scholar]
  25. Russell P., Schrock H. L., Gennis R. B. Lipid activation and protease activation of pyruvate oxidase. Evidence suggesting a common site of interaction on the protein. J Biol Chem. 1977 Nov 10;252(21):7883–7887. [PubMed] [Google Scholar]
  26. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. DNA. 1984 Dec;3(6):479–488. doi: 10.1089/dna.1.1984.3.479. [DOI] [PubMed] [Google Scholar]

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