1887

Abstract

Pyruvate dehydrogenase (PDH) of is inhibited by NADH. This inhibition is partially reversed by mutational alteration of the dihydrolipoamide dehydrogenase (LPD) component of the PDH complex (E354K or H322Y). Such a mutation in led to a PDH complex that was functional in an anaerobic culture as seen by restoration of anaerobic growth of a , double mutant of utilizing a PDH- and alcohol dehydrogenase-dependent homoethanol fermentation pathway. The glutamate at position 354 in LPD was systematically changed to all of the other natural amino acids to evaluate the physiological consequences. These amino acid replacements did not affect the PDH-dependent aerobic growth. With the exception of E354M, all changes also restored PDH-dependent anaerobic growth of and fermentation by an double mutant. The PDH complex with an LPD alteration E354G, E354P or E354W had an approximately 20-fold increase in the apparent for NADH compared with the native complex. The apparent for pyruvate or NAD for the mutated forms of PDH was not significantly different from that of the native enzyme. A structural model of LPD suggests that the amino acid at position 354 could influence movement of NADH from its binding site to the surface. These results indicate that glutamate at position 354 plays a structural role in establishing the NADH sensitivity of LPD and the PDH complex by restricting movement of the product/substrate NADH, although this amino acid is not directly associated with NAD(H) binding.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.055590-0
2012-05-01
2019-12-13
Loading full text...

Full text loading...

/deliver/fulltext/micro/158/5/1350.html?itemId=/content/journal/micro/10.1099/mic.0.055590-0&mimeType=html&fmt=ahah

References

  1. 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. [CrossRef] [PubMed]
    [Google Scholar]
  2. Ausubel F. M. , Brent R. , Kingston R. E. , Moore D. D. , Smith J. A. , Seidman J. G. , Struhl K. . ( 1987; ). Current Protocols in Molecular Biology. Brooklyn, NY:: Greene Publishing Associates and Wiley-Interscience;.
    [Google Scholar]
  3. Benen J. , van Berkel W. , Zak Z. , Visser T. , Veeger C. , de Kok A. . ( 1991; ). Lipoamide dehydrogenase from Azotobacter vinelandii: site-directed mutagenesis of the His450-Glu455 diad. Spectral properties of wild type and mutated enzymes. . Eur J Biochem 202:, 863–872. [CrossRef] [PubMed]
    [Google Scholar]
  4. Bisswanger H. . ( 1981; ). Substrate specificity of the pyruvate dehydrogenase complex from Escherichia coli . . J Biol Chem 256:, 815–822.[PubMed]
    [Google Scholar]
  5. Bisswanger H. , Henning U. . ( 1971; ). Regulatory properties of the pyruvate-dehydrogenase complex from Escherichia coli. Positive and negative cooperativity. . Eur J Biochem 24:, 376–384. [CrossRef] [PubMed]
    [Google Scholar]
  6. Bock A. , Sawers G. . ( 1996; ). Fermentation. . In Escherichia coli and Salmonella: Cellular and Molecular Biology, pp. 262–282. Edited by Neidhardt, F. C. , Curtiss R. III , Ingraham J. L. , Lin E. C. C. , Low K. B , Magasanik B. , Reznikoff W. S. , Riley M. , Schaechter M. , Umbarger H. E. . . Washington, DC:: American Society for Microbiology;.
    [Google Scholar]
  7. Bradford M. M. . ( 1976; ). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. . Anal Biochem 72:, 248–254. [CrossRef] [PubMed]
    [Google Scholar]
  8. Brautigam C. A. , Chuang J. L. , Tomchick D. R. , Machius M. , Chuang D. T. . ( 2005; ). Crystal structure of human dihydrolipoamide dehydrogenase: NAD+/NADH binding and the structural basis of disease-causing mutations. . J Mol Biol 350:, 543–552. [CrossRef] [PubMed]
    [Google Scholar]
  9. Cameron J. M. , Levandovskiy V. , Mackay N. , Raiman J. , Renaud D. L. , Clarke J. T. , Feigenbaum A. , Elpeleg O. , Robinson B. H. . ( 2006; ). Novel mutations in dihydrolipoamide dehydrogenase deficiency in two cousins with borderline-normal PDH complex activity. . Am J Med Genet A 140:, 1542–1552.[PubMed] [CrossRef]
    [Google Scholar]
  10. Cassey B. , Guest J. R. , Attwood M. M. . ( 1998; ). Environmental control of pyruvate dehydrogenase complex expression in Escherichia coli . . FEMS Microbiol Lett 159:, 325–329. [CrossRef] [PubMed]
    [Google Scholar]
  11. Clark D. P. . ( 1989; ). The fermentation pathways of Escherichia coli . . FEMS Microbiol Rev 5:, 223–234. [CrossRef] [PubMed]
    [Google Scholar]
  12. Datsenko K. A. , Wanner B. L. . ( 2000; ). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. . Proc Natl Acad Sci U S A 97:, 6640–6645. [CrossRef] [PubMed]
    [Google Scholar]
  13. Davis R. W. , Botstein D. , Roth J. R. . ( 1980; ). Advanced Bacterial Genetics. Cold Spring Harbor, NY:: Cold Spring Harbor Laboratory;.
    [Google Scholar]
  14. de Graef M. R. , Alexeeva S. , Snoep J. L. , Teixeira de Mattos M. J. . ( 1999; ). The steady-state internal redox state (NADH/NAD) reflects the external redox state and is correlated with catabolic adaptation in Escherichia coli . . J Bacteriol 181:, 2351–2357.[PubMed]
    [Google Scholar]
  15. Emsley P. , Lohkamp B. , Scott W. G. , Cowtan K. . ( 2010; ). Features and development of Coot. . Acta Crystallogr D Biol Crystallogr 66:, 486–501. [CrossRef] [PubMed]
    [Google Scholar]
  16. Guest J. R. . ( 1995; ). The Leeuwenhoek Lecture, 1995. Adaptation to life without oxygen. . Philos Trans R Soc Lond B Biol Sci 350:, 189–202. [CrossRef] [PubMed]
    [Google Scholar]
  17. Hansen H. G. , Henning U. . ( 1966; ). Regulation of pyruvate dehydrogenase activity in Escherichia coli K12. . Biochim Biophys Acta 122:, 355–358.[PubMed] [CrossRef]
    [Google Scholar]
  18. Hopkins N. , Williams C. H. Jr . ( 1995; ). Lipoamide dehydrogenase from Escherichia coli lacking the redox active disulfide: C44S and C49S. Redox properties of the FAD and interactions with pyridine nucleotides. . Biochemistry 34:, 11766–11776. [CrossRef] [PubMed]
    [Google Scholar]
  19. Kaiser M. , Sawers G. . ( 1994; ). Pyruvate formate-lyase is not essential for nitrate respiration by Escherichia coli . . FEMS Microbiol Lett 117:, 163–168. [CrossRef] [PubMed]
    [Google Scholar]
  20. Kim Y. , Ingram L. O. , Shanmugam K. T. . ( 2007; ). Construction of an Escherichia coli K-12 mutant for homoethanologenic fermentation of glucose or xylose without foreign genes. . Appl Environ Microbiol 73:, 1766–1771. [CrossRef] [PubMed]
    [Google Scholar]
  21. Kim Y. , Ingram L. O. , Shanmugam K. T. . ( 2008; ). Dihydrolipoamide dehydrogenase mutation alters the NADH sensitivity of pyruvate dehydrogenase complex of Escherichia coli K-12. . J Bacteriol 190:, 3851–3858. [CrossRef] [PubMed]
    [Google Scholar]
  22. Laemmli U. K. . ( 1970; ). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. . Nature 227:, 680–685. [CrossRef] [PubMed]
    [Google Scholar]
  23. Laskowski R. A. , MacArthur M. W. , Moss D. S. , Thornton J. M. . ( 1993; ). procheck: a program to check the stereochemical quality of protein structures. . J Appl Cryst 26:, 283–291. [CrossRef]
    [Google Scholar]
  24. Lee J. H. , Patel P. , Sankar P. , Shanmugam K. T. . ( 1985; ). Isolation and characterization of mutant strains of Escherichia coli altered in H2 metabolism. . J Bacteriol 162:, 344–352.[PubMed]
    [Google Scholar]
  25. Li de la Sierra I. , Pernot L. , Prangé T. , Saludjian P. , Schiltz M. , Fourme R. , Padrón G. . ( 1997; ). Molecular structure of the lipoamide dehydrogenase domain of a surface antigen from Neisseria meningitidis . . J Mol Biol 269:, 129–141. [CrossRef] [PubMed]
    [Google Scholar]
  26. Miller J. H. . ( 1972; ). Experiments in Molecular Genetics. Cold Spring Harbor, NY:: Cold Spring Harbor laboratory;.
    [Google Scholar]
  27. Murarka A. , Clomburg J. M. , Moran S. , Shanks J. V. , Gonzalez R. . ( 2010; ). Metabolic analysis of wild-type Escherichia coli and a pyruvate dehydrogenase complex (PDHC)-deficient derivative reveals the role of PDHC in the fermentative metabolism of glucose. . J Biol Chem 285:, 31548–31558. [CrossRef] [PubMed]
    [Google Scholar]
  28. Patel M. S. , Hong Y. S. . ( 1998; ). Lipoic Acid as an Antioxidant. The Role of Dihydrolipoamide Dehydrogenase. . In Methods in Molecular Biology. Totowa, NJ:: Humana Press;.
    [Google Scholar]
  29. Russell G. C. , Guest J. R. . ( 1990; ). Overexpression of restructured pyruvate dehydrogenase complexes and site-directed mutagenesis of a potential active-site histidine residue. . Biochem J 269:, 443–450.[PubMed]
    [Google Scholar]
  30. Sahlman L. , Williams C. H. Jr . ( 1989; ). Lipoamide dehydrogenase from Escherichia coli. Steady-state kinetics of the physiological reaction. . J Biol Chem 264:, 8039–8045.[PubMed]
    [Google Scholar]
  31. Sawers R. G. , Clark D. P. . ( 2004; ). Fermentative pyruvate and acetyl-Coenzyme A metabolism.. EcoSal-Escherichia coli and Salmonella: Cellular and Molecular Biology http://ecosal.org.
  32. Schmincke-Ott E. , Bisswanger H. . ( 1981; ). Dihydrolipoamide dehydrogenase component of the pyruvate dehydrogenase complex from Escherichia coli K12. Comparative characterization of the free and the complex-bound component. . Eur J Biochem 114:, 413–420. [CrossRef] [PubMed]
    [Google Scholar]
  33. Scouten W. H. , McManus I. R. . ( 1971; ). Microbial lipoamide dehydrogenase. Purification and some characteristics of the enzyme derived from selected microorganisms. . Biochim Biophys Acta 227:, 248–263.[PubMed] [CrossRef]
    [Google Scholar]
  34. Shen L. C. , Atkinson D. E. . ( 1970; ). Regulation of pyruvate dehydrogenase from Escherichia coli. Interactions of adenylate energy charge and other regulatory parameters. . J Biol Chem 245:, 5974–5978.[PubMed]
    [Google Scholar]
  35. Snoep J. L. , Teixeira de Mattos M. J. , Postma P. W. , Neijssel O. M. . ( 1990; ). Involvement of pyruvate dehydrogenase in product formation in pyruvate-limited anaerobic chemostat cultures of Enterococcus faecalis NCTC 775. . Arch Microbiol 154:, 50–55. [CrossRef] [PubMed]
    [Google Scholar]
  36. Snoep J. L. , de Graef M. R. , Westphal A. H. , de Kok A. , Teixeira de Mattos M. J. , Neijssel O. M. . ( 1993; ). Differences in sensitivity to NADH of purified pyruvate dehydrogenase complexes of Enterococcus faecalis, Lactococcus lactis, Azotobacter vinelandii and Escherichia coli: implications for their activity in vivo. . FEMS Microbiol Lett 114:, 279–283. [CrossRef] [PubMed]
    [Google Scholar]
  37. Underwood S. A. , Buszko M. L. , Shanmugam K. T. , Ingram L. O. . ( 2002; ). Flux through citrate synthase limits the growth of ethanologenic Escherichia coli KO11 during xylose fermentation. . Appl Environ Microbiol 68:, 1071–1081. [CrossRef] [PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.055590-0
Loading
/content/journal/micro/10.1099/mic.0.055590-0
Loading

Data & Media loading...

Supplements

Supplementary material 

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error