title: Amino acid substitutions at glutamate-354 in dihydrolipoamide dehydrogenase of Escherichia coli lower the sensitivity of pyruvate dehydrogenase to NADH.
abstract: Pyruvate dehydrogenase (PDH) of Escherichia coli 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 lpd led to a PDH complex that was functional in an anaerobic culture as seen by restoration of anaerobic growth of a pflB, ldhA double mutant of E. coli 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 ldhA, pflB double mutant. The PDH complex with an LPD alteration E354G, E354P or E354W had an approximately 20-fold increase in the apparent K(i) for NADH compared with the native complex. The apparent K(m) 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.
authors.affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611, USA.|College of Resources and Environmental Sciences, China Agricultural University, PR China.
authors.forename: Zhentao
authors.initials: Z
authors.lastname: Sun
authors.affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611, USA.
authors.forename: Phi Minh
authors.initials: PM
authors.lastname: Do
authors.affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611, USA.
authors.forename: Mun Su
authors.initials: MS
authors.lastname: Rhee
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32611, USA.
authors.forename: Lakshmanan
authors.initials: L
authors.lastname: Govindasamy
authors.affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611, USA.
authors.forename: Qingzhao
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611, USA.
authors.forename: Lonnie O
authors.initials: LO
authors.lastname: Ingram
authors.affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611, USA.
authors.forename: K T
authors.initials: KT
authors.lastname: Shanmugam
mesh_terms: D019943:Amino Acid Substitution; D008058:Dihydrolipoamide Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D000431:Ethanol / Q000378:metabolism; D005285:Fermentation; D018698:Glutamic Acid / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism*; D011768:Pyruvate Dehydrogenase Complex / Q000235:genetics / Q000378:metabolism*; D019289:Pyruvic Acid / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D011768:Pyruvate Dehydrogenase Complex; D009243:NAD; D000431:Ethanol; D018698:Glutamic Acid; D019289:Pyruvic Acid; D008058:Dihydrolipoamide Dehydrogenase