Amino acid substitutions at glutamate-354 in dihydrolipoamide dehydrogenase of Escherichia coli lower the sensitivity of pyruvate dehydrogenase to NADH
| Autor: | Qingzhao Wang, Lonnie O. Ingram, Mun Su Rhee, Phi Minh Do, Zhentao Sun, Lakshmanan Govindasamy, Keelnatham T. Shanmugam |
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| Rok vydání: | 2012 |
| Předmět: |
Models
Molecular Glutamic Acid Pyruvate Dehydrogenase Complex Biology medicine.disease_cause Microbiology chemistry.chemical_compound hemic and lymphatic diseases Pyruvic Acid Escherichia coli medicine Dihydrolipoamide Dehydrogenase chemistry.chemical_classification Dihydrolipoamide dehydrogenase Ethanol Escherichia coli Proteins Glutamic acid NAD Pyruvate dehydrogenase complex Molecular biology Amino acid Amino Acid Substitution chemistry Biochemistry Fermentation Mutagenesis Site-Directed NAD+ kinase Pyruvic acid |
| Zdroj: | Microbiology. 158:1350-1358 |
| ISSN: | 1465-2080 1350-0872 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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