Escherichia coli FabG 3-ketoacyl-ACP reductase proteins lacking the assigned catalytic triad residues are active enzymes
| Autor: | John E. Cronan, Wenhua Tong, Haihong Wang, Lei Zhu, Zhe Hu, Yicai Chen, Jin-Cheng Ma |
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| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Models Molecular ACP acyl carrier protein Mutant Reductase medicine.disease_cause Crystallography X-Ray Biochemistry 03 medical and health sciences Catalytic Domain Catalytic triad medicine Escherichia coli Amino Acid Sequence Molecular Biology Alcohol dehydrogenase fatty acid synthesis chemistry.chemical_classification Binding Sites 3-ketoacyl-ACP reductase 030102 biochemistry & molecular biology biology Chemistry Escherichia coli Proteins Fatty Acids Genetic Complementation Test Active site Cell Biology FAS fatty acid synthase short-chain alcohol dehydrogenase/reductase Acyl carrier protein Alcohol Oxidoreductases SDR short-chain alcohol dehydrogenase/reductase 030104 developmental biology Enzyme biology.protein E. coli FabG lipids (amino acids peptides and proteins) 3-Oxoacyl-(Acyl-Carrier-Protein) Reductase Oxidoreductases Protein Binding Research Article |
| Zdroj: | The Journal of Biological Chemistry |
| ISSN: | 1083-351X |
| Popis: | The FabG 3-ketoacyl-acyl carrier protein (ACP) reductase of Escherichia coli has long been thought to be a classical member of the short-chain alcohol dehydrogenase/reductase (SDR) family. FabG catalyzes the essential 3-ketoacyl-ACP reduction step in the FAS II fatty acid synthesis pathway. Site-directed mutagenesis studies of several other SDR enzymes has identified three highly conserved amino acid residues, Ser, Tyr, and Lys, as the catalytic triad. Structural analyses of E. coli FabG suggested the triad S138-Y151-K155 to form a catalytically competent active site. To test this hypothesis, we constructed a series of E. coli FabG mutants and tested their 3-ketoacyl-ACP reductase activities both in vivo and in vitro. Our data show that plasmid-borne FabG mutants, including the double and triple mutants, restored growth of E. coli and Salmonella enterica fabG temperature-sensitive mutant strains under nonpermissive conditions. In vitro assays demonstrated that all of the purified FabG mutant proteins maintained fatty acid synthetic ability, although the activities of the single mutant proteins were 20% to 50% lower than that of wildtype FabG. The S138A, Y151F, and K155A residue substitutions were confirmed by tandem mass spectral sequencing of peptides that spanned all three residues. We conclude that FabG is not a classical short-chain alcohol dehydrogenase/reductase, suggesting that an alternative mode of 3-ketoacyl-ACP reduction awaits discovery. |
| Databáze: | OpenAIRE |
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