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Biochemical and crystallographic analysis of substrate binding and conformational changes in acetyl-CoA synthetase.

Reger AS, Carney JM, Gulick AM
Biochemistry 46 6536-46 (2007)
Related entries: 2p2b, 2p2f, 2p2j, 2p2m, 2p2q

Cited: 81 times
EuropePMC logo PMID: 17497934

Abstract

The adenylate-forming enzymes, including acyl-CoA synthetases, the adenylation domains of non-ribosomal peptide synthetases (NRPS), and firefly luciferase, perform two half-reactions in a ping-pong mechanism. We have proposed a domain alternation mechanism for these enzymes whereby, upon completion of the initial adenylation reaction, the C-terminal domain of these enzymes undergoes a 140 degrees rotation to perform the second thioester-forming half-reaction. Structural and kinetic data of mutant enzymes support this hypothesis. We present here mutations to Salmonella enterica acetyl-CoA synthetase (Acs) and test the ability of the enzymes to catalyze the complete reaction and the adenylation half-reaction. Substitution of Lys609 with alanine results in an enzyme that is unable to catalyze the adenylate reaction, while the Gly524 to leucine substitution is unable to catalyze the complete reaction yet catalyzes the adenylation half-reaction with activity comparable to the wild-type enzyme. The positions of these two residues, which are located on the mobile C-terminal domain, strongly support the domain alternation hypothesis. We also present steady-state kinetic data of putative substrate-binding residues and demonstrate that no single residue plays a dominant role in dictating CoA binding. We have also created two mutations in the active site to alter the acyl substrate specificity. Finally, the crystallographic structures of wild-type Acs and mutants R194A, R584A, R584E, K609A, and V386A are presented to support the biochemical analysis.

Reviews - 2p20 mentioned but not cited (1)

  1. Conformational dynamics in the Acyl-CoA synthetases, adenylation domains of non-ribosomal peptide synthetases, and firefly luciferase. Gulick AM. ACS Chem Biol 4 811-827 (2009)

Articles - 2p20 mentioned but not cited (3)

  1. Biochemical and crystallographic analysis of substrate binding and conformational changes in acetyl-CoA synthetase. Reger AS, Carney JM, Gulick AM. Biochemistry 46 6536-6546 (2007)
  2. Succinylome analysis reveals the involvement of lysine succinylation in metabolism in pathogenic Mycobacterium tuberculosis. Yang M, Wang Y, Chen Y, Cheng Z, Gu J, Deng J, Bi L, Chen C, Mo R, Wang X, Ge F. Mol Cell Proteomics 14 796-811 (2015)
  3. Expression of the sarA family of genes in different strains of Staphylococcus aureus. Ballal A, Manna AC. Microbiology (Reading) 155 2342-2352 (2009)


Reviews citing this publication (4)

  1. Explorations of catalytic domains in non-ribosomal peptide synthetase enzymology. Hur GH, Vickery CR, Burkart MD. Nat Prod Rep 29 1074-1098 (2012)
  2. Recent advances in engineering nonribosomal peptide assembly lines. Winn M, Fyans JK, Zhuo Y, Micklefield J. Nat Prod Rep 33 317-347 (2016)
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  4. Structural advances toward understanding the catalytic activity and conformational dynamics of modular nonribosomal peptide synthetases. Patel KD, MacDonald MR, Ahmed SF, Singh J, Gulick AM. Nat Prod Rep 40 1550-1582 (2023)

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