PDBsum entry 1dpb

Dihydrolipoamide acetyltransferase

PDB id: 1dpb

Contents

Protein chain

243 a.a.

Waters ×40

References listed in PDB file

Key reference

• Title: Crystallographic and enzymatic investigations on the role of ser558, His610, And asn614 in the catalytic mechanism of azotobacter vinelandii dihydrolipoamide acetyltransferase (e2p).
• Authors: J.Hendle, A.Mattevi, A.H.Westphal, J.Spee, A.De kok, A.Teplyakov, W.G.Hol.
• Ref.: Biochemistry, 1995, 34, 4287-4298. [DOI no: 10.1021/bi00013a018]
• PubMed id: 7703242

Note In the PDB file this reference is annotated as "TO BE PUBLISHED". The citation details given above were identified by an automated search of PubMed on title and author names, giving a percentage match of 97%.

Abstract

Dihydrolipoamide acetyltransferase (E2p) is the structural and catalytic core of the pyruvate dehydrogenase multienzyme complex. In Azotobacter vinelandii E2p, residues Ser558, His610', and Asn614' are potentially involved in transition state stabilization, proton transfer, and activation of proton transfer, respectively. Three active site mutants, S558A, H610C, and N614D, of the catalytic domain of A. vinelandii E2p were prepared by site-directed mutagenesis and enzymatically characterized. The crystal structures of the three mutants have been determined at 2.7, 2.5, and 2.6 A resolution, respectively. The S558A and H610C mutants exhibit a strongly (200-fold and 500-fold, respectively) reduced enzymatic activity whereas the substitution of Asn614' by aspartate results in a moderate (9-fold) reduced activity. The decrease in enzymatic activity of the S558A and H610C mutants is solely due to the absence of the hydroxyl and imidazole side chains, respectively, and not due to major conformational rearrangements of the protein. Furthermore the sulfhydryl group of Cys610' is reoriented, resulting in a completely buried side chain which is quite different from the solvent-exposed imidazole group of His610' in the wild-type enzyme. The presence of Asn614' in A. vinelandii E2p is exceptional since all other 18 known dihydrolipoamide acyltransferase sequences contain an aspartate in this position. We observe no difference in conformation of Asp614' in the N614D mutant structure compared with the conformation of Asn614' in the wild-type enzyme. Detailed analysis of all available structures and sequences suggests two classes of acetyltransferases: one class with a catalytically essential His-Asn pair and one with a His-Asp-Arg triad as present in chloramphenicol acetyltransferase [Leslie, A. G. W. (1990) J. Mol. Biol. 213, 167-186] and in the proposed active site models of Escherichia coli and yeast E2p.

Secondary reference #1

• Title: Refined crystal structure of the catalytic domain of dihydrolipoyl transacetylase (e2p) from azotobacter vinelandii at 2.6 a resolution.
• Authors: A.Mattevi, G.Obmolova, K.H.Kalk, A.H.Westphal, A.De kok, W.G.Hol.
• Ref.: J Mol Biol, 1993, 230, 1183-1199.
• PubMed id: 8487300

Secondary reference #2

• Title: Crystallographic analysis of substrate binding and catalysis in dihydrolipoyl transacetylase (e2p).
• Authors: A.Mattevi, G.Obmolova, K.H.Kalk, A.Teplyakov, W.G.Hol.
• Ref.: Biochemistry, 1993, 32, 3887-3901. [DOI no: 10.1021/bi00066a007]
• PubMed id: 8471601

Secondary reference #3

• Title: Atomic structure of the cubic core of the pyruvate dehydrogenase multienzyme complex.
• Authors: A.Mattevi, G.Obmolova, E.Schulze, K.H.Kalk, A.H.Westphal, A.De kok, W.G.Hol.
• Ref.: Science, 1992, 255, 1544-1550. [DOI no: 10.1126/science.1549782]
• PubMed id: 1549782