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Crystallization of Escherichia coli enoyl reductase and its complex with diazaborine (1996)

Baldock, C. ; Rafferty, J. B. ; Sedelnikova, S. E. ; [et al.]

Copenhagen : International Union of Crystallography (IUCr)

Acta crystallographica 52 (1996), S. 1181-1184

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ISSN: 1399-0047

Source: Crystallography Journals Online : IUCR Backfile Archive 1948-2001

Topics: Chemistry and Pharmacology , Geosciences , Physics

Notes: Recent work has shown that the NADH-dependent enoyl acyl carrier protein reductase from Escherichia coli is the target for diazaborine, an antibacterial agent. This enzyme has been crystallized by the hanging-drop method of vapour diffusion complexed with NAD+ and in the presence and absence of a thieno diazaborine. The crystals grown in the absence of diazaborine (form A) are in the space group P21 with unit-cell dimensions a = 74.0, b = 81.2, c = 79.0 Å and β = 92.9°, and with a tetramer in the asymmetric unit, whilst those grown in the presence of diazaborine (form B) are in the space group P6122 (or P6522) with unit-cell dimensions a = b = 80.9 and c = 328.3 Å, and with a dimer in the asymmetric unit. The structure determination of this enzyme in the presence of diazaborine will provide information on the nature of the drug binding site and contribute to a programme of rational drug design.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1107/S0907444996005458

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Functional analysis of an interspecies chimera of acyl carrier proteins indicates a specialized domain for protein recognition (1998)

Ritsema, T. ; Gehring, A. M. ; Stuitje, A. R. ; [et al.]

Springer

Molecular genetics and genomics 257 (1998), S. 641-648

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ISSN: 1617-4623

Keywords: Key words Nodulation ; Rhizobium ; Lipo-chitin oligosaccharide ; NodF ; Fatty acid biosynthesis

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Abstract The nodulation protein NodF of Rhizobium shows 25% identity to acyl carrier protein (ACP) from Escherichia coli (encoded by the gene acpP). However, NodF cannot be functionally replaced by AcpP. We have investigated whether NodF is a substrate for various E. coli enzymes which are involved in the synthesis of fatty acids. NodF is a substrate for the addition of the 4′-phosphopantetheine prosthetic group by holo-ACP synthase. The Km value for NodF is 61 μM, as compared to 2 μM for AcpP. The resulting holo-NodF serves as a substrate for coupling of malonate by malonyl-CoA:ACP transacylase (MCAT) and for coupling of palmitic acid by acyl-ACP synthetase. NodF is not a substrate for β-keto-acyl ACP synthase III (KASIII), which catalyses the initial condensation reaction in fatty acid biosynthesis. A chimeric gene was constructed comprising part of the E.coliacpP gene and part of the nodF gene. Circular dichroism studies of the chimeric AcpP-NodF (residues 1–33 of AcpP fused to amino acids 43–93 of NodF) protein encoded by this gene indicate a similar folding pattern to that of the parental proteins. Enzymatic analysis shows that AcpP-NodF is a substrate for the enzymes holo-ACP synthase, MCAT and acyl-ACP synthetase. Biological complementation studies show that the chimeric AcpP-NodF gene is able functionally to replace NodF in the root nodulation process in Vicia sativa. We therefore conclude that NodF is a specialized acyl carrier protein whose specific features are encoded in the C-terminal region of the protein. The ability to exchange domains between such distantly related proteins without affecting conformation opens exciting possibilities for further mapping of the functional domains of acyl carrier proteins (i. e., their recognition sites for many enzymes).