Malonyl‐CoA: acyl carrier protein transacylase from Helicobacter pylori: Crystal structure and its interaction with acyl carrier protein

Abstract

Malonyl-CoA: acyl carrier protein transacylase (MCAT) is a critical enzyme responsible for the transfer of the malonyl moiety to holo-acyl carrier protein (ACP) forming the malonyl-ACP intermediates in the initiation step of type II fatty acid synthesis (FAS II) in bacteria. MCAT has been considered as an attractive drug target in the discovery of antibacterial agents. In this study, the crystal structure of MCAT from Helicobacter pylori (Hp) at 2.5 Å resolution is reported, and the interaction of HpMCAT with HpACP is extensively investigated by using computational docking, GST-pull-down, and surface plasmon resonance (SPR) technology-based assays. The crystal structure results reveal that HpMCAT has a compact folding composed of a large subdomain with a similar core as in a/b hydrolases, and a similar ferredoxin-like small subdomain as in acylphosphatases. The docking result suggests two positively charged areas near the entrance of the active site of HpMCAT as the ACP-binding region. Binding assay research shows that HpMCAT demonstrates a moderately binding ability against HpACP. The solved 3D structure of HpMCAT is expected to supply useful information for the structure-based discovery of novel inhibitors against MCAT, and the quantitative study of HpMCAT interaction with HpACP is hoped to give helpful hints in the understanding of the detailed catalytic mechanisms for HpMCAT.Keywords: Helicobacter pylori; Manoyl-CoA: acyl carrier protein transacylase (MCAT); acyl carrier protein (ACP); fatty acid biosynthesis; crystal structure Supplemental material: see www.proteinscience.orgThe biosynthesis of fatty acid (FAS) is an essential process for the survival of the organism (Magnuson et al. 1993;White et al. 2005). In bacteria, the process of FAS is completed by a series of individual enzymes, while in animals, only a single enzyme with several distinct domains is involved in all the reactions. Such a major difference between the animal and bacterial systems therefore makes the enzymes involved in the FAS process potential drug targets for the discovery of antibacterial agents (Campbell and Cronan Jr. 2001;Miesel et al. 2003;White et al. 2005).Malonyl-CoA: acyl carrier protein transacylase (fabD; MCAT, EC2.3.1.39) is responsible for the transfer of malonyl moiety to holo-ACP forming malonyl-ACP intermediates to participate in fatty acid biosynthesis (Ruch and Vagelos 1973). It is reported that MCAT might also be involved in polyketide biosynthesis, producing one of the largest Reprint requests to: Hualiang Jiang or Xu Shen, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China; e-mail: hljiang@mail.shcnc.ac.cn or xshen@mail.shcnc.ac.cn; fax: 86-21-50806918.Abbreviations: FAS, fatty acid biosynthesis; ACP, acyl carrier protein; IPTG, isopropyl-b-D-thiogalasctopyranoside; SPR, surface plasmon resonance; FabZ, b-hydroxyacyl-ACP dehydratase; MCAT, manoyl-CoA: acyl carrier protein transacylase; FabG, b-ketoacyl-acyl carrier protein reductase; FabH, b-keto...

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