Structure and substrate specificity of β‐ketoacyl‐acyl carrier protein synthase III from Acinetobacter baumannii

Abstract

Originally annotated as the initiator of fatty acid synthesis (FAS), β-ketoacyl-acyl carrier protein synthase III (KAS III) is a unique component of the bacterial FAS system. Novel variants of KAS III have been identified that promote the de novo use of additional extracellular fatty acids by FAS. These KAS III variants prefer longer acyl-groups, notably octanoyl-CoA. Acinetobacter baumannii, a clinically important nosocomial pathogen, contains such a multifunctional KAS III (AbKAS III). To characterize the structural basis of its substrate specificity, we determined the crystal structures of AbKAS III in the presence of different substrates. The acyl-group binding cavity of AbKAS III and co-crystal structure of AbKAS III and octanoyl-CoA confirmed that the cavity can accommodate acyl groups with longer alkyl chains. Interestingly, Cys264 formed a disulfide bond with residual CoA used in the crystallization, which distorted helices at the putative interface with acyl-carrier proteins. The crystal structure of KAS III in the alternate conformation can also be utilized for designing novel antibiotics.

Structural Comparison of Acinetobacter baumannii β-ketoacyl-acyl Carrier Protein Reductases in Fatty Acid and Aryl Polyene Biosynthesis

“…Crystal structure of the FAS β-ketoacyl ACP reductase AbFabG As a member of Gammaproteobacteria, A. baumannii is closely related to P. aeruginosa and E. coli. In particular, P. aeruginosa and A. baumannii have similar FAS systems, whereby the host fatty acids can be incorporated for de novo fatty acid synthesis 17,27 . FabG of P. aeruginosa (PaFabG) was identi ed as a potential drug target with allosteric inhibition targeted to multimeric interfaces 25 .…”

“…A. baumannii FabD enzyme was cloned as previously described 18 . A. baumannii holo-ACP synthase (AbAcpS) was prepared as previously described 17 .…”

“…For the generation of holo-ACPs, ACP1 and ACP2 were co-expressed with AbAcpS as previously described 17 . In brief, either the ACP1 or ACP2 gene was cloned into the NdeI/XhoI sites of MCS2 in the pETDuet-1 vector containing the AbAcpS sequence in MCS1 to generate two expression vectors: holo-ACP1 and holo-ACP2.…”

“…The FAS of A. baumannii has been relatively less explored compared to that of other notable pathogens such as Pseudomonas aeruginosa or Mycobacterium tuberculosis. Our previous studies focusing on the FAS proteins of A. baumannii led to the development of antibiotics against MDR strains [16][17][18] . Moreover, amino acid sequence homology analyses revealed that the majority of MDR A. baumannii strains harbor APE biosynthesis genes in contrast to the non-virulent standard strain.…”