Dataset Information

Structural modeling and site-directed mutagenesis of the actinorhodin beta-ketoacyl-acyl carrier protein synthase.

ABSTRACT: A three-dimensional model of the Streptomyces coelicolor actinorhodin beta-ketoacyl synthase (Act KS) was constructed based on the X-ray crystal structure of the related Escherichia coli fatty acid synthase condensing enzyme beta-ketoacyl synthase II, revealing a similar catalytic active site organization in these two enzymes. The model was assessed by site-directed mutagenesis of five conserved amino acid residues in Act KS that are in close proximity to the Cys169 active site. Three substitutions completely abrogated polyketide biosynthesis, while two replacements resulted in significant reduction in polyketide production. (3)H-cerulenin labeling of the various Act KS mutant proteins demonstrated that none of the amino acid replacements affected the formation of the active site nucleophile.

SUBMITTER: He M

PROVIDER: S-EPMC111329 | biostudies-literature | 2000 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Structural modeling and site-directed mutagenesis of the actinorhodin beta-ketoacyl-acyl carrier protein synthase.

He M M Varoglu M M Sherman D H DH

Journal of bacteriology 20000501 9

A three-dimensional model of the Streptomyces coelicolor actinorhodin beta-ketoacyl synthase (Act KS) was constructed based on the X-ray crystal structure of the related Escherichia coli fatty acid synthase condensing enzyme beta-ketoacyl synthase II, revealing a similar catalytic active site organization in these two enzymes. The model was assessed by site-directed mutagenesis of five conserved amino acid residues in Act KS that are in close proximity to the Cys169 active site. Three substituti ...[more]

PMID: 10762267

Similar Datasets

Project description:Mycolic acids are long chain alpha-alkyl branched, beta-hydroxy fatty acids that represent a characteristic component of the Mycobacterium tuberculosis cell wall. Through their covalent attachment to peptidoglycan via an arabinogalactan polysaccharide, they provide the basis for an essential outer envelope membrane. Mycobacteria possess two fatty acid synthases (FAS); FAS-I carries out de novo synthesis of fatty acids while FAS-II is considered to elongate medium chain length fatty acyl primers to provide long chain (C(56)) precursors of mycolic acids. Here we report the crystal structure of Mycobacterium tuberculosis beta-ketoacyl acyl carrier protein synthase (ACP) II mtKasB, a mycobacterial elongation condensing enzyme involved in FAS-II. This enzyme, along with the M. tuberculosis beta-ketoacyl ACP synthase I mtKasA, catalyzes the Claisen-type condensation reaction responsible for fatty acyl elongation in FAS-II and are potential targets for development of novel anti-tubercular drugs. The crystal structure refined to 2.4 A resolution revealed that, like other KAS-II enzymes, mtKasB adopts a thiolase fold but contains unique structural features in the capping region that may be crucial to its preference for longer fatty acyl chains than its counterparts from other bacteria. Modeling of mtKasA using the mtKasB structure as a template predicts the overall structures to be almost identical, but a larger entrance to the active site tunnel is envisaged that might contribute to the greater sensitivity of mtKasA to the inhibitor thiolactomycin (TLM). Modeling of TLM binding in mtKasB shows that the drug fits the active site poorly and results of enzyme inhibition assays using TLM analogues are wholly consistent with our structural observations. Consequently, the structure described here further highlights the potential of TLM as an anti-tubercular lead compound and will aid further exploration of the TLM scaffold towards the design of novel compounds, which inhibit mycobacterial KAS enzymes more effectively.

Project description:Staphylococcus aureus is a versatile and dangerous pathogen and one of the major causes of community-acquired and hospital-acquired infections. The rise of multidrug-resistant strains of S. aureus requires the development of new antibiotics with previously unexploited mechanisms of action, such as inhibition of the beta-ketoacyl-acyl carrier protein (ACP) synthase III (FabH). This enzyme initiates fatty acid biosynthesis in a bacterial type II fatty acid synthase, catalyzing a decarboxylative condensation between malonyl-ACP and an acyl coenzyme A (CoA) substrate and is essential for viability. We have identified only one fabH in the genome of S. aureus and have shown that it encodes a protein with 57, 40, and 34% amino acid sequence identity with the FabH proteins of Bacillus subtilis (bFabH1), Escherichia coli (ecFabH), and Mycobacterium tuberculosis (mtFabH). Additional genomic sequence analysis revealed that this S. aureus FabH (saFabH) is not mutated in certain methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA) strains. saFabH was expressed in E. coli with an N-terminal polyhistidine tag and subsequently purified by metal chelate and size exclusion chromatography. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a molecular mass of 37 kDa, while gel filtration demonstrated a mass of 66.7 kDa, suggesting a noncovalent homodimeric structure for saFabH. The apparent K(m) for malonyl-ACP was 1.76 +/- 0.40 microM, and the enzyme was active with acetyl-CoA (k(cat), 16.18 min(-1); K(m), 6.18 +/- 0.9 microM), butyryl-CoA (k(cat), 42.90 min(-1); K(m), 2.32 +/- 0.12 microM), and isobutyryl-CoA (k(cat), 98.0 min(-1); K(m), 0.32 +/- 0.04 microM). saFabH was weakly inhibited by thiolactomycin (50% inhibitory concentration [IC50], >100 microM) yet was efficiently inhibited by two new FabH inhibitors, 5-chloro-4-phenyl-[1,2]-dithiol-3-one (IC50, 1.87 +/- 0.10 microM) and 4-phenyl-5-phenylimino-[1,2,4]dithiazolidin-3-one (IC50, 0.775 +/- 0.08 microM).

Project description:Mycolic acids are hallmark features of the Mycobacterium tuberculosis cell wall. They are synthesized by the condensation of two fatty acids, a C56-64-meromycolyl chain and a C24-26-fatty acyl chain. Meromycolates are produced via the combination of type I and type II fatty acid synthases (FAS-I and FAS-II). The beta-ketoacyl-acyl carrier protein (ACP) synthase III (mtFabH) links FAS-I and FAS-II, catalyzing the condensation of FAS-I-derived acyl-CoAs with malonyl-ACP. Because mtFabH represents a potential regulatory key point of the mycolic acid pathway, we investigated the hypothesis that phosphorylation of mtFabH controls its activity. Phosphorylation of proteins by Ser/Thr protein kinases (STPKs) has recently emerged as a major physiological mechanism of regulation in prokaryotes. We demonstrate here that mtFabH was efficiently phosphorylated in vitro by several mycobacterial STPKs, particularly by PknF and PknA, as well as in vivo in mycobacteria. Analysis of the phosphoamino acid content indicated that mtFabH was phosphorylated exclusively on threonine residues. Mass spectrometry analyses using liquid chromatography-electrospray ionization/tandem mass spectrometry identified Thr45 as the unique phosphoacceptor. This was further supported by complete loss of PknF- or PknA-dependent phosphorylation of a mtFabH mutant. Mapping Thr45 on the crystal structure of mtFabH illustrates that this residue is located at the entrance of the substrate channel, suggesting that the phosphate group may alter accessibility of the substrate and thus affect mtFabH enzymatic activity. A T45D mutant of mtFabH, designed to mimic constitutive phosphorylation, exhibited markedly decreased transacylation, malonyl-AcpM decarboxylation, and condensing activities compared with the wild-type protein or the T45A mutant. Together, these findings not only represent the first demonstration of phosphorylation of a beta-ketoacyl-ACP synthase III enzyme but also indicate that phosphorylation of mtFabH inhibits its enzymatic activity, which may have important consequences in regulating mycolic acid biosynthesis.

Dataset Information

Structural modeling and site-directed mutagenesis of the actinorhodin beta-ketoacyl-acyl carrier protein synthase.

Publications

Structural modeling and site-directed mutagenesis of the actinorhodin beta-ketoacyl-acyl carrier protein synthase.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets