Structural Insight into the Reaction Mechanism of Ketosynthase-Like Decarboxylase in a Loading Module of Modular Polyketide Synthases

Abstract

Ketosynthase-like decarboxylase (KSQ) domains are widely distributed in the loading modules of modular polyketide synthases (PKSs) and are proposed to catalyze the decarboxylation of a malonyl or methylmalonyl unit for the construction of the PKS starter unit. KSQ domains have high sequence similarity to ketosynthase (KS) domains, which catalyze transacylation and decarboxylative condensation in polyketide and fatty acid biosynthesis, except that the catalytic Cys residue of KS domains is replaced by Gln in KSQ domains. Here, we present biochemical analyses of GfsA KSQ and CmiP4 KSQ, which are involved in the biosynthesis of FD-891 and cremimycin, respectively. In vitro analysis showed that these KSQ domains catalyze the decarboxylation of malonyl and methylmalonyl units. Furthermore, we determined the crystal structure of GfsA KSQ in complex with a malonyl thioester substrate analogue, which enabled identification of key amino acid residues involved in the decarboxylation reaction. The importance of these residues was confirmed by mutational analysis. On the basis of these findings, we propose a mechanism of the decarboxylation reaction catalyzed by GfsA KSQ. GfsA KSQ initiates decarboxylation by fixing the substrate in a suitable conformation for decarboxylation. The formation of enolate upon decarboxylation is assisted by two conserved threonine residues. Comparison of the structure of GfsA KSQ with those of KS domains suggests that the Gln residue in the active site of the KSQ domain mimics the acylated Cys residue in the active site of KS domains.

Mechanism-based crosslinking probes capture E. coli ketosynthase FabB in conformationally-distinct catalytic states

“…This network might assist the decarboxylation of malonyl-ACP by providing such a water molecule needed for bicarbonate formation, as well as for accommodating the bicarbonate product. This observation supports the bicarbonate-forming mechanism, as opposed to the mechanism that posits release of carbon dioxide (Chisuga et al ., 2022). More biochemical and structural studies are required to definitively address this long-standing question (Heil et al ., 2019).…”

Protein‐Protein Recognition Involved in the Intermodular Transacylation Reaction in Modular Polyketide Synthase in the Biosynthesis of Vicenistatin

“…We expected that in vitro analysis of the transacylation reaction of the VinP2 KS 4 domain is suitable for evaluation of the protein‐protein recognition involved in the intermodular transacylation reaction between polypeptides. We expressed recombinant VinP2 NDD 4 KS 4 protein that contained the neighboring AT 4 domain (Met1‐Gly931) in Escherichia coli because it was reported that type I PKS KS recombinant protein expressed as a single domain is often obtained as an insoluble form [11,12] . To exclude the effect of the VinP2 AT 4 domain, we prepared the VinP2 NDD 4 KS 4 AT 4 S684G mutant in which the catalytic Ser684 residue of the AT 4 domain was mutated.…”

“…The vinP1 ACP 3 CDD 3 fragment was amplified by PCR using cosmid K1B10 as the template DNA with the oligonucleotides shown in Table S5. The amplified fragment was cloned into the expression vector pColdW (modified vector derived from pColdI) [12] using Nde I and Xho I restriction sites to form pColdW‐ vinP1 ACP 3 CDD 3 . For the expression of the VinP1 ACP 3 CDD 3 , E. coli BL21(DE3) (Nippon Gene Co., Ltd.) cells harboring pColdW‐ vinP1 ACP 3 CDD 3 were grown at 37 °C in LB broth containing ampicillin (50 μg/mL).…”

“…We expressed recombinant VinP2 NDD 4 KS 4 protein that contained the neighboring AT 4 domain (Met1-Gly931) in Escherichia coli because it was reported that type I PKS KS recombinant protein expressed as a single domain is often obtained as an insoluble form. [11,12] To exclude the effect of the VinP2 AT 4 domain, we prepared the VinP2 NDD 4 KS 4 AT 4 S684G mutant in which the catalytic Ser684 residue of the AT 4 domain was mutated. We also expressed recombinant VinP1 ACP 3 CDD 3 protein (Asp5651-Asp5826) (Figure S3) in E. coli.…”

Mechanism-based cross-linking probes capture the Escherichia coli ketosynthase FabB in conformationally distinct catalytic states

“…This network might assist the decarboxylation of malonyl-ACP by providing the water molecule needed for bicarbonate formation, as well as by accommodating the bicarbonate product. This observation would support the bicarbonate-forming mechanism, as opposed to the mechanism that posits the release of carbon dioxide (Chisuga et al, 2022). Further biochemical and structural studies are required to definitively address this longstanding question (Heil et al, 2019).…”