Acyltransferases (ATs) are responsible for the selection and incorporation of acyl building blocks in the biosynthesis of various polyketide natural products. The trans-AT modular polyketide synthases have a discrete trans-acting AT for the loading of an acyl unit onto the acyl carrier protein (ACP) located within each module. Despite the importance of protein-protein interactions between ATs and ACPs in trans-AT assembly lines, the dynamic actions of ACPs and trans-acting ATs remain largely uncharacterized because of the inherently transient nature of ACP-enzyme interactions. Herein, we report the crystal structure of the AT-ACP complex of disorazole trans-AT polyketide synthase. We used a bromoacetamide pantetheine cross-linking probe in combination with a Cys mutation to trap the transient AT-ACP complex, allowing the determination of the crystal structure of the disorazole AT-ACP complex at 2.03 Å resolution. On the basis of the cross-linked AT-ACP complex structure, ACP residues recognized by trans-acting AT were identified and validated by mutational studies, which demonstrated that the disorazole AT recognizes the loop 1 and helix III' residues of disorazole ACP. The disorazole AT-ACP complex structure presents a foundation for defining the dynamic processes associated with trans-acting ATs and provides detailed mechanistic insights into their ability to recognize ACPs.
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.
The unique five-membered aminocyclitol core of the antitumor antibiotic pactamycin originates from d-glucose, so unprecedented enzymatic modifications of the sugar intermediate are involved in the biosynthesis. However, the order of the modification reactions remains elusive. Herein, we examined the timing of introduction of an amino group into certain sugar-derived intermediates by using recombinant enzymes that were encoded in the pactamycin biosynthesis gene cluster. We found that the NAD -dependent alcohol dehydrogenase PctP and pyridoxal 5'-phosphate dependent aminotransferase PctC converted N-acetyl-d-glucosaminyl-3-aminoacetophonone into 3'-amino-3'-deoxy-N-acetyl-d-glucosaminyl-3-aminoacetophenone. Further, N-acetyl-d-glucosaminyl-3-aminophenyl-β-oxopropanoic acid ethyl ester was converted into the corresponding 3'-amino derivative. However, PctP did not oxidize most of the tested d-glucose derivatives, including UDP-GlcNAc. Thus, modification of the GlcNAc moiety in pactamycin biosynthesis appears to occur after the glycosylation of aniline derivatives.
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