Abstract
Many microbial secondary metabolites are produced by multienzyme complexes comprising nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). The ketosynthase (KS) domains of polyketide synthase normally catalyze the decarboxylative Claisen condensation of acyl and malonyl blocks to extend the polyketide chain. However, the terminal KS domain in tenuazonic acid synthetase 1 (TAS1) from the fungus Pyricularia oryzae conducts substrate cyclization. Here, we report on the unique features of the KS domain in TAS1. We observed that this domain is monomeric, not dimeric as is typical for KSs. Analysis of a 1.68-Å resolution crystal structure suggests that the substrate cyclization is triggered via proton abstraction from the active methylene moiety in the substrate by a catalytic His-322 residue. Additionally, we show that TAS1 KS promiscuously accepts aminoacyl substrates and that this promiscuity can be increased by a single amino acid substitution in the substrate-binding pocket of the enzyme. These findings provide insight into a KS domain that accepts the amino acid-containing substrate in an NRPS-PKS hybrid enzyme and provide hints to the substrate cyclization mechanism performed by the KS domain in the biosynthesis of the mycotoxin tenuazonic acid.
Highlights
We previously reported that tetramic acid (2,4-pyrrolidinedione) derivative mycotoxin tenuazonic acid (TeA) is synthesized by tenuazonic acid synthetase 1 (TAS1) in Pyricularia oryzae
To determine whether this state is distinctive to TAS1 KS or common to amino acid–containing substrate-accepting KS, we performed size-exclusion chromatography analysis of OzmQ-KS1 protein, which accepts aminoacyl substrate but preforms traditional elongation in the hybrid oxazolomycin biosynthetic gene cluster [18]
Tetramic acid– containing natural products are generally biosynthesized by a polyketide synthase (PKS)–nonribosomal peptide synthetase (NRPS) hybrid enzyme, and tetramic acid ring structure formation is reportedly related to the terminal cyclization domain that has a sequence similarity to short-chain dehydrogenase/reductases [29, 30]
Summary
A gene construct encoding only the KS domain from TAS1 was heterologously expressed in Escherichia coli. TAS1 KS shows two substitutions of conserved residues compared with type I PKS KS, His to Asn-376 and Thr to Ser-324 To determine whether these replacements affect cyclization, we tested N376H and S324T single mutants and N376H/S324T double mutant. Based on the crystal structure and docking simulation of TAS1 KS, we hypothesize that covalently bound Nacetoacetyl-L-Ile onto Cys-179 is properly positioned by hydrogen bonding to Ser-324, and the methylene proton abstraction by His-322 triggers a nucleophilic attack on the thioester carbonyl to produce the tetramic acid ring and TeA release (Fig. 4C). Bound protein–substrate complexes were detected in WT and H322F, whereas no complex was observed in C179A These results show the possibilities that the H322F mutant can load but not cyclize the substrate (Fig. 5), and that proton abstraction from the active methylene moiety is a crucial step for cyclization. We cannot discriminate between substrate-loaded and -unloaded protein using this
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