Structural and computational insights into the substrate specificity of acyltransferase domains from modular polyketide synthases.

Polyketides are natural products synthesized by polyketide synthases (PKSs), where acyltransferase (AT) domains play a crucial role in selection of extender units. Engineering of AT domains enables the site-specific incorporation of non-natural extender units, leading to generation of novel derivatives. Here, we determined the crystal structures of AT domains from the fifth module of tylosin PKS (TylAT5) derived from Streptomyces fradiae and the eighth module of spinosad PKS (SpnAT8) derived from Saccharopolyspora spinosa, and combined them with molecular dynamics simulations and enzyme kinetic studies to elucidate the molecular basis of substrate selection. The ethylmalonyl-CoA-specific conserved motif TAGH of TylAT5 and the MMCoA-specific conserved motif YASH of SpnAT8 were identified within the substrate-binding pocket, and several key residues close to the substrate acyl moiety were located. Through site-directed mutagenesis of four residues near the active site, we successfully reprogrammed the specificity of these two AT domains toward malonyl-CoA. Mutations in TylAT5 enhanced its catalytic activity 2.6-fold toward malonyl-CoA, and mutations in SpnAT8 eliminated the substrate promiscuity. These results extend our understanding of AT substrate specificity and would benefit the engineering of PKSs.

Structural and computational insights into the regioselectivity of SpnK involved in rhamnose methylation of spinosyn.

Rhamnose methylation of spinosyn critical for insecticidal activity is orchestrated by substrate specificity of three S-adenosyl-L-methionine (SAM) dependent methyltransferases (MTs). Previous in vitro enzymatic assays indicate that 3'-O-MT SpnK accepts the rhamnosylated aglycone (RAGL) and 2'-O-methylated RAGL as substrates, but does not tolerate the presence of a methoxy moiety at the O-4' position of the rhamnose unit. Here we solved the crystal structures of apo and ligand-bound SpnK, and used molecular dynamic (MD) simulations to decipher the molecular basis of substrate specificity. SpnK assembles into a tetramer, with each set of three monomers forming an integrated substrate binding pocket. The MD simulations of SpnK complexed with RAGL or 2'-O-methylated RAGL revealed that the 4'-hydroxyl of the rhamnose unit formed a hydrogen bond with a conserved Asp299 of the catalytic center, which is disrupted in structures of SpnK complexed with 4'-O-methylated RAGL or 2',4'-di-O-methylated RAGL. Comparison with SpnI methylating the C2'-hydroxyl of RAGL reveals a correlation between a DLQT/DLWT motif and the selectivity of rhamnose O-MTs. Together, our structural and computational results revealed the structural basis of substrate specificity of rhamnose O-MTs and would potentially help the engineering of spinosyn derivatives.

The COMPASS Complex Regulates Fungal Development and Virulence through Histone Crosstalk in the Fungal Pathogen Cryptococcus neoformans.

The Complex of Proteins Associated with Set1 (COMPASS) methylates lysine K4 on histone H3 (H3K4) and is conserved from yeast to humans. Its subunits and regulatory roles in the meningitis-causing fungal pathogen Cryptococcus neoformans remain unknown. Here we identified the core subunits of the COMPASS complex in C. neoformans and C. deneoformans and confirmed their conserved roles in H3K4 methylation. Through AlphaFold modeling, we found that Set1, Bre2, Swd1, and Swd3 form the catalytic core of the COMPASS complex and regulate the cryptococcal yeast-to-hypha transition, thermal tolerance, and virulence. The COMPASS complex-mediated histone H3K4 methylation requires H2B mono-ubiquitination by Rad6/Bre1 and the Paf1 complex in order to activate the expression of genes specific for the yeast-to-hypha transition in C. deneoformans. Taken together, our findings demonstrate that putative COMPASS subunits function as a unified complex, contributing to cryptococcal development and virulence.

The Streptomyces viridochromogenes product template domain represents an evolutionary intermediate between dehydratase and aldol cyclase of type I polyketide synthases.

The product template (PT) domains act as an aldol cyclase to control the regiospecific aldol cyclization of the extremely reactive poly-ß-ketone intermediate assembled by an iterative type I polyketide synthases (PKSs). Up to now, only the structure of fungal PksA PT that mediates the first-ring cyclization via C4-C9 aldol cyclization is available. We describe here the structural and computational characterization of a bacteria PT domain that controls C2-C7 cyclization in orsellinic acid (OSA) synthesis. Mutating the catalytic H949 of the PT abolishes production of OSA and results in a tetraacetic acid lactone (TTL) generated by spontaneous O-C cyclization of the acyl carrier protein (ACP)-bound tetraketide intermediate. Crystal structure of the bacterial PT domain closely resembles dehydrase (DH) domains of modular type I PKSs in the overall fold, dimerization interface and His-Asp catalytic dyad organization, but is significantly different from PTs of fungal iterative type I PKSs. QM/MM calculation suggests that the catalytic H949 abstracts a proton from C2 and transfers it to C7 carbonyl to mediate the cyclization reaction. According to structural similarity to DHs and functional similarity to fungal PTs, we propose that the bacterial PT represents an evolutionary intermediate between the two tailoring domains of type I PKSs.

Hexachlorobenzene Monooxygenase Substrate Selectivity and Catalysis: Structural and Biochemical Insights.

Hexachlorobenzene (HCB), as one of the persistent organic pollutants (POPs) and a possible human carcinogen, is especially resistant to biodegradation. In this study, HcbA1A3, a distinct flavin-N5-peroxide-utilizing enzyme and the sole known naturally occurring aerobic HCB dechlorinase, was biochemically characterized. Its apparent preference for HCB in binding affinity revealed that HcbA1 could oxidize only HCB rather than less-chlorinated benzenes such as pentachlorobenzene and tetrachlorobenzenes. In addition, the crystal structure of HcbA1 and its complex with flavin mononucleotide (FMN) were resolved, revealing HcbA1 to be a new member of the bacterial luciferase-like family. A much smaller substrate-binding pocket of HcbA1 than is seen with its close homologues suggests a requirement of limited space for catalysis. In the active center, Tyr362 and Asp315 are necessary in maintaining the normal conformation of HcbA1, while Arg311, Arg314, Phe10, Val59, and Met12 are pivotal for the substrate affinity. They are supposed to place HCB at a productive orientation through multiple interactions. His17, with its close contact with the site of oxidation of HCB, probably fixes the target chlorine atom and stabilizes reaction intermediates. The enzymatic characteristics and crystal structures reported here provide new insights into the substrate specificity and catalytic mechanism of HcbA1, which paves the way for its rational engineering and application in the bioremediation of HCB-polluted environments.IMPORTANCE As an endocrine disrupter and possible carcinogen to human beings, hexachlorobenzene (HCB) is especially resistant to biodegradation, largely due to difficulty in its dechlorination. The lack of knowledge of HCB dechlorinases limits their application in bioremediation. Recently, an HCB monooxygenase, HcbA1A3, representing the only naturally occurring aerobic HCB dechlorinase known so far, was reported. Here, we report its biochemical and structural characterization, providing new insights into its substrate selectivity and catalytic mechanism. This research also increases our understanding of HCB dechlorinases and flavin-N5-peroxide-utilizing enzymes.

Structural and Biochemical Insight into the Recruitment of Acyl Carrier Protein-Linked Extender Units in Ansamitocin Biosynthesis.

A few acyltransferase (AT) domains of modular polyketide synthases (PKSs) recruit acyl carrier protein (ACP)-linked extender units with unusual C2 substituents to confer functionalities that are not available in coenzyme A (CoA)-linked ones. In this study, an AT specific for methoxymalonyl (MOM)-ACP in the third module of the ansamitocin PKS was structurally and biochemically characterized. The AT uses a conserved tryptophan residue at the entrance of the substrate binding tunnel to discriminate between different carriers. A W275R mutation switches its carrier specificity from the ACP to the CoA molecule. The acyl-AT complex structures clearly show that the MOM-ACP accepted by the AT has the 2S instead of the opposite 2R stereochemistry that is predicted according to the biosynthetic derivation from a d-glycolytic intermediate. Together, these results reveal the structural basis of ATs recognizing ACP-linked extender units in polyketide biosynthesis.

Structural Insights into the Substrate Specificity of Acyltransferases from Salinomycin Polyketide Synthase.

Salinomycin with antibacterial and anticoccidial activities is a commercial polyether polyketide widely used in animal husbandry as a food additive. Malonyl-CoA (MCoA), methylmalonyl-CoA (MMCoA), and ethylmalonyl-CoA (EMCoA) are used as extension units in its biosynthesis. To understand how the salinomycin modular polyketide synthase (PKS) strictly discriminates among these extension units, the acyltransferase (AT) domains selecting MCoA, MMCoA, and EMCoA were structurally characterized. Molecular dynamics simulations of the AT structures helped to reveal the key interactions involved in enzyme-substrate recognitions, which enabled the engineering of AT mutants with switched specificity. The catalytic efficiencies ( kcat/ Km) of these AT mutants are comparable with those of the wild-type AT domains. These results set the stage for engineering the AT substrate specificity of modular PKSs.