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 (k
cat/K
m) 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.
Af ew acyltransferase( AT)d omains of modular polyketide synthases (PKSs) recruit acyl carrierp rotein (ACP)-linked extender units with unusual C2 substituents to confer functionalities that are not available in coenzyme A( CoA)-linked ones. In this study,a nA Ts pecific for methoxymalonyl (MOM)-ACP in the third moduleo ft he ansamitocin PKS was structurallya nd biochemically characterized. The AT uses ac onserved tryptophan residue at the entrance of the substrate bindingt unnelt o discriminate betweend ifferent carriers. AW 275R mutation switchesi ts carriers pecificity from the ACP to the CoA molecule. The acyl-ATc omplex structuresc learly showt hat the MOM-ACP accepted by the AT hast he 2S instead of the opposite 2R stereochemistry that is predicted according to the biosynthetic derivation from a d-glycolytic intermediate. To gether, these results revealt he structural basis of ATsr ecognizing ACPlinked extender units in polyketide biosynthesis.
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 determined that HcbA1 could only oxidize HCB rather than less chlorinated benzenes such as pentachlorobenzene and tetrachlorobenzenes. In addition, the crystal structure of HcbA1 and its complex with FMN were resolved, revealing HcbA1 as a new member of the bacterial luciferase-like family. Compared with its close homologues, a much smaller substrate-binding pocket of HcbA1 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 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 its difficulty in dechlorination. The lack of knowledge for HCB dechlorinases limits their application in bioremediation. Recently, an HCB monooxygenase HcbA1A3 was reported, which is the only known naturally occurring aerobic HCB dechlorinase so far. Here we report its biochemical and structural characterizations, which provide new insights into its substrate selectivity and catalytic mechanism. This research also increases our understanding of HCB dechlorinases and flavin-N5-peroxide utilizing enzymes.
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.
Polyketides are a large group of natural products with diverse chemical structures and biological activities. They are biosynthesized by modular polyketide synthases (PKSs) from coenzyme A (CoA) thioesters of short-chain...
The product template (PT) domains act as an aldol cyclase to control the regiospecific aldol cyclization of the extremely reactive poly-β-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.
The product template (PT) domains act as an aldol cyclase to control the regiospecific aldol cyclization of the extremely reactive poly-β-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 His949 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 catalytic “His-Asp” dyad organization, but is significantly different from PTs of fungal iterative type I PKSs. QM/MM calculation reveals that the catalytic His949 abstracts a proton from C2 and transfers it to C7 carbonyl to mediate the cyclization reaction. According to the structural similarity to DHs and the functional similarity to fungal PTs, we propose that the bacterial PT represents an evolutionary intermediate between the two tailoring domains of type I PKSs.
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