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Mutational Analysis of the Myxovirescin Biosynthetic Gene Cluster Reveals Novel Insights into the Functional Elaboration of Polyketide Backbones
Author(s) -
Simunovic Vesna,
Müller Rolf
Publication year - 2007
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200700153
Subject(s) - mutant , polyketide , hydroxylation , cytochrome p450 , chemistry , gene , biochemistry , stereochemistry , gene cluster , biology , genetics , biosynthesis , enzyme
It has been proposed that two acyl carrier proteins (ACPs)—TaB and TaE—and two 3‐hydroxy‐3‐methylglutaryl synthases (HMGSs)—TaC and TaF—could constitute two functional ACP‐HMGS pairs (TaB/TaC and TaE/TaF) responsible for the incorporation of acetate and propionate units into the myxovirescin A scaffold, leading to the formation of β‐methyl and β‐ethyl groups, respectively. It has been suggested that three more proteins—TaX and TaY, which are members of the superfamily of enoyl‐CoA hydratases (ECHs), and a variant ketosynthase (KS) TaK—are shared between two ACP‐HMGS pairs, to give the complete set of enzymes required to perform the β‐alkylations. The β‐methyl branch is presumably further hydroxylated (by TaH) and methylated to produce the methoxymethyl group observed in myxovirescin A. To substantiate this hypothesis, a series of gene‐deletion mutants were created, and the effects of these mutations on myxovirescin production were examined. As predicted, Δ taB and Δ taE ACP mutants revealed similar phenotypes to their associated HMGS mutants Δ taC and Δ taF , respectively, thus providing direct evidence for the role of TaE/TaF in the formation of the β‐ethyl branch and implying a role for TaB/TaC in the formation of the β‐methyl group. Production of myxovirescin A was dramatically reduced in a Δ taK mutant and abolished in both the Δ taX and the Δ taY mutant backgrounds. Analysis of a Δ taH mutant confirmed the role of the cytochrome P450 TaH in hydroxylation of the β‐methyl group. Taken together, these experiments support a model in which the discrete ACPs TaB and TaE are compatible only with their associated HMGSs TaC and TaF, respectively, and function in a substrate‐specific manner. Both TaB and TaC are essential for myxovirescin production, and the TaB/TaC pair can rescue antibiotic production in the absence of either TaE or TaF. Finally, the reduced level of myxovirescin production in the Δ taE mutant, relative to the Δ taF strain, suggests an additional function of the TaE ACP.