Substitution of a Single Amino Acid Reverses the Regiospecificity of the Baeyer–Villiger Monooxygenase PntE in the Biosynthesis of the Antibiotic Pentalenolactone

In the biosynthesis of pentalenolactone (1), PenE and PntE, orthologous proteins from Streptomyces exfoliatus and S. arenae, respectively, catalyze the flavin-dependent Baeyer–Villiger oxidation of 1-deoxy-11-oxopentalenic acid (4) to the lactone pentalenolactone D (5), in which the less-substituted methylene carbon has migrated. By contrast, the paralogous PtlE enzyme from S. avermitilis catalyzes the oxidation of 4 to neopentalenolactone D (6), in which the more substituted methane substitution has undergone migration. We report the design and analysis of 13 single and multiple mutants of PntE mutants to identify the key amino acids that contribute to the regiospecificity of these two classes of Baeyer–Villiger monooxygenases. The L185S mutation in PntE reversed the observed regiospecificity of PntE such that all recombinant PntE mutants harboring this L185S mutation acquired the characteristic regiospecificity of PtlE, catalyzing the conversion of 4 to 6 as the major product. The recombinant PntE mutan...