Ketonization of proline residues in the peptide chains of actinomycins by a 4-Oxoproline synthase.

X-type actinomycins (Acms) contain 4-hydroxyproline (Acm X-0) or 4-oxoproline (Acm X-2) in their -pentapeptide lactone rings, whereas their ring contains proline. We demonstrate that these Acms are formed through asymmetric condensation of Acm half molecules (Acm halves) containing proline with 4-hydroxyproline- or 4-oxoproline-containing Acm halves. In turn, we showusing an artificial Acm half analogue (PPL1) with proline in its peptide chaintheir conversion into the 4-hydroxyproline- and 4-oxoproline-containing Acm halves, PPL0 and PPL2, in mycelial suspensions of Streptomyces antibioticus. Two responsible genes of the AcmX biosynthetic gene cluster of S.antibioticus, saacmM and saacmN, encoding a cytochrome P450 monooxygenase (Cyp) and a ferredoxin were identified. After coexpression in Escherichia coli, their gene products converted PPL1 into PPL0 and PPL2 in vivo as well as in situ in permeabilized cell of the transformed E.coli strain in conjunction with the host-encoded ferredoxin reductase in a NADH (NADPH)-dependent manner. saAcmM has high sequence similarity to the Cyp107Z (Ema) family of Cyps, which can convert avermectinB1 into its keto derivative, 4-oxoavermectinB1. Determination of the structure of saAcmM reveals high similarity to the Ema structure but with significant differences in residues decorating their active sites, which defines saAcmM and its orthologues as a distinct new family of peptidylprolineketonizing Cyp.