Ketonization of Proline Residues in the Peptide Chains of Actinomycins by a 4‐Oxoproline Synthase

Abstract

X-type actinomycins (Acms) contain 4-hydroxyproline (Acm X ) or 4-oxoproline (Acm X ) in their β-pentapeptide lactone rings, whereas their α 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 show-using an artificial Acm half analogue (PPL 1) with proline in its peptide chain-their conversion into the 4-hydroxyproline- and 4-oxoproline-containing Acm halves, PPL 0 and PPL 2, in mycelial suspensions of Streptomyces antibioticus. Two responsible genes of the Acm X 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 PPL 1 into PPL 0 and PPL 2 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 avermectin B1 into its keto derivative, 4''-oxoavermectin B1. 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.

Identification of the Actinomycin D Biosynthetic Pathway from Marine-Derived Streptomyces costaricanus SCSIO ZS0073

“…Previous studies demonstrated the importance of a 4-oxoproline synthase within the actinomycin BGC of S. antibioticus . This enzyme catalyzes proline oxidation in each of the actinomycin halves (prior to dimerization) to form 4-hydroxyproline or 4-oxoproline; condensation of each of the hydroxylated actinomycin halves affords actinomycin X oβ or actinomycin X 2 [42]. Within the actinomycin BGC in S. costaricanus SCSIO ZS0073, we identified a cytochrome P450 gene, acnP , that encodes a 436 aa protein.…”

Comparison of actinomycin peptide synthetase formation in Streptomyces chrysomallus and Streptomyces antibioticus

“…Actinomycin D also contains D‐valine in the 2‐positions of its pentapeptide lactone rings, but carries proline residues in the 3‐positions of both pentapeptide lactone rings. Actinomycin D and X have a common biosynthetic origin . Moreover, actinomycin synthesis in actinomycin D‐producing S. parvulus like in actinomycin X‐producing S. antibioticus was shown to be repressible by glucose .…”

Structure-guided manipulation of the regioselectivity of the cyclosporine A hydroxylase CYP-sb21 from Sebekia benihana

“…2 A and B). Collectively, our structure reflects an open conformation [ 18 , 31 ]] and the missing BC-loop appears to be a common characteristic for the P450 enzymes that accommodate bulky substrates [ [18] , [31] ].

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“…, MeLeu 9 rather than MeLeu 4 is the preferred hydroxylation site) when compared with CYP-sb21 [ 7 ]. saAcmM, which was identified from S. antibioticus, catalyzes a specific hydroxylation of bicyclic chromopeptide lactones [ 31 ]. The sequence identities and similarities between CYP-sb21 and CYP-pa1, CYP-sb21 and saAcmM, and CYP-pa1 and saAcmM are 55% and 67%, 52% and 64%, and 48% and 58%, respectively ( Fig.…”