Abstract
Streptovaricin C is a naphthalenic ansamycin antibiotic structurally similar to rifamycins with potential anti-MRSA bioactivities. However, the formation mechanism of the most fascinating and bioactivity-related methylenedioxy bridge (MDB) moiety in streptovaricins is unclear. Based on genetic and biochemical evidences, we herein clarify that the P450 enzyme StvP2 catalyzes the MDB formation in streptovaricins, with an atypical substrate inhibition kinetics. Furthermore, X-ray crystal structures in complex with substrate and structure-based mutagenesis reveal the intrinsic details of the enzymatic reaction. The mechanism of MDB formation is proposed to be an intramolecular nucleophilic substitution resulting from the hydroxylation by the heme core and the keto-enol tautomerization via a crucial catalytic triad (Asp89-His92-Arg72) in StvP2. In addition, in vitro reconstitution uncovers that C6-O-methylation and C4-O-acetylation of streptovaricins are necessary prerequisites for the MDB formation. This work provides insight for the MDB formation and adds evidence in support of the functional versatility of P450 enzymes.
Highlights
Streptovaricin C is a naphthalenic ansamycin antibiotic structurally similar to rifamycins with potential anti-methicillin-resistant S. aureus (MRSA) bioactivities
We demonstrate that reduced form of damavaricin C as a vital precursor is transformed into methylenedioxy bridge (MDB)-contained mature streptovaricins by cytochrome P450 enzyme StvP2 after sequential methylation and acetylation
StvP2 is responsible for the MDB formation of streptovaricins
Summary
StvP2 is responsible for the MDB formation of streptovaricins. MDB is a characteristic moiety mainly existing in phenylpropanoids such as (−)-pluviatolide[12], (+)-pluviatilol[13,14], (+)-sesamin[13], etc. and alkaloids such as (S)-cheilanthifoline[15], (S)-canadine[16,17], (S)stylopine[18], etc. from higher plants and in some microbiala. The NH2 group and the O atom of carbonyl in Ile[390] are bond to the H atom of hydroxyl at C-23 and C-27, respectively (Fig. 4b) These interactions are contributed to bind the substrate molecule to a reasonable conformation to facilitate the enzymatic reaction. The carboxyl group of Asp[89] is hydrogen bonded to the N-2 atom of imidazole group in His[92], and the N-4 atom of imidazole group in His[92] is hydrogen bonded to the O atom of the water molecule, which further forms a bifurcated hydrogen bond with the hydroxyl group at the C-1 of the substrate and the imine group of the guanidyl in Arg[72], respectively These three amino acid residues are almost in a straight line, approximately facing and paralleling with the naphthol plane. The carboxyl group of Asp[89] is hydrogen bonded to the imidazole of His[92] to protonate the N-2 atom of the imidazole group, which further provided a proton to the water molecule, a
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