The Structural Basis for Substrate Anchoring, Active Site Selectivity, and Product Formation by P450 PikC from Streptomyces venezuelae

Abstract

The pikromycin (Pik)/methymycin biosynthetic pathway of Streptomyces venezuelae represents a valuable system for dissecting the fundamental mechanisms of modular polyketide biosynthesis, aminodeoxysugar assembly, glycosyltransfer, and hydroxylation leading to the production of a series of macrolide antibiotics, including the natural ketolides narbomycin and pikromycin. In this study, we describe four x-ray crystal structures and allied functional studies for PikC, the remarkable P450 monooxygenase responsible for production of a number of related macrolide products from the Pik pathway. The results provide important new insights into the structural basis for the C10/C12 and C12/C14 hydroxylation patterns for the 12-(YC-17) and 14-membered ring (narbomycin) macrolides, respectively. This includes two different ligand-free structures in an asymmetric unit (resolution 2.1 Å ) and two co-crystal structures with bound endogenous substrates YC-17 (resolution 2.35 Å ) or narbomycin (resolution 1.7 Å ). A central feature of the enzymesubstrate interaction involves anchoring of the desosamine residue in two alternative binding pockets based on a series of distinct amino acid residues that form a salt bridge and a hydrogen-bonding network with the deoxysugar C3 dimethylamino group. Functional significance of the salt bridge was corroborated by site-directed mutagenesis that revealed a key role for Glu-94 in YC-17 binding and Glu-85 for narbomycin binding. Taken together, the x-ray structure analysis, site-directed mutagenesis, and corresponding product distribution studies reveal that PikC substrate tolerance and product diversity result from a combination of alternative anchoring modes rather than an induced fit mechanism.Macrolide antibiotics comprise a large group of medicinal agents characterized by a macrocyclic lactone ring, to which one or more sugar residues are covalently linked. Despite considerable structural variation, macrolides represent a homogeneous group of therapeutic drugs with similar activity spectra and mode of action as anti-infective agents. The success of macrolide antibiotics is attributed to their propensity to bind to the large subunit of prokaryotic ribosomes and inhibit protein synthesis, thereby preventing bacterial growth (1, 2). The first generation macrolide introduced into clinical practice over 50 years ago was erythromycin. Since then, macrolide antibiotics have been further optimized, resulting in improved 14-, 15-, and 16-membered ring macrolides (second generation), acylides, and ketolides (third generation) (3).Most of the natural product macrolide antibiotics are produced by Streptomyces sp. and related bacteria, in which assembly of polyketides from simple carboxylic acid precursors is catalyzed by modular polyketide synthases. Over the past 15 years, advances in understanding the modular architecture of polyketide biosynthetic machinery has enabled development of metabolic engineering approaches for production of new antibiotics (4 -7). However, significant additional structural...

Mycobacterium tuberculosis CYP130

“…7C), contrasts with the almost exclusively hydrophobic environment of the rest of the CYP130 active site. This pocket could serve as a landmark for substrate (or inhibitor) recognition, similar to that observed in the macrolide monooxygenase PikC, where a salt bridge formed between the positively charged tertiary amino group of the macrolide substrate, and a negatively charged carboxylic amino acid residue is essential to achieve catalytically competent binding (50).…”

Crystal Structures of Cytochrome P450 105P1 from Streptomyces avermitilis : Conformational Flexibility and Histidine Ligation State

“…These facts indicate significant flexibility of BC loop of CYP105P1, unlike those of MoxA and P450nor. Among bacterial P450 enzymes similar to CYP105P1, OxyB (CYP165B3), P450 PikC, and P450 SU-1 have a partially disordered BC loop for three to seven amino acids in ligand-free forms (47,49,55). In contrast, there are a number of examples that the FG helices displace between ligand-free and ligand-bound forms, e.g., CYP119 from Sulfolobus solfataricus (36), P450nor (31), and CYP 158A2 (56).…”

“…The hydroxylase activity of these enzymes for at least one or two macrolide compounds has been confirmed in vitro. Recently, the crystal structure of P450 PikC (CYP107L1), which is responsible for the production of a number of macrolide antibiotics related to narbomycin and pikromycin, has been reported (47). Moreover, the crystal structures of macrolide monooxygenase P450epoK, which catalyzes epoxidation in the epothilone biosynthesis, have been determined in both the substrate-free and the substrate-bound forms (26).…”

Use of Chemical Auxiliaries to Control P450 Enzymes for Predictable Oxidations at Unactivated C-H Bonds of Substrates

“…Based on the X-ray crystal structure of PiKC complexed with different macrolides, the authors reasoned that the desosamine moiety might position the macrolide substrates in the active site. This reasoning stemmed from the observation that the majority of hydrogen bonds and electrostatic interactions within the active site are formed by the desosamine group of the natural substrates, while the macrolactones are held in place merely through more general hydrophobic interactions [64,65]. In support of their hypothesis, they first synthesized a series of carbocyclic rings ranging in size from 12 to 15 carbons and linked to a desosamine glycoside, altogether referred to as carbolides.…”