Regulation of the P450 Oxygenation Cascade Involved in Glycopeptide Antibiotic Biosynthesis

Abstract

Glycopeptide antibiotics (GPAs) are nonribosomal peptides rich in modifications introduced by external enzymes. These enzymes act on the free peptide aglycone or intermediates bound to the nonribosomal peptide synthetase (NRPS) assembly line. In this process the terminal module of the NRPS plays a crucial role as it contains a unique recruitment platform (X-domain) interacting with three to four modifying Cytochrome P450 (P450) enzymes that are responsible for cyclizing bound peptides. However, whether these enzymes share the same binding site on the X-domain and how the order of the cyclization steps is orchestrated has remained elusive. In this study we investigate the first two reactions in teicoplanin aglycone maturation catalyzed by the enzymes OxyBtei and OxyAtei. We demonstrate that both enzymes interact with the X-domain via the identical interaction site with similar affinities, irrespective of the peptide modification stage, while their catalytic activity is restricted to the correctly cross-linked peptide. On the basis of steady state kinetics of the OxyBtei-catalyzed reaction, we propose a model for P450 recruitment and peptide modification that involves continuous association/dissociation of the P450 enzymes with the NRPS, followed by specific recognition of the peptide cyclization state by the P450 (scanning). This leads to an induced conformational change that enhances the affinity of the enzyme/substrate complex and initiates catalysis; product release then occurs, with the product itself becoming the substrate for the second enzyme in the pathway. This model rationalizes our experimental findings for this complex enzyme cascade and provides insights into the orchestration of the sequential peptide tailoring reactions on the terminal NRPS module in GPA biosynthesis.

Biochemical and Structural Characterization of MycCI, a Versatile P450 Biocatalyst from the Mycinamicin Biosynthetic Pathway

“…Recent work carried out by Cryle and coworkers has demonstrated the importance of the non-ribosomal peptide synthetase X-domain in helping to coordinate the P450-mediated oxidative cascade that leads to fully cyclized aglycones in the biosynthesis of vancomyin, teicoplanin, and other glycopeptides. 69–71 While the P450 OxyB from the vancomycin pathway (OxyB van ) has long been recognized as a versatile biocatalyst with the ability to accept a wide range of peptidyl carrier protein (PCP)-bound substrates, 72–75 the homolog from the teicoplanin pathway (OxyB tei , 74% sequence identity) exhibits a much narrower substrate scope when acting on PCP-bound peptides in the absence of the X-domain. 76 However, OxyB tei and homologs from other pathways are able to act on a broader range of substrates when the X-domain is present, although they tend to exhibit lower overall activity compared to OxyB van .…”

Biochemical and structural characterisation of the second oxidative crosslinking step during the biosynthesis of the glycopeptide antibiotic A47934

“…More importantly, with the exception of OxyB van , the activity of Oxy enzymes in vitro has also been shown to be highly dependent on the presence of the X-domain fused to the peptidyl carrier protein domain [16]. This in turn has, for the first time, allowed the characterisation of the second cyclisation step, catalysed by OxyA, from the teicoplanin system [13,16–17]. These results showed that OxyA, in contrast to OxyB, is highly selective for the correct stereochemistry of the peptide C-terminal residue and generally displays a higher selectivity for the structure of the substrate peptides [13,17].…”

Unrivalled diversity: the many roles and reactions of bacterial cytochromes P450 in secondary metabolism

“…[250][251][252] More recent work has shown that the process of P450 recruitment to the NRPS-bound peptide is more complicated than is the case for PCP-bound amino acids, with GPA biosynthesis relying on the X-domain, a conserved (albeit modied) condensation domain found between the PCP 253 and TE-domains in the nal NRPS module. 243,[254][255][256][257] The X-domain has an interaction interface that specically binds to the Oxy enzymes, with these continually binding to and releasing from the X-domain to ensure that complete crosslinking of the peptide occurs. 254 The X-domain is also required in the majority of cases to support efficient P450-catalysed crosslinking in vitro, with this requirement strictly enforced for OxyA and OxyE that act aer OxyB.…”

“…243,[254][255][256][257] The X-domain has an interaction interface that specically binds to the Oxy enzymes, with these continually binding to and releasing from the X-domain to ensure that complete crosslinking of the peptide occurs. 254 The X-domain is also required in the majority of cases to support efficient P450-catalysed crosslinking in vitro, with this requirement strictly enforced for OxyA and OxyE that act aer OxyB. 243,255,256,[258][259][260][261][262][263] Complestatin 264 and kistamicin 265 are structurally similar to GPAs, and also display several oxidative crosslinks whose generation has been ascribed to P450 enzymes, i.e.…”