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

Abstract

The chemical complexity and biological activity of the glycopeptide antibiotics (GPAs) stems from their unique crosslinked structure, which is generated by the actions of cytochrome P450 (Oxy) enzymes that affect the crosslinking of aromatic side chains of amino acid residues contained within the GPA heptapeptide precursor. Given the crucial role peptide cyclisation plays in GPA activity, the characterisation of this process is of great importance in understanding the biosynthesis of these important antibiotics. Here, we report the cyclisation activity and crystal structure of StaF, the D-O-E ring forming Oxy enzyme from A47934 biosynthesis. Our results show that the specificity of StaF is reduced when compared to Oxy enzymes catalysing C-O-D ring formation and that this activity relies on interactions with the non-ribosomal peptide synthetase via the X-domain. Despite the interaction of StaF with the A47934 X-domain being weaker than for the preceding Oxy enzyme StaH, StaF retains higher levels of in vitro activity: we postulate that this is due to the ability of the StaF/X-domain complex to allow substrate reorganisation after initial complex formation has occurred. These results highlight the importance of testing different peptide/protein carrier constructs for in vitro GPA cyclisation assays and show that different Oxy homologues can display significantly different catalytic propensities despite their overall similarities.

Cytochromes P450 for natural product biosynthesis in Streptomyces: sequence, structure, and function

“…76–78 This finding suggests that this cluster of P450s (>180 members) may all work on ACP- or PCP-tethered substrates and gives insight into the putative endogenous function of CYP125A2, a P450 shown to hydroxylate a xenobiotic flavonoid. 171 Not all characterized P450s that act on ACP- or PCP-tethered substrates are found within this cluster, with AcmG8, 172 StaF, and StaH being notable exceptions, 99,106 suggesting there is more than one type of sequence that can utilize protein-protein interactions for substrate selectivity.…”

“…3). 99,106 Adding to the complexity and interest of this family of P450s, they utilize PCP-tethered substrates and interact with the so-called X-domain, found in the last NRPS module, for P450 recruitment and cyclization efficiency. 97,98,107 …”

“…Finally, (v) the biaryl ring coupling-catalyzing P450s involved in glycopeptide biosynthesis, are splintering off of the CYP105 family. These include the aforementioned StaH, StaG, StaF, and StaJ 99,106 and ComI and ComJ from complestatin biosynthesis. 165 Although only StaH and StaF were characterized in vitro showing a requirement for the PCP-tethered substrate and the X-domain, 107 it is plausible that the other four enzymes, along with the other unidentified members in this splintered group follow the same reactivity requirements.…”

A route to diastereomerically pure phenylglycine thioester peptides: crucial intermediates for investigating glycopeptide antibiotic biosynthesis

“…12 In GPA biosynthesis, we have recently demonstrated the necessity of maintaining the correct stereochemistry of C-terminal phenylglycine residues, where the activity of the second essential P450 in the GPA cyclisation cascade -OxyA -was shown to depend on the (L)-configuration of the terminal residue. [13][14][15][16] As complete racemisation of this residue occurred under previous Fmocsynthetic conditions, 10,11 access to enantiomerically pure peptides was restricted and based purely on the ability to affect their chromatographic separation. Whilst resolution of simplified GPA peptides -those containing a C-terminal Hpg residue -was possible, resolution of those containing the natural Dpg residue was not.…”

“…We then tested the level of cyclisation seen in these peptides using the proven enzymatic coupling of OxyB van and OxyA tei , 7,[13][14][15]24,25 with the peptides themselves loaded onto a PCP-X di-domain construct excised from the final module of the teicoplanin NRPS machinery. 7 The results of the Oxy-catalysed turnover of peptides 4-5 demonstrated how vital the L-configuration of the C-terminal residue is for effective enzymatic crosslinking.…”