S T R U C T U R A L B I O L O G Y

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5 2 H I G H L I G H T S 2 0 2 1 I

Fig. 39: a) Structure of bottromycin A2. b) Oxidative decarboxylation reaction catalysed by SalCYP.

Fig. 40: a) Crystal structure of SalCYP, which makes contact with a symmetry mates heme via its N-terminus (magenta sphere). b) Surface representation of the modified SalCYP crystal structure showing its active site. Heme shown as sticks, iron as orange sphere. c) LC-MS analysis of the SalCYP substrate without (top) and with (bottom) SalCYP added. d) Addition of the epimerase BotH leads to complete turnover of the SalCYP reaction.

Building an antibiotic scaffold the final step

Biological systems are extraordinary chemists and can create highly complex, efficacious molecules from simple building blocks. X-ray diffraction revealed the final step in the biosynthesis of the core scaffold of a potent antibiotic, Bottromycin, which is effective against multi-drug-resistant human pathogens.

Natural products, or secondary metabolites, have had a profound impact on human health. About 70% of anti- infectives used today are natural products, their derivatives, or synthetic molecules inspired by them. It is therefore not surprising that significant efforts are being made to identify candidate natural products that could help to avert the looming antibiotic crisis. Bottromycins (Figure 39a) were first discovered as antibiotic molecules in the 1950s [1], and were later shown to be effective against antibiotic- resistant, problematic human pathogens [2]. It took almost 60 years to find the genes responsible for bottromycin production [3-6]. This work set out to understand how a small set of enzymes assembles these molecules, in order to produce derivatives for antibiotic testing.

Biosynthesis of the bottromycin core scaffold was reconstituted and the crystal structures of several key enzymes were described, but the final step, the oxidation of a cysteine-derived thiazoline to a thiazole (Figure 39b) presented a number of difficulties. Previous work had shown that a P450 enzyme catalysed this reaction [7]. All attempts to crystallise this protein had failed, but a close homologue, SalCYP, was crystallised, with micro-seeding yielding diffraction-quality, single crystals. Due to the bound heme group, X-ray diffraction datasets were collected at the Fe-edge at beamline ID29 and the SalCYP crystal structure was determined using Fe-SAD. However, surprisingly, the disordered N-termini were coordinated to the heme-iron via their N-terminal amino groups (Figure 40a). This precluded an unbiased structural analysis so the protein was partially truncated at the N-terminus and a single point mutation of an N-terminal residue was introduced to engineer a new crystal contact. This version of the protein yielded high-quality crystals (studied at beamline ID30-3) with an unoccupied active-site, which is much wider than found in regular P450 enzymes (Figure 40b).

Suitable redox partners were found for SalCYP, making it possible to reconstitute SalCYP s activity in vitro, and it was shown that the enzyme is sufficient to catalyse the oxidative decarboxylation reaction required to complete