Abstract
Polyketide synthases encode a distinct number of enzymes which catalyse different biological reactions i.e., priming, chain elongation, reduction and tailoring reactions and produce a remarkable number of structurally diverse polyketide products from bacteria, fungi, and some plants. Mupirocin and thiomarinol are polyketide antibiotics produced by Pseudomonas fluorescens and Pseudo-alteromonas sp. SANK73390, respectively. Both of these natural products contain a polyketide backbone esterified with a fatty acid derived side chain. The esterification of these two components (via the free carboxyl group of the polyketide and a hydroxyl group present on the fatty acid) represents a key biosynthetic step but the enzyme that catalyses this reaction is unknown. MupB/TmlB are potential candidates for catalysing this reaction and share 37% sequence identity with each other. The aim of this work has been the mechanistic and structural characterization of both enzymes from these pathways.Cloning, expression, purification, and crystallization of MupB and TmlB allowed the determination of high-resolution crystal structures of both enzymes (2.4 Å and 1.5 Å respectively) which revealed a thiolase-fold common to the FabH like enzymes (KAS-III) but containing an alternate catalytic triad comprised of Cys-His-Asp rather than Cys-His-Asn typical of KAS-III enzymes. The orientation of these catalytic residues was non-canonical but matched a single example (CerJ) from the cervimycin pathway that has been proven to catalyse an esterification reaction. Extensive structural analysis revealed that MupB/TmlB possesses two alkyl channels along with a phosphopantetheine binding channel required to bind the substrates for the esterification reaction.
In vitro self-acylation assays confirmed that TmlB selectively bound branched substrates (pre-monic acid mimic, 3-methylbut-2-enoyl (MBE) although MupB non-selectively self-acylated with branched and nonbranched substrates to a low extent. Site directed mutagenesis of the proposed active site cysteine of MupB (C116A) resulted in a surprising increase in self-acylation activity, suggesting the presence of a surrogate nucleophile which is proposed to be a neighbouring serine residue (S224). A co-crystal structure of TmlB in complex with MBE was also solved to 1.59 Å that not only confirmed the presence of hydrophobic alkyl channel but suggested the potentially incorrect orientation of this substrate mimic may indicate the requirement of a more authentic polar substrate as well as cognate ACPs for a successful esterification reaction.
1H-15N HSQC NMR screening experiments showed that only the modular MmpE_ACP, MmpB_ACP5 and ACP7 interacted with MupB and are predicted to deliver the hydroxylated fatty acid chains to the esterification reaction. The in vitro acyltransferase assay of MBE loaded M/TmpE_ACP confirmed MmpE_ACP as the cognate ACP to deliver the polyketide substrate to MupB. Both MupB and TmlB efficiently hydrolysed acyl groups off the ACP to produce holo-ACP which interestingly covalently complexed with MupB/TmlB. Covalent complex formation was far more efficient with MBE present compared to mixing free proteins and may be a future route to capturing an elusive ACP:KAS-III type complex for structure determination.
Although both cognate ACPs for this reaction have been identified, extensive in vitro esterification assay attempts with varied substrates failed to produce any detectable esterified product in presence of acylated MupB/TmlB, producing consistent hydrolysis of 9-hydroxynonanoic acid mediated M/TmpB_ACPs to produce holo-ACPs. Nonetheless, MupB and TmlB remain strong candidates for mediating esterification but appears to suggest that both enzymes have a strict requirement for the presence of a more authentic polyketide substrate.
| Date of Award | 3 Oct 2023 |
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| Original language | English |
| Awarding Institution |
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| Sponsors | Commonwealth Scholarship Commission |
| Supervisor | Ian D Bull (Supervisor) & Matthew P Crump (Supervisor) |