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Structural Insights into Mycobacterial Protein-Mannosyltransferase: Evidence of a WW-Domain-Like Substrate Recognition Mechanism.

ABSTRACT: We have previously demonstrated that protein-O-mannosylation (POM), a widespread post-translational glycosyl modification of proteins, is a key virulence factor of Mycobacterium tuberculosis (Mt), the world’s deadliest infectious agent. Here, we report a detailed analysis of the structure-function relationship of MtPMT, the enzyme that catalyzes POM in Mtb. Using mutagenesis and in cellulo monitoring of POM activity, we demonstrate that, despite notable structural differences, MtPMT shares functional homologies with yeasts’ PMTs in the mechanism of the sugar transfer from lipidic donors. Furthermore, we provide evidence that the selectivity for proline-rich target glycosylation sites that differentiates MtPMT from its eukaryotic homologues, relies on a WW-like domain, which preferentially interacts with proline-rich acceptor substrate analogues. This first identification of a functional WW-like domain in a prokaryotic protein raises questions about its potential evolutionary linkage with eukaryotic WW modules and provides new insights into PMT’s acceptor-substrate recognition mechanism paving the way for the development selective inhibitors of MtPMT with potential therapeutic application against tuberculosis. This dataset consists in the entire protein analysis of the Fasciclin reporter protein produced in a strain of Mycobacterium smegmatis (Ms) devoid of MsPMT (ΔMsPMT) and expressing either WT-MtPMT, mCherry-Nter-MtPMT or MtPMT-Cter-PhoA.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Escherichia Coli

SUBMITTER: Julien Marcoux

LAB HEAD: Julien Marcoux

PROVIDER: PXD065654 | Pride | 2025-07-22

REPOSITORIES: Pride

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Project description:Background: Among the nineteen polyketide synthases encoded by the genome of Mycobacterium tuberculosis, Pks13 was identified as the condensase required for the final condensation step of two long acyl chains. This leads to mycolic acids, which are essential components of the cell envelope of Corynebacterineae species. The single polypeptide chain of Pks13 comprises five catalytic domains, including two functional acyl carrier protein domains, separated by linkers comprising up to ca. 200 amino acid residues. In many aspects, Pks13 is an atypical polyketide synthase. It has been validated as a promising druggable target and its structure is badly needed to speed up drug discovery to fight against tuberculosis. Results: We report here a quasi-atomic model of Pks13 obtained using small angle X-ray scattering of the entire protein and various molecular subspecies combined with known high-resolution structures of Pks13 domains or structural homologues. In addition, the structural changes induced by the loading of a substrate analogue have been challenged. As a comparison, the low-resolution structures of two other mycobacterial polyketide synthases, Mas and PpsA from Mycobacterium bovis BCG, are also presented. This study highlights an identical and elongated structure of the apo and holo forms at the resolution probed. Catalytic domains are segregated into two parts, which correspond to the condensation reaction per se and to the release of the product, a pivot for the enzyme flexibility being at the interface. The two acyl carrier protein domains are found at opposite sides of the ketosynthase domain and display distinct characteristics in terms of flexibility. Apo-Pks13 is in our conditions in a monomeric state, but loading of a substrate analogue push the enzyme towards the functional dimeric state. Conclusions: The Pks13 model provides the first structural information on the molecular mechanism of this complex enzyme and open up new perspectives to develop inhibitors that target the interactions with its enzymatic partners or between catalytic domains within Pks13 itself.

			Action	DRS
	FasC.fasta	Fasta
	Fascicline_20220126.pptx	Other
	Mass-Spec-FasC.xlsx	Xlsx
	OFJMX220119_17.raw	Raw
	OFJMX220119_18.raw	Raw

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Structural Insights into Mycobacterial Protein-Mannosyltransferase: Evidence of a WW-Domain-Like Substrate Recognition Mechanism.

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