Structural snapshots catch an enzyme in the act

27 July 2016
Thiamin diphosphate (ThDP)-dependent enzymes perform crucial C–C bond forming and breaking reactions
Structure coverpage:Thiamin diphosphate (ThDP)-dependent enzymes perform crucial C–C bond forming and breaking reactions

It is rare to be able to capture an intermediate in an enzymatic reaction and visualize it in full atomic detail, let alone to do so for the full series of key intermediates in a catalytic cycle. Yet this is just what a team in SBS has done, reported in the Cell Press journal Structure, together with a coveted cover figure.

The enzyme in question was MenD, an essential gatekeeper in the biosynthesis of menaquinone in Mycobacterium tuberculosis, and an important TB drug target. Dr. Jodie Johnston initiated the project, which Ehab Jirgis, now a general staff member in SBS, undertook as his MSc project, supervised by Jodie, Dr. Ghader Bashiri and Distinguished Professor Ted Baker and Dr Esther Bulloch also made a crucial contribution to NMR aspects. 

Some critical roadblocks had to be overcome. To get pure, soluble enzyme, expression in Mycobacterium smegmatis had to be used, instead of the usual E. coli. A reproducible crystallization protocol was required, after the first high-quality crystals grew accidentally in the fridge! One substrate, isochorismate, was unstable and unobtainable commercially. It had to be made enzymatically, monitored by NMR, then used as a crude mixture with its precursor chorismate. Fortunately, the enzyme selected only the correct substrate.

Altogether 7 crystal structures were determined as the cofactor, thiamine diphosphate, and the two substrates α-ketoglutarate and isochorismate were successively added. Two key covalent intermediates were actually obtained by reactions carried out in the crystals themselves, as substrates were added. The outcome is a remarkable series of structural snapshots, revealing each stage in the catalytic cycle – the first time this has been done for any such enzyme. 

The success of this team effort has enabled Jodie Johnston to win AMRF funding  and led to a full Marsden proposal with Drs. Johnston and Bashiri as co-PIs. The work detailed in the paper was funded by Ted Baker’s HRC and MBIE grants and is being continued within the TB Flagship program of the Maurice Wilkins Centre.