Mutagenesis of threonine to serine in the active site of Mycobacterium tuberculosis fructose-1,6-bisphosphatase (Class II) retains partial enzyme activity

•Fructose-1,6-bisphosphatase (class II) is an essential enzyme and attractive drug target in M. tuberculosis using structure-based drug design.•T84A mutagenesis in the active site of MtFBPase shows no enzyme activity and no change in binding affinity.•T84S mutagenesis in the active site of MtFBPase results in reduced enzyme activity (lower Vmax), but retains lithium sensitive.•The optimal position of the catalytic OH- from the essential Thr84 has likely been subtly altered, resulting in lower enzymatic efficiency.

The glpX gene encodes for the Class II fructose-1,6-bisphosphatase enzyme in Mycobacterium tuberculosis (Mt), an essential enzyme for pathogenesis. We have performed site directed mutagenesis to introduce two mutations at residue Thr84, T84A and T84S, to explore the binding affinity of the substrate and the catalytic mechanism. The T84A mutant fully abolishes enzyme activity while retaining substrate binding affinity. In contrast, the T84S mutant retains some activity having a 10 times reduction in Vmax and exhibited similar sensitivity to lithium when compared to the wildtype. Homology modeling using the Escherichia coli enzyme structure suggests that the replacement of the critical nucleophile OH− in the Thr84 residue of the wildtype of MtFBPase by Ser84 results in subtle alterations of the position and orientation that reduce the catalytic efficiency. This mutant could be used to trap reaction intermediates, through crystallographic methods, facilitating the design of potent inhibitors via structure-based drug design.