Journal:Acta Cryst D:S2059798319004169

Structural and functional insights into phosphomannose isomerase

Mamata Bangera, Giri Gowda K., S.R. Sagurthi and M.R.N. Murthy ^[1]

Molecular Tour
Phosphomannose isomerase is a zinc binding enzyme that catalyses the reversible isomerization of mannose 6-phosphate and fructose 6-phosphate. These substrates could exist in two conformations. They are covalently closed (cyclic form) in one conformation while a covalent bond is disrupted in the other linear form. The reaction most likely proceeds by binding of the cyclic form of substrate, conversion of its closed to open form, transfer of protons between atoms of the open form of substrate by a suitable base followed by its cyclisation to form the cyclic form of the product.

Structure of phosphomannose isomerase from Salmonella typhimurium

The monomer is made up of three domains: an N terminal ־±- helical domain, a central catalytic domain and a C terminal domain. The polypeptide fold of the C terminal domain and catalytic domain are very similar to that of the cupin domain found in proteins belonging to different catalytic classes. The central catalytic domain contains the zinc binding site and has longer loops than the C terminal domain

Zinc binding site

Zinc is bound by His99, Glu134, His255 and a water molecule in a tetrahedral coordination. Mutation in any of these residues interferes with zinc binding, which in turn leads to a non-functional enzyme. Isothermal calorimetric experiments with mutants showed that loss of zinc binding is associated with lack of substrate binding as well. Hence zinc plays an important role in anchoring the substrate in the active site.

Active site and catalytic base

Examination of conservation of residues in the protein showed that active site colocalises with the zinc binding site. In order to determine the most important residues for catalysis, site directed mutagenesis and activity studies were carried out on the mutants. Based on location in active site, conservation across species and complete loss of activity upon mutation, Lys86 was proposed to be the catalytic base.

Concerted movement of residues

Lys132 and Arg274 were also identified to be important for the catalytic reaction by this and other studies. However, Arg274 is located far from the active site and the side chain of Lys132 is flipped away from the active site pocket in the native enzyme. Comparison of structures of mutants with native enzyme revealed open and closed conformational states of the enzyme regulated by these residues. These states might be important for the binding of both the open and closed forms of the substrate and product in the catalytic cycle.

References

1. ↑ Bangera M, Gowda K G, Sagurthi SR, Murthy MRN. Structural and functional insights into phosphomannose isomerase: the role of zinc and catalytic residues. Acta Crystallogr D Struct Biol. 2019 May 1;75(Pt 5):475-487. doi:, 10.1107/S2059798319004169. Epub 2019 Apr 29. PMID:31063150 doi:http://dx.doi.org/10.1107/S2059798319004169