The structural characterisation of a glucosylglycerate hydrolase provides insights into the molecular mechanism of mycobacterial recovery from nitrogen starvation
Tatiana Barros Cereija, Susana Alarico, Eva C. Lourenço, José António Manso, M. Rita Ventura, Nuno Empadinhas, Sandra Macedo-Ribeiro and Pedro José Barbosa Pereira [1]
Molecular Tour
When facing nitrogen-limiting conditions, mycobacteria accumulate glucosylglycerate, which is rapidly mobilized when nitrogen levels are restored. In Mycolicibacterium hassiacum (basonym Mycobacterium hassiacum), glucosylglycerate mobilization was concomitant with the up-regulation of the gene coding for glucosylglycerate hydrolase (GgH). Highly conserved among unrelated phyla, GgH is able to hydrolyse glucosylglycerate to glycerate and glucose and is likely involved in bacterial reactivation following nitrogen starvation.
Using X-ray crystallography, high-resolution structural models of different forms of GgH could be obtained. These detailed views of GgH revealed several important aspects of its mode of action, including its oligomeric organization, with the homotetramer present in the crystals being fully compatible with the architecture of the active form of GgH in solution, assessed by small-angle X-ray scattering (SAXS) using synchrotron radiation. The crystallographic structures of unliganded and substrate-bound GgH further revealed the existence of a coordinated movement of several surface loops in the vicinity of the active site during the catalytic cycle of the enzyme. Therefore, the active site of GgH is only completely structured upon substrate binding, with the mobile loops shielding the bound compounds from the solvent and facilitating the enzymatic reaction. Reversal of this movement opens up the active site of GgH, allowing product release and readying the enzyme for another catalytic cycle.
Finally, the different complexes of GgH with substrates and substrate analogues allowed to infer the molecular details of the reaction mechanism of this inverting hydrolase and to ascribe the functional roles of highly conserved residues in this class of enzymes.
. Monomers are coloured green (molecule A), wheat (molecule B), cyan (molecule C) and blue (molecule D). Interfaces A:B and A:C are indicated. The serine molecules found in the active site region are represented by salmon spheres. Approximate dimensions of the homotetramer are indicated.
. Open (lighter hues) and closed (darker hues) states of monomeric MhGgH are shown. The A’-region (flexible segment), and loops A, B, D and E are coloured salmon, yellow, blue, brown and green, respectively. Some of the substrate-interacting residues present in the highlighted regions [Tyr36 (loop A), Tyr88 (loop B), Arg216, Tyr222 (A’-region), Tyr375, Trp376 (loop D) and Gln434 (loop E)] are represented as ball-and-sticks.
Close-up of the movement of the catalytic residues. . Catalytic residues (yellow ball-and-sticks) are pointing away from the active site cavity (salmon spheres), stabilised by direct hydrogen bonds and by water (w)-mediated contacts (dashed lines) with the neighbour residues.
Closed and open conformations of MhGgH.
- . Arg and Lys residues are colored blue, His in deepskyblue; Asp and Glu in red. A negatively charged tunnel is colored crimson.
- . The residues which correspond to a negatively charged tunnel is colored crimson.
- . Substrate-binding residues are highlighted in yellow. In the open state, an opening leading to an acidic cavity is observed; a negatively charged tunnel connects the active site cavity to the exterior of the molecule.
- . In the closed state, the active site cavity (colored in salmon) becomes inaccessible to the solvent.
References
- ↑ Cereija TB, Alarico S, Lourenco EC, Manso JA, Ventura MR, Empadinhas N, Macedo-Ribeiro S, Pereira PJB. The structural characterization of a glucosylglycerate hydrolase provides insights into the molecular mechanism of mycobacterial recovery from nitrogen starvation. IUCrJ. 2019 May 8;6(Pt 4):572-585. doi: 10.1107/S2052252519005372. eCollection, 2019 Jul 1. PMID:31316802 doi:http://dx.doi.org/10.1107/S2052252519005372
