Glycoside hydrolases (GHs) are a large group of enzymes that catalyze the hydrolysis of glycosidic bonds. Among the 168 reported GH families, all except two, use a shared mechanism involving an oxocarbenium ion-like intermediate state and two acidic catalytic residues, acting as general acid/base. The GH family 4 (GH4) employs an exceptional redox-based NAD(H)-dependent reaction mechanism involving anionic intermediates and an obligatory divalent metal ion. However, the structural basis of mechanism and substrate recognition, binding and specificity, remains poorly described in this family. This thesis reports the first structure-function characterization of a GH4 α-glucuronidase using the T. maritima enzyme TmAgu4B, as a model system.

Biochemical studies of TmAgu4B established the substrate specificity and the sequential-order bi-substrate kinetics and the hyperthermostability properties. The crystal structure of TmAgu4B in complex with Co2+ and citrate (competitive inhibitor) showed that a metal-bound conserved water molecule is likely to act as the general base, a rate-limiting step in the mechanism [1]. To elucidate the structural basis of enzyme function, crystal structures of the apo form, the NADH bound holo form, and the ternary complex (NADH and reaction product α-D-glucuronic acid) were determined. These structures revealed the step-wise route of conformational rearrangements to achieve the catalytically competent state, and illustrated the direct role of residues that determine the reaction mechanism and the strict substrate specificity. Mutational studies provided evidence for the overlapping roles of specific residues in catalysis and substrate recognition [2]. Unlike most GH families, GH4 enzymes display both α- and β-anomeric specificities within the same family. The structural basis of the stringent α-anomeric specificity of TmAgu4B was established using docking studies.

TmAgu4B contains a single turn π-helix interspersed within an α-helix in the dimeric interface. The π-helix, although energetically unstable, is evolutionarily conserved within a GH4 subfamily, suggesting that this event is a likely mechanism for an adaptive gain-of-function. To test this, a single residue deletion variant was generated and the crystal structure determined. Comparisons of the structures and temperature-dependent protein stability profiles suggest that specific non-covalent interactions and the presence of residual structure in the unfolded state are crucial structural determinants of hyperthermostability in TmAgu4B. These features permit TmAgu4B to balance the preservation of structure at 90 °C with the thermodynamic stability required for optimum catalysis [3]. In summary, these studies highlighted hitherto unreported molecular features and associated dynamics that determine the structure-function relationships within the unique GH4 family.



Publications:

S.B. Mohapatra, N. Manoj, Structure of an α-glucuronidase in complex with Co2+ and citrate provides insights into the mechanism and substrate recognition in the family 4 glycosyl hydrolases, Biochem. Biophys. Res. Commun. 518 (2019), 197–203.
S.B. Mohapatra, N. Manoj, Structural basis of catalysis and substrate recognition by the NAD(H)-dependent α-glucuronidase from the glycoside hydrolase family 4, Biochem J. 478 (2021), 943-959.
S.B. Mohapatra, N. Manoj, A conserved π-helix plays a key role in thermoadaptation of catalysis in the glycoside hydrolase family 4, BBA-Proteins & Proteomics. 1869 (2021), 140523.