Structural basis of catalysis and substrate recognition by the NAD(H)-dependent α-d-glucuronidase from the glycoside hydrolase family 4

Abstract

Members of the glycoside hydrolase family 4 (GH4) employ an unusual glycosidic bond cleavage mechanism utilizing NAD(H) and a divalent metal ion, under reducing conditions. These enzymes act upon a wide diverse range of glycosides, and unlike most other GH families, homologs here are known to accommodate both α- and β-anomeric specificities within the same active site. Here, we report the catalytic properties and the crystal structures of TmAgu4B, an α-d-glucuronidase from the hyperthermophile Thermotoga maritima. The structures in three different states include the apo form, the NADH bound holo form, and the ternary complex with NADH and the reaction product D-glucuronic acid, at 2.15. 1.97 and 1.85 Å resolutions, respectively. These structures reveal the step-wise route of conformational changes required in the active site to achieve the catalytically competent state, and illustrate the direct role of residues that determine the reaction mechanism. Furthermore, a structural transition of a helical region in the active site to a turn geometry resulting in the rearrangement of a unique arginine residue governs the exclusive glucopyranosiduronic acid recognition in TmAgu4B. Mutational studies show that modifications of the glycone binding site geometry lead to catalytic failure and indicate overlapping roles of specific residues in catalysis and substrate recognition. The data highlight hitherto unreported molecular features and associated active site dynamics that determine the structure-function relationships within the unique GH4 family.

NADP(H)-dependent biocatalysis without adding NADP(H)

“…S1 A and B ) ( 14 , 19 – 22 ). Other proteins that copurify with bound NAD(H) ( 23 – 32 ) and NADP(H) ( 33 – 39 ) include the nicotinoproteins ( 28 – 32 , 37 , 38 , 40 ) that apparently use NAD(P)(H) as a permanently bound prosthetic group (instead of an exchangeable cofactor) to perform a variety of reactions. The limited examples of NAD(P)(H)-dependent dehydrogenases copurifying with nicotinamide cofactors ( 23 , 24 , 32 – 35 ) suggest that extremely tight cofactor binding may be unusual.…”

Accurate structure prediction of biomolecular interactions with AlphaFold 3

“…We also used the Chemical Components Dictionary downloaded on 19 October 2023 (https://www.wwpdb.org/data/ccd). We show experimental structures from the PDB with accession numbers 7PZB 52,53 , 7PNM 54,55 , 7TQL 56,57 , 7AU2 58,59 , 7U8C 60,61 , 7URD 62,63 , 7WUX 64,65 , 7QIE 66,67 , 7T82 68,69 , 7CTM 70,71 , 8CVP 44,72 , 8D7U 44,73 , 7F60 74,75 , 8BTI 76,77 , 7KZ9 78,79 , 7XFA 80,81 , 7PEU 82,83 , 7SDW 84,85 , 7TNZ 86,87 , 7R6R 88,89 , 7USR 90,91 , and 7Z1K. 92,93 We also used the following publicly available databases for training or evaluation.…”