Removal of substrate inhibition of Acinetobacter baumannii xanthine oxidase by point mutation at Gln-201 enables efficient reduction of purine content in fish sauce

Abstract

Xanthine oxidase is an oxidase that has a molybdopterin structure with substrate inhibition. Here, we show that a single point mutation (Q201) in the Acinetobacter baumannii xanthine oxidase (AbXOD) obtained mutant Q201E (k _cat =799.44 s^-1, no inhibition) with high enzyme activity and decrease of substrate inhibition in 5 mmol/L high substrate model, and which cause two loops structure change at active center, characterized by complete loss of substrate inhibition without reduction of enzymatic activity. Molecular docking results showed that the change of flexible loop increased the affinity between substrate and enzyme, and the formation of a π-π bond and two hydrogen bonds made the substrate more stable in the active center. Ultimately, Q201E can still maintain better enzyme activity under high purine content (an approximately 7-fold improvement over the wild-type), indicating a broader application prospect in the manufacture of low-purine food.

Keywords: Acinetobacter baumannii; Enzyme engineering; Fish sauce; Point mutation; Substrate inhibition; Xanthine oxidase.

Fig. 1

Enzyme activity in two substrate concentrations. Two different xanthine substrate concentration models, commonly used standard substrate concentration model — 0.1 mmol/L and high substrate concentration model —5 mmol/L.

Fig. 2

Characterization of the enzymatic properties of the mutants. (A) Temperature-activity profile. (B) pH-activity profile. (C) Temperature-stability profile. (D) pH-stability profile. Enzymatic assays were carried out with 5 mmol/L xanthine.

Fig. 3

Conformational fluctuations in the predict structures of XOD. The entire model in the green cartoon, molybdopterin structure in red spheres, and Q201 is represented in the red stick. The Q201 mutant interacts with surrounding amino acid hydrogen bonds in the red frame, Q201 in A is shown by red stick, the residue has hydrogen bonding interact with Q201showed by orange stick, and the hydrogen bonding in yellow dotted line. (A)Wide-type (B)Q201C (C) Q201E (D)Q201N (E) Q201L. The conformational changes in the active center in the blue frame, the wild-type show as the blue flexible loop (loop^234-245 (Ser234-Arg235-Arg236-Met237-Gly238-Gly239-Gly249-Phe241-Gly242-Lys243-Glu245) and loop^523-530 (Ser523-Ala524-Thr525-Ala526-Ala527-Ser528-Ser529-Gly530)), (a) two flexible loops are shown by purple in Q201C; (b) two flexible loops are shown by turquoise blue in Q201E; (c) two flexible loops are shown by orange in Q201N; (d) two flexible loops are shown by yellow in Q201L.

Fig. 4

Docking interaction plots of xanthine with flexible loop residues of Q201E. (A) interacting residues of Q201E were shown in sticks colored by atom type, the carbon in green, hydrogen in white, oxygen in red, and nitrogen in blue. Interactions included non-covalent bonds, π interaction and contacts, the hydrogen bonds in blue, and π-cation in yellow. But xanthine is shown in yellow sticks. (B) 2D interaction schematic of Xanthine molecule with Q201E.

Fig. 5

HPLC analysis of uric acid and other purine contents of fish sauce (pH=9.0) with WT (A), and fish sauce (pH=9.0) with Q201E (B). The numbers indicate purine standards, uric acid (1), hypoxanthine, and xanthine. The samples with and without treatment of Q201E (1.6 U/mL) at 40℃ for 30 min are shown in line, short dot, and short dash-dot, respectively. Control, the centrifuged supernatant of Q201E enzyme inactivated in boiling water for 20 min was used to replace the reaction enzyme solution in the enzymatic reaction system.