Enzymatic Characterization and Directed Evolution of Agrobacterium tumefaciens O-demethylase Atu1420

doi:10.13560/j.cnki.biotech.bull.1985.2024-0993

Abstract

Abstract:

Objective Protocatechuic acid (PCA) is a widely used phenolic compound. Biosynthesis method of PCA is a potential alternative to the highly-polluting chemical synthesis. Finding and improving enzymes for synthesizing PCA is the key to biosynthesis. Agrobacterium tumefaciens O-demethylase Atu1420 is an enzyme that can convert vanillic acid (VA) to PCA. This work aims to characterize the enzymatic properties of Atu1420 and improve it. Method Atu1420 was expressed in Escherichia coli, and the enzymatic properties of Atu1420 were determined by methods such as high-performance liquid chromatography (HPLC). Based on the predicted structure and catalytic mechanism of Atu1420, 19 sites were selected to conduct site-directed mutagenesis on Atu1420. A visual method for detecting the catalytic activity of Atu1420 was developed by using 4-aminoantipyrine to screen variants of Atu1420. The structure of Atu1420 variants was predicted by alphaFold, and the potential mechanism for the improvement of catalytic efficiency of variants was analyzed. Result The V_max of Atu1420 was determined to be 33.5±1.6 nmol/(L·s), the K_m is 82.7±3.5 μmol/L, the k_cat is (6.7±0.3)×10^-1 s^-1, k_cat/K_m is 8.1×10^-3 L/(μmol·s), the optimal pH is between 7 and 8, and the optimal temperature is 30°C. Five variants with enhanced enzyme activity were screened out from the variants obtained by site-directed mutagenesis. The variant with the strongest activity is G35S, and its catalytic activity is 66.0% higher than that of the wild type. Combined mutations at these five sites did not produce a significant additive effect on enzyme activity. Comparative analysis of the structures indicates that the deflection of the arginine residue at position 121 may be the reason for the improvement of the catalytic efficiency of the variants. Conclusion The enzymatic properties of Atu1420 have been characterized, and five Atu1420 variants with improved catalytic efficiency have been obtained. The increase in the catalytic efficiency of the variants may be caused by the deflection of the arginine residue at position 121.

Key words: O-demethylase, enzymatic properties, catalytic efficiency, protocatechuic acid, vanillic acid

WANG Hao, CAO An-ni, GAO Xin-yi, GUO Min-liang. Enzymatic Characterization and Directed Evolution of Agrobacterium tumefaciens O-demethylase Atu1420[J]. Biotechnology Bulletin, 2025, 41(3): 319-329.

Figures/Tables 6

Fig. 1 Identification of Atu1420 expression and catalytic activityA: Schematic diagram of the reaction catalyzed by Atu1420. B: SDS-PAGE electrophoretogram of Atu1420. C: Detecting peak of Atu1420 reaction mixture via HPLC. The blue curve inidicates the reaction mixture without Atu1420, and the red curve indicates the reaction mixture with Atu1420 added. D: Enzyme kinetics curve of Atu1420. The Vmax and Km values are obtained through the double reciprocal plot method

Fig. 2 Effects of pH and temperature on the activities of Atu1420

Fig. 3 Establishment of the detection method for VA using 4-aminoantipyrineA: Schematic diagram of the reaction between 4-aminoantipyrine and VA. B: Colors in the reaction between 4-aminoantipyrine and VA of different concentrations. C: HPLC analysis of VA metabolism by A. tumefaciens. CK indicates the untreated control, while the other samples were treated with A. tumefaciens for 6 h. Curves shows the remaining VA levels. D: Detection of VA degradation by A. tumefaciens C58 and ∆atu1420 using the 4-aminoantipyrine method

Fig. 4 Semi-rational design and screening of variants of Atu1420A: Molecular docking of VA with Atu1420. The green molecule refers to Atu1420, with four amino acid residues involved in VA binding and catalysis shown in stick form, where blue indicates nitrogen atoms. The cyan molecule indicates VA, and red indicates oxygen atoms. B: Sequence alignment of Atu1420 homologous proteins. 1 is the Atu1420 homologous protein from Microbacterium trichothecenolyticum, 2 from Sinomonas atrocyanea, 3 from Citricoccus sp. K5, 4 from Sphingobium sp., 5 from Rhizobium leguminosarum, 6 from Betaproteobacteria bacterium, and 7 is Atu1420 itself. Blue triangular arrows indicate the four key amino acid residues from Fig. A, while purple asterisks indicate amino acid residues spatially adjacent to these four key residues. Numbers indicate amino acid positions in Atu1420. C: Catalytic activity of Atu1420 and its variants in the bacteria. CK indicates the control without adding A. tumefaciens, while the other samples were treated with A. tumefaciens for 6 h. D: Catalytic activity of Atu1420 and its variants using proteins. CK indicates the control without adding protein, while the other samples were treated with the protein for 30 min. E: Catalytic activity of multi-points combined variants in the bacteria. CK indicates the control without bacteria, while the other samples were treated with A. tumefaciens for 6 h. F: Catalytic activity of multi-points combined variants using proteins. CK indicates the control without adding protein, while the other samples were treated with the protein for 30 min

Fig. 5 Enzyme kinetic curve of G35S

Fig. 6 Structural analysis of Atu1420 mutated variantsThe green carbon skeleton indicate the four key amino acid residues of Atu1420, and the carbon skeletons with other colors indicate the four key amino acid residues of other variants. The cyan carbon skeleton molecule indicates VA

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