Rational design to enhance the catalytic activity of 2-deoxy-D-ribose-5-phosphate aldolase from Pseudomonas syringae pv. syringae B728a.

Autor: He FF; College of Life Sciences and Medicine, Zhejiang Sci-Tech University, 310018, China; Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, Zhejiang Sci-Tech University, Hangzhou, 310018, China., Xin YY; College of Life Sciences and Medicine, Zhejiang Sci-Tech University, 310018, China., Ma YX; College of Life Sciences and Medicine, Zhejiang Sci-Tech University, 310018, China; Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, Zhejiang Sci-Tech University, Hangzhou, 310018, China., Yang S; College of Life Sciences and Medicine, Zhejiang Sci-Tech University, 310018, China. Electronic address: yangswm@zstu.edu.cn., Fei H; College of Life Sciences and Medicine, Zhejiang Sci-Tech University, 310018, China; Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, Zhejiang Sci-Tech University, Hangzhou, 310018, China. Electronic address: feihui@zju.edu.cn.
Jazyk: angličtina
Zdroj: Protein expression and purification [Protein Expr Purif] 2021 Jul; Vol. 183, pp. 105863. Date of Electronic Publication: 2021 Mar 05.
DOI: 10.1016/j.pep.2021.105863
Abstrakt: The 2-Deoxy-d-ribose-5-phosphate aldolase (DERA) enzyme in psychrophilic bacteria has gradually attracted the attention of researchers. A novel gene, deoC (681 bp), encoding DERA Psy , was identified in Pseudomonas syringae pv. syringae B728a, recombinantly expressed in E. coli BL21 and purified via affinity chromatography, which yielded a homodimeric enzyme of 23 kDa. The specific activity of DERA Psy toward 2-deoxy-d-ribose-5-phosphate (DR5P) was 7.37 ± 0.03 U/mg, and 61.32% of its initial activity remained after incubation in 300 mM acetaldehyde at 25 °C for 2 h. Based on the calculation results (dock binding free energy) with the ligand chloroacetaldehyde (CAH), five target substitutions (T16L, F69R, V66K, S188V, and G189R) were identified, in which the DERA Psy mutant (G189R) exhibited higher catalytic activity toward DR5P than DERA Psy . Only the DERA Psy mutant (V66K) exhibited 12% higher activity toward chloroacetaldehyde and acetaldehyde condensation reactions than DERA Psy . Fortunately, the aldehyde tolerance of these mutants exhibited no significant decline compared with the wild type. These results indicate an effective strategy for enhancing DERA activity.
(Copyright © 2021 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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