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 DERAPsy, 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 DERAPsy 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 DERAPsy mutant (G189R) exhibited higher catalytic activity toward DR5P than DERAPsy. Only the DERAPsy mutant (V66K) exhibited 12% higher activity toward chloroacetaldehyde and acetaldehyde condensation reactions than DERAPsy. 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.
Keywords: 2-deoxy-d-ribose-5-phosphate aldolase; Binding free energy; Catalytic activity; Molecular simulation; Pseudomonas syringae pv. Syringae B728a.
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