Figure 3. Structure-guided engineering of HmaS to reduce byproduct formation. (IMAGE)
Caption
a. Schematic representation of the structure-guided engineering strategy. Mutation of HmaS at S201F was designed to block the formation of unwanted aromatic byproducts such as (S)-mandelate (S-MA) and (S)-4-hydroxymandelate (S-HMA) by steric hindrance. b. SDS‒PAGE analysis of wild-type HmaS and the S201F variant. Protein bands corresponding to each HmaS variant (35–40 kDa) are indicated by asterisks. c. HPLC analysis of byproduct formation from L-phenylalanine in whole-cell systems expressing either wild-type HmaS or the S201F variant. d. Molecular docking of 2-keto-4-methyl-pentanoate (2-KMP) into the active site of the HmaS (S201F) variant. The docking results show that the S201F mutation does not impair substrate binding to 2-KMP, as indicated by comparable binding poses and docking scores. e. Bioconversion of L-leucine to S-HIV by the HmaS (S201F)-expressing strain.
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