Abstract
2,4-Dichlorophenol hydroxylase (2,4-DCP hydroxylase) is a key enzyme in the degradation of 2,4-dichlorophenoxyacetic acid in the hydroxylation step in many bacteria. Our previous study demonstrated that a 2,4-DCP hydroxylase (TfdB-JLU) exhibits broad substrate specificity for chlorophenols (CPs) and their homologues. In this study, TfdB-JLU has been engineered by rational design to further broaden its substrate scope towards CPs. We dissect the architectures of enzymes from oxidoreductase families to discover their underlying structural sources of substrate promiscuity. A homology model of TfdB-JLU has been built and docking experiments of this homology model with its natural substrate 2,4-DCP reveal that the phenyl rings of 2,4-DCP form strong interactions with residues His47, Ile48, Trp222, Pro316, and Phe424. These residues are found to be important for substrate binding in the active site. Then, the site-directed mutagenesis strategy has been applied for redesigning substrate promiscuity in TfdB-JLU. The TfdB-JLU-P316Q variant obtained shows a significant enhancement of activity (up to 3.4-fold) toward 10 CP congeners compared to wild-type TfdB-JLU. Interestingly, the active improvements of TfdB-JLU-P316Q toward CP congeners show significant difference, especially for active improvements of positional congeners such as 3-CP (1.1-fold) compared to 4-CP (3.0-fold), as well as 2,3-DCP (1.2-fold) compared to 2,5-DCP (3.4-fold). Structural analysis results indicate that the improvement in substrate promiscuity of the variant enzyme compared to the wild-type enzyme is possibly due to the increase of non-bonding interaction. The results suggest that exploiting enzyme–substrate promiscuity is promising, which would provide a starting point for designing and engineering novel biological catalysts for pollution removal.
Highlights
Enzymes are attractive catalysts because of their promiscuity and their ability to perform highly regio, chemo- and stereoselective transformations
This study mainly aims to discover underlying structural basis of substrate promiscuity of TfdB-JLU toward CPs and to engineer TfdB-JLU variants with higher activity toward CPs
In order to engineer the promiscuity of the enzyme toward the CPs by rational protein design, it is mandatory to understand the mechanism of this reaction and to have a reliable structural model of this enzyme
Summary
Enzymes are attractive catalysts because of their promiscuity and their ability to perform highly regio-, chemo- and stereoselective transformations. Enzymes that display promiscuous behavior can be classi ed into reaction conditions of promiscuity, substrate promiscuity, catalytic promiscuity and alternate-site promiscuity.[1] Enzyme promiscuity is the engine of evolutionary innovation which can dramatically enhance the utility of biocatalysis in biotechnology. It has attracted signi cant attention from chemists and biochemists.[2]. Enzyme with substrate promiscuity displays several advantages since they can be used to transform substrates apart from the native
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