Abstract
Deoxyxylulose 5-phosphate synthase (DXS), a thiamine diphosphate (ThDP) dependent enzyme, plays a regulatory role in the methylerythritol 4-phosphate (MEP) pathway. Isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), the end products of this pathway, inhibit DXS by competing with ThDP. Feedback inhibition of DXS by IDP and DMADP constitutes a significant metabolic regulation of this pathway. The aim of this work was to experimentally test the effect of key residues of recombinant poplar DXS (PtDXS) in binding both ThDP and IDP. This work also described the engineering of PtDXS to improve the enzymatic activity by reducing its inhibition by IDP and DMADP. We have designed and tested modifications of PtDXS in an attempt to reduce inhibition by IDP. This could possibly be valuable by removing a feedback that limits the usefulness of the MEP pathway in biotechnological applications. Both ThDP and IDP use similar interactions for binding at the active site of the enzyme, however, ThDP being a larger molecule has more anchoring sites at the active site of the enzyme as compared to the inhibitors. A predicted enzyme structure was examined to find ligand-enzyme interactions, which are relatively more important for inhibitor-enzyme binding than ThDP-enzyme binding, followed by their modifications so that the binding of the inhibitors can be selectively affected compared to ThDP. Two alanine residues important for binding ThDP and the inhibitors were mutated to glycine. In two of the cases, both the IDP inhibition and the overall activity were increased. In another case, both the IDP inhibition and the overall activity were reduced. This provides proof of concept that it is possible to reduce the feedback from IDP on DXS activity.
Highlights
The 2-methyl-3-erythritol-4-phosphate (MEP) pathway is one of the biochemical pathways that lead to the biosynthesis of isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), the building blocks of isoprenoids [1,2,3,4,5]
Any mutations involving these polar interactions may affect the binding of thiamine diphosphate (ThDP) as well as IDP and would be unlikely to improve the kinetics of the enzyme
To select interactions essential for IDP binding, but not as important for the binding of ThDP we focused on those residues of the enzyme that are important for binding the carbon chain of IDP, reasoning that the diphosphate binding was likely to be similar between ThDP and IDP
Summary
The 2-methyl-3-erythritol-4-phosphate (MEP) pathway is one of the biochemical pathways that lead to the biosynthesis of isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), the building blocks of isoprenoids [1,2,3,4,5]. Natural sources of some isoprenoids is limited [7, 9, 12], the biotechnological production of commercially important isoprenoids has attracted widespread attention as a valuable industrial target [11]. Metabolic engineering of the MEP pathway has become a focus to improve the bio-production of downstream isoprenoids [13]. 1-Deoxy-D-xylulose-5-phosphate synthase (DXS), the first enzyme of the MEP pathway, has been one of the main targets for engineering. It has been shown that coexpression of DXS with other downstream enzymes improved the yield of taxadiene, lycopene, sesquiterpenes, various mono and diterpenes, carotenoids including β-carotene, and zeaxanthin in E. coli [15,16,17,18,19,20,21]
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