Engineering of Recombinant Poplar Deoxy-D-Xylulose-5-Phosphate Synthase (PtDXS) by Site-Directed Mutagenesis Improves Its Activity

• Published in: PloS one Vol. 11; no. 8; p. e0161534
• Main Authors: Banerjee, Aparajita, Preiser, Alyssa L, Sharkey, Thomas D
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 22-08-2016; Public Library of Science (PLoS)
• Subjects: Alanine; Alanine - metabolism; Amino Acid Sequence; Anchoring; Antifungal agents; Binding; Biochemistry; Biology and Life Sciences; Biosynthesis; Biotechnology; Carotenoids; Catalytic Domain; E coli; Engineering; Engineering and Technology; Enzymatic activity; Enzyme Inhibitors - chemistry; Enzymes; Escherichia coli; Feedback; Feedback inhibition; Feedback, Physiological; Gene Expression; Genetic aspects; Glycine; Glycine - metabolism; Hemiterpenes - metabolism; Inhibitors; Isopentenyl diphosphate; Kinetics; Ligands; Metabolic regulation; Metabolism; Models, Molecular; Molecular biology; Molecular chains; Mutagenesis; Mutagenesis, Site-Directed; Mutation; Organophosphorus Compounds - metabolism; Phosphates; Physical Sciences; Physiological aspects; Plant Proteins - genetics; Plant Proteins - metabolism; Plasmids; Poplar; Populus; Populus - enzymology; Populus - genetics; Populus trichocarpa; Protein Binding; Protein Engineering; Protein Interaction Domains and Motifs; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Research and Analysis Methods; Residues; Sequence Alignment; Sequence Homology, Amino Acid; Site-directed mutagenesis; Substrate Specificity; Thiamine; Thiamine diphosphate; Thiamine Pyrophosphate - metabolism; Transferases - antagonists & inhibitors; Transferases - genetics; Transferases - metabolism; Xylulose; United States > US; East Lansing Michigan; Michigan
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0161534

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.