Structural and biochemical characterization of α/β-Hydrolase fold enzymes in biotin and menaquinone biosynthesis

Abstract

α/β-Hydrolase fold enzymes are extensively involved in natural product biosynthesis due to their powerful catalysis of diverse reactions. Biotin serve as an essential prosthetic cofactor for enzymatic carbon dioxide transfer reactions in all three domains of life and biotin biosynthesis generally involves an α/β-hydrolase fold enzyme, pimeloyl-acyl carrier protein (ACP) methyl estersase like BioH in E. coli, to cleave the methyl ester bond of pimeloyl-ACP methyl ester and release pimeloyl-ACP. Meanwhile, menaquinone and phylloquinone are important electron carriers in the respiratory system of bacteria and photosystem of algae and plants. The conversion from 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1carboxylate (SEPHCHC) to 2-succinyl-6-hydroxy-2,4-cyclohexaadiene-1carboxylate (SHCHC) in biosynthesis of both quinones engages an α/β-hydrolase fold enzyme, SHCHC synthase or MenH. Both α/β-Hydrolase fold enzymes are investigated in this thesis study. Firstly, the crystal structure of pimeloyl-ACP methyl estersase, BioG, from Haemophilus influenzae is determined at 1.26 Å resolution. The BioG structure is similar to the BioH structure and is composed of an α-helical lid domain and a core domain that contains a central seven-stranded β-pleated sheet. However, four of the six α-helices that flank both sides of the BioH core β-sheet are replaced with long loops in BioG, thus forming an unusual α/β-hydrolase fold. This structural variation results in a significantly decreased thermal stability of the enzyme, evidenced by both PISA calculation and thermal denaturation. Concomitantly the interface between the α-layer and β-sheet of BioG was rather diminished in comparison with BioH. The melting temperature of BioG was determined to be 50.4 ℃, significantly lower than that of BioH determined as 58.3 ℃. Nevertheless, the lid domain and the residues at the lid-core interface are well conserved between BioH and BioG, in which an analogous hydrophobic pocket for pimelate binding as well as similar ionic interactions with the ACP moiety is retained. Biochemical characterization of site-directed mutants of the residues hypothesized to interact with the ACP moiety supports a similar substrate interaction mode for the two enzymes. Consequently, these enzymes package the identical catalytic function under a considerably different protein surface. Secondly, the MenH M93P mutant from Escherichia coli is characterized by both HPLC and kinetic assays. The mutant protein retains its enzymatic activity with a relatively lower catalytic efficiency in comparison with the wild type MenH. Limited by the protein concentration or stability, the changes of the protein in secondary structures are unable to be unambiguously identified from the circular dichroism spectra. Based on these structural and kinetic analysis, the lethal effect of PHYLLO L1502P in apple is proposed to be due to the structural instability although the impaired activity might influence synergistically.

Date of Award	2017
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology