Dataset Information

Protein and ligand dynamics in 4-hydroxybenzoate hydroxylase.

ABSTRACT: para-Hydroxybenzoate hydroxylase catalyzes a two-step reaction that demands precise control of solvent access to the catalytic site. The first step of the reaction, reduction of flavin by NADPH, requires access to solvent. The second step, oxygenation of reduced flavin to a flavin C4a-hydroperoxide that transfers the hydroxyl group to the substrate, requires that solvent be excluded to prevent breakdown of the hydroperoxide to oxidized flavin and hydrogen peroxide. These conflicting requirements are met by the coordination of multiple movements involving the protein, the two cofactors, and the substrate. Here, using the R220Q mutant form of para-hydroxybenzoate hydroxylase, we show that in the absence of substrate, the large beta alpha beta domain (residues 1-180) and the smaller sheet domain (residues 180-270) separate slightly, and the flavin swings out to a more exposed position to open an aqueous channel from the solvent to the protein interior. Substrate entry occurs by first binding at a surface site and then sliding into the protein interior. In our study of this mutant, the structure of the complex with pyridine nucleotide was obtained. This cofactor binds in an extended conformation at the enzyme surface in a groove that crosses the binding site of FAD. We postulate that for stereospecific reduction, the flavin swings to an out position and NADPH assumes a folded conformation that brings its nicotinamide moiety into close contact with the isoalloxazine moiety of the flavin. This work clearly shows how complex dynamics can play a central role in catalysis by enzymes.

SUBMITTER: Wang J

PROVIDER: S-EPMC117353 | biostudies-literature | 2002 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Protein and ligand dynamics in 4-hydroxybenzoate hydroxylase.

Wang Jian J Ortiz-Maldonado Mariliz M Entsch Barrie B Massey Vincent V Ballou David D Gatti Domenico L DL

Proceedings of the National Academy of Sciences of the United States of America 20020101 2

para-Hydroxybenzoate hydroxylase catalyzes a two-step reaction that demands precise control of solvent access to the catalytic site. The first step of the reaction, reduction of flavin by NADPH, requires access to solvent. The second step, oxygenation of reduced flavin to a flavin C4a-hydroperoxide that transfers the hydroxyl group to the substrate, requires that solvent be excluded to prevent breakdown of the hydroperoxide to oxidized flavin and hydrogen peroxide. These conflicting requirements ...[more]

PMID: 11805318

Similar Datasets

Project description:BackgroundMicrobial production of aromatic chemicals is an attractive method for obtaining high-performance materials from biomass resources. A non-proteinogenic amino acid, 4-amino-3-hydroxybenzoic acid (4,3-AHBA), is expected to be a precursor of highly functional polybenzoxazole polymers; however, methods for its microbial production have not been reported. In this study, we attempted to produce 4,3-AHBA from glucose by introducing 3-hydroxylation of 4-aminobenzoic acid (4-ABA) into the metabolic pathway of an industrially relevant bacterium, Corynebacterium glutamicum.ResultsSix different 4-hydroxybenzoate 3-hydroxylases (PHBHs) were heterologously expressed in C. glutamicum strains, which were then screened for the production of 4,3-AHBA by culturing with glucose as a carbon source. The highest concentration of 4,3-AHBA was detected in the strain expressing PHBH from Caulobacter vibrioides (CvPHBH). A combination of site-directed mutagenesis in the active site and random mutagenesis via laccase-mediated colorimetric assay allowed us to obtain CvPHBH mutants that enhanced 4,3-AHBA productivity under deep-well plate culture conditions. The recombinant C. glutamicum strain expressing CvPHBHM106A/T294S and having an enhanced 4-ABA biosynthetic pathway produced 13.5 g/L (88 mM) 4,3-AHBA and 0.059 g/L (0.43 mM) precursor 4-ABA in fed-batch culture using a nutrient-rich medium. The culture of this strain in the chemically defined CGXII medium yielded 9.8 C-mol% of 4,3-AHBA from glucose, corresponding to 12.8% of the theoretical maximum yield (76.8 C-mol%) calculated using a genome-scale metabolic model of C. glutamicum.ConclusionsIdentification of PHBH mutants that could efficiently catalyze the 3-hydroxylation of 4-ABA in C. glutamicum allowed us to construct an artificial biosynthetic pathway capable of producing 4,3-AHBA on a gram-scale using glucose as the carbon source. These findings will contribute to a better understanding of enzyme-catalyzed regioselective hydroxylation of aromatic chemicals and to the diversification of biomass-derived precursors for high-performance materials.

Dataset Information

Protein and ligand dynamics in 4-hydroxybenzoate hydroxylase.

Publications

Protein and ligand dynamics in 4-hydroxybenzoate hydroxylase.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets