Dataset Information

Structure-guided steric hindrance engineering of Bacillus badius phenylalanine dehydrogenase for efficient L-homophenylalanine synthesis.

ABSTRACT:

Background

Direct reductive amination of prochiral 2-oxo-4-phenylbutyric acid (2-OPBA) catalyzed by phenylalanine dehydrogenase (PheDH) is highly attractive in the synthesis of the pharmaceutical chiral building block L-homophenylalanine (L-HPA) given that its sole expense is ammonia and that water is the only byproduct. Current issues in this field include a poor catalytic efficiency and a low substrate loading.

Results

In this study, we report a structure-guided steric hindrance engineering of PheDH from Bacillus badius to create an enhanced biocatalyst for efficient L-HPA synthesis. Mutagenesis libraries based on molecular docking, double-proximity filtering, and a degenerate codon significantly increased catalytic efficiency. Seven superior mutants were acquired, and the optimal triple-site mutant, V309G/L306V/V144G, showed a 12.7-fold higher k_cat value, and accordingly a 12.9-fold higher k_cat/K_m value, than that of the wild type. A paired reaction system comprising V309G/L306V/V144G and glucose dehydrogenase converted 1.08 M 2-OPBA to L-HPA in 210 min, and the specific space-time conversion was 30.9 mmol g^-1 L^-1 h^-1. The substrate loading and specific space-time conversion are the highest values to date. Docking simulation revealed increases in substrate-binding volume and additional degrees of freedom of the substrate 2-OPBA in the pocket. Tunnel analysis suggested the formation of new enzyme tunnels and the expansion of existing ones.

Conclusions

Overall, the results show that the mutant V309G/L306V/V144G has the potential for the industrial synthesis of L-HPA. The modified steric hindrance engineering approach can be a valuable addition to the current enzyme engineering toolbox.

SUBMITTER: Wu T

PROVIDER: S-EPMC8543943 | biostudies-literature | 2021 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Structure-guided steric hindrance engineering of Bacillus badius phenylalanine dehydrogenase for efficient L-homophenylalanine synthesis.

Wu Tao T Mu Xiaoqing X Xue Yuyan Y Xu Yan Y Nie Yao Y

Biotechnology for biofuels 20211024 1

<h4>Background</h4>Direct reductive amination of prochiral 2-oxo-4-phenylbutyric acid (2-OPBA) catalyzed by phenylalanine dehydrogenase (PheDH) is highly attractive in the synthesis of the pharmaceutical chiral building block L-homophenylalanine (L-HPA) given that its sole expense is ammonia and that water is the only byproduct. Current issues in this field include a poor catalytic efficiency and a low substrate loading.<h4>Results</h4>In this study, we report a structure-guided steric hindrance ...[more]

PMID: 34689801

Similar Datasets

Project description:BackgroundIn computational structural biology, structure comparison is fundamental for our understanding of proteins. Structure comparison is, e.g., algorithmically the starting point for computational studies of structural evolution and it guides our efforts to predict protein structures from their amino acid sequences. Most methods for structural alignment of protein structures optimize the distances between aligned and superimposed residue pairs, i.e., the distances traveled by the aligned and superimposed residues during linear interpolation. Considering such a linear interpolation, these methods do not differentiate if there is room for the interpolation, if it causes steric clashes, or more severely, if it changes the topology of the compared protein backbone curves.ResultsTo distinguish such cases, we analyze the linear interpolation between two aligned and superimposed backbones. We quantify the amount of steric clashes and find all self-intersections in a linear backbone interpolation. To determine if the self-intersections alter the protein's backbone curve significantly or not, we present a path-finding algorithm that checks if there exists a self-avoiding path in a neighborhood of the linear interpolation. A new path is constructed by altering the linear interpolation using a novel interpretation of Reidemeister moves from knot theory working on three-dimensional curves rather than on knot diagrams. Either the algorithm finds a self-avoiding path or it returns a smallest set of essential self-intersections. Each of these indicates a significant difference between the folds of the aligned protein structures. As expected, we find at least one essential self-intersection separating most unknotted structures from a knotted structure, and we find even larger motions in proteins connected by obstruction free linear interpolations. We also find examples of homologous proteins that are differently threaded, and we find many distinct folds connected by longer but simple deformations. TM-align is one of the most restrictive alignment programs. With standard parameters, it only aligns residues superimposed within 5 Ångström distance. We find 42165 topological obstructions between aligned parts in 142068 TM-alignments. Thus, this restrictive alignment procedure still allows topological dissimilarity of the aligned parts.ConclusionsBased on the data we conclude that our program ProteinAlignmentObstruction provides significant additional information to alignment scores based solely on distances between aligned and superimposed residue pairs.

Dataset Information

Structure-guided steric hindrance engineering of Bacillus badius phenylalanine dehydrogenase for efficient L-homophenylalanine synthesis.

Background

Results

Conclusions

Publications

Structure-guided steric hindrance engineering of Bacillus badius phenylalanine dehydrogenase for efficient L-homophenylalanine synthesis.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets