Enhanced Catalytic Efficiency of L‐amino Acid Deaminase Achieved by a Shorter Hydride Transfer Distance

Wu, Yaoyun; Zhang, Sheng; Song, Wei; Liu, Jia; Chen, Xiulai; Hu, Guipeng; Zhou, Yiwen; Li, Liming; Wu, Jing

doi:10.1002/cctc.202101067

Cited by 8 publications

(21 citation statements)

References 51 publications

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“…Consequently, the catalytic ability of PmiLAAD was enhanced from a new perspective of the substrate spectrum, rather than by merely increasing the enzyme activity. The variants with broader substrate specificity obtained in our research have promising application prospects for the synthesis of various keto acids [28]. Additionally, the variants of PmiLAAD have potential in the production of different kinds of D-amino acids when constructing multi-enzyme systems with other catalysts such as D-amino acid dehydrogenase [19,20], ammonia lyase [18], and D-amino acid transferase [21].…”

Section: Site-directed Saturation Mutagenesis At Potential Key Residuesmentioning

confidence: 77%

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Semi-Rational Design of Proteus mirabilis l-Amino Acid Deaminase for Expanding Its Substrate Specificity in α-Keto Acid Synthesis from l-Amino Acids

Fan

Wang

et al. 2022

Catalysts

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l-amino acid deaminases (LAADs) are flavoenzymes that catalyze the stereospecific oxidative deamination of l-amino acids into α-keto acids, which are widely used in the pharmaceutical, food, chemical, and cosmetic industries. However, the substrate specificity of available LAADs is limited, and most substrates are concentrated on several bulky or basic l-amino acids. In this study, we employed a LAAD from Proteus mirabilis (PmiLAAD) and broadened its substrate specificity using a semi-rational design strategy. Molecular docking and alanine scanning identified F96, Q278, and E417 as key residues around the substrate-binding pocket of PmiLAAD. Site-directed saturation mutagenesis identified E417 as the key site for substrate specificity expansion. Expansion of the substrate channel with mutations of E417 (E417L, E417A) improved activity toward the bulky substrate l-Trp, and mutation of E417 to basic amino acids (E417K, E417H, E417R) enhanced the universal activity toward various l-amino acid substrates. The variant PmiLAADE417K showed remarkable catalytic activity improvement on seven substrates (l-Ala, l-Asp, l-Ile, l-Leu, l-Phe, l-Trp, and l-Val). The catalytic efficiency improvement obtained by E417 mutation may be attributed to the expansion of the entrance channel and its electrostatic interactions. These PmiLAAD variants with a broadened substrate spectrum can extend the application potential of LAADs.

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Section: Site-directed Saturation Mutagenesis At Potential Key Residuesmentioning

confidence: 77%

“…The structural analysis showed that residue E417 is located in the loop structure of the substrate entrance channel of PmiLAAD (Figure S2), and is thus expected to play an important role in substrate entry and product release [28].…”

Section: Structural Analysis Of E417 and Its Mutationsmentioning

confidence: 99%

Semi-Rational Design of Proteus mirabilis l-Amino Acid Deaminase for Expanding Its Substrate Specificity in α-Keto Acid Synthesis from l-Amino Acids

Fan

Wang

et al. 2022

Catalysts

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“…The N1 and N3 atoms of FAD play vital roles in transferring electrons and protons, which are crucial for catalyzing the desaturation of 10-hydroxydecanoyl coenzyme A. 33 Therefore, we calculated the minimum catalytic distance between the N1 atom on FAD and the desaturation site of the ligand (C2 and C3 atoms). The average distance was 2.9 Å in ACOX (Supporting Information, Figure S3A) and over 5.6 Å in FadE (Supporting Information, Figure S3B), making 10-hydroxydecanoyl coenzyme A desaturation unfavorable.…”

Section: Acox-catalyzed Desaturation Of 10-hydroxydecanoyl Coenzyme Amentioning

confidence: 99%

Biosynthesis of 10-Hydroxy-2-decenoic Acid through a One-Step Whole-Cell Catalysis

Fang,

Xu,

Yang

et al. 2024

J. Agric. Food Chem.

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10-Hydroxy-2-decenoic acid (10-HDA) is an important component of royal jelly, known for its antimicrobial, antiinflammatory, blood pressure-lowering, and antiradiation effects. Currently, 10-HDA biosynthesis is limited by the substrate selectivity of acyl-coenzyme A dehydrogenase, which restricts the technique to a two-step process. This study aimed to develop an efficient and simplified method for synthesizing 10-HDA. In this study, ACOX from Candida tropicalis 1798, which catalyzes 10hydroxydecanoyl coenzyme A desaturation for 10-HDA synthesis, was isolated and heterologously coexpressed with FadE, Macs, YdiI, and CYP in Escherichia coli/SK after knocking out FadB, FadJ, and FadR genes. The engineered E. coli/AKS strain achieved a 49.8% conversion of decanoic acid to 10-HDA. CYP expression was improved through ultraviolet mutagenesis and high-throughput screening, increased substrate conversion to 75.6%, and the synthesis of 10-HDA was increased to 0.628 g/L in 10 h. This is the highest conversion rate and product concentration achieved in the shortest time to date. This study provides a simple and efficient method for 10-HDA biosynthesis and offers an effective method for developing strains with high product yields.

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“…S1a). A previously reported highly active LAAD mutant from Proteus mirabilis (PmLAAD M2 , H295S/V437S) (Wu et al 2021) and phenylpyruvate reductase from Lactobacillus sp. CGMCC 9967 (LaPPR) (Xu et al 2016) were selected based on its speci c activity (Additional le 1: Table S3-S4); and the FDH from Candida boidinii (CbFDH)was used to regenerate NADH.…”

Section: Design Of a Tri-enzymatic Cascade Pathway To Synthesize Saa ...mentioning

confidence: 99%

Efficient production of salvianic acid A from L-dihydroxyphenylalanine through a tri-enzyme cascade

Yang

Wei

Gao

et al. 2023

Preprint

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Salvianic acid A (SAA), used for treating cardiovascular and cerebrovascular diseases, possesses several pharmacological properties. However, the current methods for the enzymatic synthesis of SAA show low efficiency. Here, we constructed a three-enzyme cascade pathway in Escherichia coli BL21(DE3) to produce SAA from L-dihydroxyphenylalanine (L-DOPA). The phenylpyruvate reductase (LaPPR) from Lactobacillus sp. CGMCC 9967 is a rate-limiting enzyme in this process. Therefore, we employed a mechanism-guided protein engineering strategy to shorten the transfer distances of protons and hydrides, generating an optimal LaPPR mutant, LaPPRMu2 (H89M/H143D/P256C), with a 2.8-fold increase in specific activity and 9.3-time increase in kcat/Km value compared to that of the wild type. Introduction of the mutant LaPPRMu2 into the cascade pathway and the optimization of enzyme levels and transformation conditions allowed the obtainment of the highest SAA titer (82.55 g L− 1) ever reported in vivo, good conversion rate (91.3%), excellent ee value (99%) and the highest productivity (6.88 g L− 1 h− 1) from 90 g L− 1 L-DOPA in 12 h. This successful strategy provides a potential new method for the industrial production of SAA.

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Enhanced Catalytic Efficiency of L‐amino Acid Deaminase Achieved by a Shorter Hydride Transfer Distance

Cited by 8 publications

References 51 publications

Semi-Rational Design of Proteus mirabilis l-Amino Acid Deaminase for Expanding Its Substrate Specificity in α-Keto Acid Synthesis from l-Amino Acids

Semi-Rational Design of Proteus mirabilis l-Amino Acid Deaminase for Expanding Its Substrate Specificity in α-Keto Acid Synthesis from l-Amino Acids

Biosynthesis of 10-Hydroxy-2-decenoic Acid through a One-Step Whole-Cell Catalysis

Efficient production of salvianic acid A from L-dihydroxyphenylalanine through a tri-enzyme cascade

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