Jul 2026· Journal of Physical Chemistry B· Vol 130, pp. 7562 - 7577· 0 citations· 51 references
Medicine
Abstract
The specificity and catalytic efficiency of enzymes make them attractive for applications ranging from therapeutics to chemical manufacturing. However, it remains challenging to identify specific structural and dynamic mechanisms by which enzymes achieve their catalytic rate enhancements as well as to re-engineer enzymes to improve their catalytic properties. In earlier work we reported the design and selection of KARI mutants with calculated increases in specific activity (i.e., k cat) relative to wild type (WT) for the isomerization step of one of its native substrates: 2-acetolactate (ACL), which leads to the synthesis of the amino acids valine and leucine. Eight mutants were identified with computed improvements in k cat of up to 4 orders of magnitude. In the current study, we investigate the effects of these same mutations on the isomerization of the other native substrate, 2-aceto-2-hydroxybutyrate (AHB, leading to the synthesis of isoleucine). Paralleling our previous work, we use the computational statistical mechanical method transition interface sampling (TIS) to simulate reaction kinetics and compute reaction rate constants. We find that the mutants selected for increased efficiency on ACL had varied levels of activity on AHB–some enhancing reactivity and others diminishing it–with the range in computed AHB rate constants spanning more than 7 orders of magnitude. Analysis of the simulations for WT-AHB revealed that only some of the structural mechanisms associated with mutants’ improved ACL catalysis were expected to transfer to, and thereby improve, AHB catalysis. For two mutants with significantly lower catalytic efficiency on AHB than WT, further analysis identified unique conformational changes that may explain their low activity on AHB.
Glucose dehydrogenase (GDH) is a key enzyme for NAD(P)H cofactor regeneration in industrial biocatalysis. However, conventional engineering approaches are frequently constrained by limited catalytic efficiency and excessive enzyme loading, which collectively compromise process economics and hinder large-scale deployment. In this study, we performed structure-guided rational design by targeting three functionally discrete domains of GDH, including the substrate-binding region, the cofactor-binding pocket, and the interdomain communication interface. An engineered variant GDH-M6 was constructed, which manifests a 35-fold enhancement in catalytic efficiency relative to the wild-type enzyme. In the biocatalytic synthesis of the pivotal chiral intermediate for R-lipoic acid, GDH-M6 reduced enzyme loading by more than 90% and allowed a doubling of the substrate concentration. As a result, overall reaction productivity was substantially increased and the GDH-M6 outperformed wild-type GDH as well as all previously reported mutants under comparable conditions. Notably, the domain-engineering paradigm established herein provides a broadly applicable toolkit for augmenting the catalytic performance of dehydrogenases, and offers a structural blueprint for resolving analogous kinetic bottlenecks that commonly arise in NAD(P)⁺-dependent enzymes utilized for industrial cofactor regeneration.
Min Cao, Xin Hao, Mingjian Zhu et al.· Enzyme and Microbial Technol...· 0 citations
Aspartase from Escherichia coli (AspA) catalyzes the direct conversion of acrylic acid to β-alanine; however, its substrate specificity and low catalytic efficiency limit its broader application. We engineered an AspA mutant capable of efficiently catalyzing the amination of acrylic acid for β-alanine synthesis, using Rosetta Enzyme Design to computationally redesign the Cβ-binding region of the acrylic acid binding site in AspA. Based on energy scores, structural configurations, and hydrogen bonding networks, 51 candidate variants with penalty scores below 30 were selected for mutant construction and performance testing; >70% of these variants exhibited enhanced catalytic activity in acrylic acid’s hydrogen amination. Four mutants achieved over 3-fold improved activity. The optimal mutant, M1 (T190I-M324I-K327L-N329C), demonstrated a 7.3-fold increased specific enzyme activity and a 13.0-fold improved kcat/Km compared with the wild type. Conformational changes in the S-loop and enhanced hydrophobic interactions near the active site contributed significantly to M1’s enhanced activity. Upon reaction optimization, the conversion of β-alanine synthesis using M1 in whole-cell catalysis increased from 5% with the wild type to 90% with M1. This study provides a reference for the biocatalytic synthesis of β-alanine, significantly enhancing the conversion of acrylic acid and demonstrating the enzyme’s potential for industrial applications.
Long-Xian Li, Baodi Ma, Yi Xu· Catalysts· 0 citations
We report the discovery and engineering of a new (R)-selective transaminase (RTA-223, UniProt W9Z089), identified from Capronia coronata. The wild-type enzyme was found to display broad activity on a range of bulky aryl and alkyl ketone substrates, with high enantioselectivity using both d-alanine and isopropyl amine as amine donors. We then investigated activity towards pro-sitagliptin ketone, a well-known challenging pharmaceutical target of industrial significance. Although no forward amination of pro-sitagliptin was initially detected by wild-type RTA-223, low-level activity in the reverse deamination reaction enabled engineering without the need for truncated sitagliptin analogues. Adopting this reverse screening strategy, two active-site mutations unlocked forward amination activity, and subsequent rounds of directed evolution delivered a quadruple mutant (H53L/V60G/F113A/V148A) capable of converting pro-sitagliptin to (R)-sitagliptin with high levels of stereoselectivity (e.r. 93 : 7). We further performed a kinetic analysis of the candidates from across the evolution process, using a previously reported coupled assay system linked to d-alanine oxidation. This work validates reverse screening as an effective approach for evolving transaminases toward sterically hindered substrates and highlights RTA-223 as a promising candidate for further biocatalyst development.
Matthew Treadell, Gareth R E Surman, G. Ford et al.· RSC Chemical Biology· 0 citations
The rational engineering of an α-L-rhamnosidase (DthRha) to address limitations and enhance its performance for flavonoid production highlights the R783A mutant as a robust and thermally stable biocatalyst with great potential for the sustainable production of bioactive flavonoids.
Haoyu Jia, Luran Wang, Tong Yan et al.· International Journal of Bio...· 0 citations
Fe(II)/α-ketoglutarate (αKG)-dependent halogenases that catalyze site-selective C-H halogenation of free substrates without carrier proteins are attractive biocatalysts for diversifying pharmaceuticals and agrochemicals. However, their application remains limited by the narrow diversity of natural halogenases, poor stability, and restricted substrate scope. Protein stabilization is a common strategy to enhance mutational tolerance during enzyme engineering; however, Fe(II)/αKG-dependent halogenases are structurally closely related to hydroxylases, and consensus-based stabilizing mutations risk shifting activity toward competing hydroxylation. To address this challenge, a workflow was designed to improve structural stability while preserving substrate and product specificity by combining computational identification of substrate-recognition residues with Rosetta-based stabilization. This approach was applied to the l-lysine 4-chlorinase BesD from Streptomyces cattleya as a model enzyme. The resulting variants exhibited a T50 increase of more than 45 °C with no loss of substrate specificity or regioselective chlorination activity and served as stable seed enzymes for subsequent substrate scope expansion. This strategy, which systematically excludes substrate recognition- and/or reaction-selectively related residues from the mutation space to preserve native enzyme function, may provide a versatile platform for stabilizing enzymes without substantially compromising catalytic activity.
Teppei Niide, Keita Miyawaki, Hyuga Miyamoto et al.· ACS Chemical Biology· 0 citations
Bioreduction of prochiral
N
-(3-oxobutyl)- and
N
-(2-oxopropyl)heterocycles—comprising (partially) saturated, flexible rings—was explored, using microbial whole-cell ketoreductases, including selected wild-type yeast strains and enantiocomplementary recombinant alcohol dehydrogenases (ADHs). Initial screening reactions with yeast strains resulted in (
S
)-alcohols with low to moderate conversions. Optimization of the reaction conditions (substrate and cosubstrate concentration) of the yeast-catalyzed bioreductions was performed using design of experiments (DOE), resulting in significant increases of conversion for most substrate–yeast strain combinations. Thus, several substrates could be fully or almost fully converted to the corresponding enantiopure (
S
)-alcohols with wild-type yeasts. Extending the scope of biocatalysts to recombinant ADHs [two (
S
)-selective ADHs from
Candida parapsilosis
(CpADH) and
Rhodococcus aetherivorans
(RaADH) and an (
R
)-selective one from
Lactobacillus kefir
(LkADH)] resulted in further improvements. Of the investigated biocatalysts, CpADH proved to be the most versatile, converting the more challenging shorter sidechain
N
-(2-oxopropyl)heterocycles as well. The (
R
)-selective
Lactobacillus kefir
ADH provided enantiopure (
R
)-alcohols, enabling the efficient enantiocomplementary synthesis of the target alcohols. The preparative-scale bioreductions under optimal conditions usually showed superior conversions compared to the screening reactions, resulting in enantiopure (
S
)- and (
R
)-enantiomers (ee > 99%) of these synthetically valuable chiral fragments. Molecular docking studies with the substrates and the recombinant ADHs supported the observed stereoselectivity and confirmed the absolute configuration of the alcohol products. In summary, the present study provides efficient access to valuable drug-like enantiopure alcohols suitable for inclusion in chiral fragment-based drug discovery (FBDD) libraries.
Microbial whole-cell ketoreductases were investigated in bioreductions.
Optimization of the reaction conditions greatly increased conversions.
A docking study confirmed the enantiotopic selectivity of the ADHs.
Máté Gergő Honvári, Levente András Mócza, Máté Laurinyecz et al.· Applied Microbiology and Bio...· 0 citations