Aug 2026· RSC Chemical Biology· 0 citations· 24 references
Medicine
TL;DR
R reverse screening as an effective approach for evolving transaminases toward sterically hindered substrates is 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.
Abstract
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.
The direct asymmetric synthesis of chiral amides remains a focal point in green manufacturing, particularly for (S)-2-aminobutyramide whose traditional production is often hampered by low atom economy and arduous chiral resolution steps. Here, we report the creation of a non-native bio-amidation activity in Escherichia coli asparagine synthetase A (AsnA) for the proof-of-concept one-step biosynthesis of (S)-2-aminobutyramide, a critical intermediate for anti-epileptic drugs. To overcome the bottleneck of high-throughput screening, we engineered the AmiC–AmiR transcription factor into a highly sensitive biosensor (RC-4-Y83H) by remodeling its ligand-binding pocket, achieving a 23-fold induction response specifically toward (S)-2-AB. Utilizing this biosensor to screen saturation mutagenesis libraries, we identified an evolved variant (K77L/E120L/R255W) that showed a reversed substrate preference between the two substrates tested, converting (S)-2-aminobutyric acid to (S)-2-AB with retention of configuration. Molecular dynamics simulations and free-energy landscape analysis reveal that the mutations fundamentally remodel the active site, inducing a 180° flip of the substrate binding mode. This structural reorientation positions the non-native carboxyl group in a catalytically productive geometry relative to ATP, a conformation inaccessible in the wild-type enzyme. As a proof of concept, this work does not aim to deliver an industrially competitive biocatalyst; instead, it shows that the specificity of a native amide-forming enzyme can be reprogrammed toward a non-native alpha-amino acid, and it provides a generalizable, biosensor-guided framework for such reprogramming.
Qianpeng Lai, Yiwei Meng, Zhongmei Liu et al.· Chemical Science· 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
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
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.
Elijah Karvelis, Bruce Tidor· Journal of Physical Chemistr...· 0 citations
The extensive use of polyethylene terephthalate (PET) has resulted in severe environmental pollution and ecological stress. Despite advances in PET recycling, current processes struggle to achieve high product value, as the complete conversion to terephthalic acid remains energetically demanding and economically inefficient. The catalytic promiscuity inherent in natural enzyme evolution holds great promise for providing novel candidates to accelerate PET biodegradation and upcycling. Herein, we report for the first time that the acyltransferase metA from Mycobacterium tuberculosis (MtMetA) catalyzes the conversion of bis(2-hydroxyethyl) terephthalate (BHET), an intermediate of PET hydrolysis, into the high-value monomer mono(2-hydroxyethyl) terephthalate (MHET). Guided by molecular dynamics (MD) simulations, we applied a catalytic barrier-minimization strategy to optimize the active-site environment of MtMetA, yielding engineered variants, notably ΔBarrier2 and ΔBarrier3. Specifically, ΔBarrier2 achieved a 3.1-fold increase in MHET yield, while ΔBarrier3 demonstrated a 3.2-fold enhancement in catalytic efficiency (kcat/KM) relative to the wild-type. The truncated MtMetA variants also exhibited enhanced robustness, showing improved thermostability (3.5-fold increase in residual activity at 60 °C for ΔBarrier3), as well as higher tolerance to metal ions and organic solvents. Specifically, ΔBarrier2 displayed a 7.2-fold increase in product yield in Ca2+-containing systems, while ΔBarrier3 retained 2.3-fold higher residual activity in the presence of 50 % (v/v) isopropanol. MD simulations revealed that an enlarged active pocket and a shortened nucleophilic attack distance synergistically govern the enhanced catalytic activity and robustness. This work expands the enzymatic toolbox for PET recycling and targeted BHET degradation, advancing sustainable plastic waste management through biocatalytic innovations.
Jie Qiao, Yibo Song, Nan Zhao et al.· Bioresource Technology· 0 citations
The meta-selective C–H functionalization of aromatic substrates lacking meta-directing electron-withdrawing groups remains challenging. To engineer the regioselective enzymatic conversion of biphenyl (BP) to 3,3′-dihydroxybiphenyl (33DHBP), an industrial precursor to high-performance polymers, substrate positioning within the active site must be precisely controlled. Here, we report the regioselective catalytic promiscuity engineering of toluene/o-xylene monooxygenase (ToMO) to achieve sequential, meta-selective hydroxylation of the non-natural biaryl BP, where distinct ToMO variants were engineered to catalyze meta-selective hydroxylation of BP, 3-hydroxybiphenyl (3HBP), or both. Variants to catalyze the meta-selective hydroxylation of BP were designed using an automated docking workflow, with predicted binding poses consistent with the observed regioselectivity; this resulted in the development of the I100V-E103V-F205G variant that hydroxylated BP with 100% apparent meta-selectivity. Additional substitutions, especially L268A and L402A, were introduced to widen the long hydrophobic active-site access channel, further improving meta-selective BP hydroxylation in Pseudomonas putida. Variants to catalyze the meta-selective hydroxylation of 3HBP were selected through a structure-based residue scan of 437 active-site substitutions, resulting in the identification of the I100V-E103V-F176H variant that hydroxylated 3HBP with over 90% apparent meta-selectivity and no activity toward BP. In addition, the combined I100V-E103V-I162Y-F205G variant hydroxylated 3HBP with over 90% apparent meta-selectivity and improved hydroxylation of BP. This study demonstrates structural and computational monooxygenase engineering for the regioselective hydroxylation of non-natural biaryls, enabling the production of 33DHBP as a valuable precursor to specialized polymers.
C. Vavricka, Takeshi Matsui, Satoshi Yuzawa et al.· ACS Catalysis· 0 citations
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