Molecular Evolution of an Aminotransferase Based on Substrate–Enzyme Binding Energy Analysis for Efficient Valienamine Synthesis

Abstract

Valienamine is a valuable building block for active pharmaceuticals and agrochemicals because of its aminocyclitol structure and glucosidase inhibitory activity. Straightforward amino chiral center construction on valienone using a sugar aminotransferase (SAT) to produce valienamine achieved strict stereo-specificity; however, low transamination activity owing to an unfavorable binding conformation of the non-natural substrate valienone in the oversized substrate-binding pocket currently limits SAT-based valienamine production. Here, we employed a tailored combinatorial active-site saturation test/iterative saturation mutagenesis (CAST/ISM) strategy to engineer a recently identified SAT (RffA_Kpn) to optimize the binding of valienone in the large binding pocket and thus improve transamination activity. In silico analyses predicting mutation binding energies and assessing evolutionary conservation identified 9 of 62 contact residues positioned within 8 Å of the valienone–PMP external aldimine transition state as hotspots for destabilizing unfavorable binding. Four of these residues were subsequently confirmed to improve activity using site-directed saturation mutagenesis, and combinatorial mutations were prepared in further iterative cycles. The quadruple mutant M4 (K209W/Y321F/V318Q/K25W) displayed a 35.59-fold improvement in valienamine synthesis activity over wild-type RffA_Kpn and achieved 12.18% conversion toward valienone in 100 mg preparative-scale reaction. Our demonstration of asymmetric biosynthesis for a valuable chiral aminocyclitol illustrates how an evolution model can help engineer enzymes that handle small, non-natural substrates in oversized binding pockets.

Sequence- and Structure-Based Mining of Thermostable D-Allulose 3-Epimerase and Computer-Guided Protein Engineering To Improve Enzyme Activity

“…If the predicted binding energy of a mutant is negative, then it indicates a higher affinity of binding between the protein and ligand, which may improve the enzymatic activity . The prediction result shows that 251 negative ΔΔ G mut values were widely distributed among the analyzed residues.…”

“…If the predicted binding energy of a mutant is negative, then it indicates a higher affinity of binding between the protein and ligand, which may improve the enzymatic activity. 48 The prediction result shows that 251 negative ΔΔG mut values were widely distributed among the analyzed residues. The average mutation binding energy ( G mut ) of each mutation site was calculated and compared to that of WT TtDAE to identify residues that may increase substrate-ligand affinity.…”

Rational Design of l-Threonine Transaldolase-Mediated System for Enhanced Florfenicol Intermediate Production

“…fluorescens LTTA (ObiH) as the template (PDB code: 7K34). 97.2% of total residues in the model were regarded as Ramachandran favored by SWISS-MODEL structure assessment (Figure S3), indicating that the quality of the model was sufficient . The structural model of Bu LTTA showed the typical aspartate aminotransferase superfamily type I fold of a PLP-dependent enzyme.…”

“…97.2% of total residues in the model were regarded as Ramachandran favored by SWISS-MODEL structure assessment (Figure S3), indicating that the quality of the model was sufficient. 39 The structural model of BuLTTA showed the typical aspartate aminotransferase superfamily type I fold of a PLP-dependent enzyme. BuLTTA was modeled as a domainswapped homodimer with the C-terminus extending into the dimer interface (Figure S4).…”

Reshaping Substrate-Binding Pocket of Leucine Dehydrogenase for Bidirectionally Accessing Structurally Diverse Substrates

“…However, the above studies were limited to using unidirectional enzymatic activity or substrate specificity traits of the difficult-to-accommodate bulky substrates as a measure of fitness. ,, It is of interest to note that the sterically small substrates can be easily accommodated into the pocket compared with bulky substrates, while the catalytic activity of enzymes toward these non-natural small substrates is usually lower than that of their natural substrates or even no specificity. − This phenomenon indicates that the non-natural small substrates accommodated into the pocket are still difficult to capture rationally and efficiently by enzymes. To the best of our knowledge, unlocking the enzyme acceptance toward the non-natural small substrates through steric hindrance strengthening-based pocket reshaping engineering has not been systematically explored.…”