Abstract:Active-site loops play essential roles in various catalytically important enzyme properties like activity, selectivity, and substrate scope. However, their high flexibility and diversity makes them challenging to incorporate into rational enzyme engineering strategies. Here, we report the engineering of hot-spots in loops of the cumene dioxygenase from Pseudomonas fluorescens IP01 with high impact on activity, regio- and enantioselectivity. Libraries based on alanine scan, sequence alignments, and deletions al… Show more
“…This observation demonstrated that the formation of new tunnels and widening of existing ones can modulate the catalytic activity of enzymes. It also resembled the findings reported for prior studies [ 46 , 47 ]. Fig.…”
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 kcat value, and accordingly a 12.9-fold higher kcat/Km 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.
“…This observation demonstrated that the formation of new tunnels and widening of existing ones can modulate the catalytic activity of enzymes. It also resembled the findings reported for prior studies [ 46 , 47 ]. Fig.…”
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 kcat value, and accordingly a 12.9-fold higher kcat/Km 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.
“…The mutations in the course of evolution of Bs DyP were inserted in flexible loops and in a small helix close to the heme pocket ( Figure 3 a,b). Flexible loops in the proximity of active sites frequently play roles in catalysis as they interact with solvent and substrates [ 53 , 54 ]; it is widely accepted that their pronounced conformational flexibility may contribute to their function, in substrate selectivity and recognition, and the facilitation of substrate binding, as well as protein evolution, as they represent molecular elements of variability [ 55 ]. Loop 1 (222–250) and loop 3 (334–342) are predominantly located at the distal side of the heme and comprise the catalytic residues D240 and R339, respectively.…”
Bacillus subtilis BsDyP belongs to class I of the dye-decolorizing peroxidase (DyP) family of enzymes and is an interesting biocatalyst due to its high redox potential, broad substrate spectrum and thermostability. This work reports the optimization of BsDyP using directed evolution for improved oxidation of 2,6-dimethoxyphenol, a model lignin-derived phenolic. After three rounds of evolution, one variant was identified displaying 7-fold higher catalytic rates and higher production yields as compared to the wild-type enzyme. The analysis of X-ray structures of the wild type and the evolved variant showed that the heme pocket is delimited by three long conserved loop regions and a small α helix where, incidentally, the mutations were inserted in the course of evolution. One loop in the proximal side of the heme pocket becomes more flexible in the evolved variant and the size of the active site cavity is increased, as well as the width of its mouth, resulting in an enhanced exposure of the heme to solvent. These conformational changes have a positive functional role in facilitating electron transfer from the substrate to the enzyme. However, they concomitantly resulted in decreasing the enzyme’s overall stability by 2 kcal mol−1, indicating a trade-off between functionality and stability. Furthermore, the evolved variant exhibited slightly reduced thermal stability compared to the wild type. The obtained data indicate that understanding the role of loops close to the heme pocket in the catalysis and stability of DyPs is critical for the development of new and more powerful biocatalysts: loops can be modulated for tuning important DyP properties such as activity, specificity and stability.
“… 1 In particular, the importance of flexibility of loops for protein function and enzyme catalysis has gained increased attention. 15 For some enzymes such as terpene cyclases, 16 loop dynamics is a necessity for expedient catalysis by capping the active site following substrate binding, shielding reactive intermediates from the solvent, and enabling efficient transition-state stabilization. 16 Proteins with flexible loops have previously been shown to exhibit allokairic regulation.…”
Thermostability is
the key to maintain the structural integrity
and catalytic activity of enzymes in industrial biotechnological processes,
such as terpene cyclase-mediated generation of medicines, chiral synthons,
and fine chemicals. However, affording a large increase in the thermostability
of enzymes through site-directed protein engineering techniques can
constitute a challenge. In this paper, we used ancestral sequence
reconstruction to create a hyperstable variant of the
ent
-copalyl diphosphate synthase PtmT2, a terpene cyclase involved in
the assembly of antibiotics. Molecular dynamics simulations on the
μs timescale were performed to shed light on possible molecular
mechanisms contributing to activity at an elevated temperature and
the large 40 °C increase in melting temperature observed for
an ancestral variant of PtmT2.
In silico
analysis
revealed key differences in the flexibility of a loop capping the
active site, between extant and ancestral proteins. For the modern
enzyme, the loop collapses into the active site at elevated temperatures,
thus preventing biocatalysis, whereas the loop remains in a productive
conformation both at ambient and high temperatures in the ancestral
variant. Restoring a Pro loop residue introduced in the ancestral
variant to the corresponding Gly observed in the extant protein led
to reduced catalytic activity at high temperatures, with only moderate
effects on the melting temperature, supporting the importance of the
flexibility of the capping loop in thermoadaptation. Conversely, the
inverse Gly to Pro loop mutation in the modern enzyme resulted in
a 3-fold increase in the catalytic rate. Despite an overall decrease
in maximal activity of ancestor compared to wild type, its increased
thermostability provides a robust backbone amenable for further enzyme
engineering. Our work cements the importance of loops in enzyme catalysis
and provides a molecular mechanism contributing to thermoadaptation
in an ancestral enzyme.
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