Rational hinge engineering of carboxylic acid reductase from Mycobacterium smegmatis enhances its catalytic efficiency in biocatalysis.

BACKGROUND: Carboxylic acid reductases (CARs) represent useful tools for the production of aldehydes from ubiquitous organic carboxylic acids. However, the low catalytic efficiency of these enzymes hampers their application. METHODS: Herein, a CAR originating from Mycobacterium smegmatis was redesigned through rational hinge engineering to enhance the catalytic efficiency. RESULTS: Based on the unique domain architecture of CARs and their superfamily, a mutagenesis library of the hinge region was designed. The best mutant R505I/N506K showed a 6.57-fold improved catalytic efficiency. Molecular dynamics simulations showed the increased catalytic efficiency was due to the strong binding of the acyl-AMP complex with it. Meanwhile, the ε-nitrogen atom of Lys610 frequently interacted with the ribose-ring oxygen atom of the complex, the distance (d1) between them represents a great indicator for that. The d1 value was used as a nimble indicator to evaluate unexplored mutants of that region for enhanced activity by in silico mutational experiments. Overall, eight mutants were identified to show higher enhanced activity compared with wild-type enzyme and R505F/N506G showed the highest catalytic efficiency. CONCLUSION: Altogether, the two-step strategy used here provided useful references for the engineering of CARs and other similar multiple-domain enzymes.

Switching the secondary and natural activity of Nitrilase from Acidovorax facilis 72 W for the efficient production of 2-picolinamide.

OBJECTIVES: Catalytic promiscuity, or the ability to catalyze a secondary reaction, provides new opportunities for industrial biocatalysis by expanding the range of biocatalytic reactions. Some nitrilases converting nitriles to amides, referred to as the secondary activity, show great potential for amides production. And our goal was exploiting the amide-forming potential of nitrilases. RESULTS: In this study, we characterized and altered the secondary activity of nitrilase from Acidovorax facilis 72 W (Nit72W) towards different substrates. We increased the secondary activity of Nit72W towards 2-cyanopyridine by 196-fold and created activity toward benzonitrile and p-nitrophenylacetonitrile by modifying the active pocket. Surprisingly, the best mutant, W188M, completely converted 250 mM 2-cyanopyridine to more than 98% 2-picolinamide in 12 h with a specific activity of 90 U/mg and showed potential for industrial applications. CONCLUSIONS: Nit72W was modified to increase its secondary activity for the amides production, especially 2-picolinamide.

[Customization of enzyme molecular machine and cell factory, leading the future of biomanufacturing industry].

The development and application of industrial enzymes have penetrated major industrial fields. China faces a major challenge as a large country in applying enzyme but a small one in producing enzyme. Biocatalysis has become an important technology and strategy of industrial development in the world since chemical catalysis encounters the crises from resource, energy and environment. The application of efficient and clean biocatalysis is one of the important ways to realize the sustainable development of chemical industry and to modernize the fermentation industry. From perspective of the industry-university-research cooperation, we reviewed the current status and the future development of enzyme engineering from the aspects of enzyme resources, customization of enzyme molecular machine and cell factory.

Crius: A novel fragment-based algorithm of de novo substrate prediction for enzymes.

The study of enzyme substrate specificity is vital for developing potential applications of enzymes. However, the routine experimental procedures require lot of resources in the discovery of novel substrates. This article reports an in silico structure-based algorithm called Crius, which predicts substrates for enzyme. The results of this fragment-based algorithm show good agreements between the simulated and experimental substrate specificities, using a lipase from Candida antarctica (CALB), a nitrilase from Cyanobacterium syechocystis sp. PCC6803 (Nit6803), and an aldo-keto reductase from Gluconobacter oxydans (Gox0644). This opens new prospects of developing computer algorithms that can effectively predict substrates for an enzyme.

The Important Role of Halogen Bond in Substrate Selectivity of Enzymatic Catalysis.

The use of halogen bond is widespread in drug discovery, design, and clinical trials, but is overlooked in drug biosynthesis. Here, the role of halogen bond in the nitrilase-catalyzed synthesis of ortho-, meta-, and para-chlorophenylacetic acid was investigated. Different distributions of halogen bond induced changes of substrate binding conformation and affected substrate selectivity. By engineering the halogen interaction, the substrate selectivity of the enzyme changed, with the implication that halogen bond plays an important role in biosynthesis and should be used as an efficient and reliable tool in enzymatic drug synthesis.

Identification of novel thermostable taurine-pyruvate transaminase from Geobacillus thermodenitrificans for chiral amine synthesis.

ω-Transaminases (ω-TAs) are one of the most popular candidate enzymes in the biosynthesis of chiral amines. Determination of yet unidentified ω-TAs is important to broaden their potential for synthetic application. Taurine-pyruvate TA (TPTA, EC 2.6.1.77) is an ω-TA belonging to class III of TAs. In this study, we cloned a novel thermostable TPTA from Geobacillus thermodenitrificans (TPTAgth) and overexpressed it in Escherichia coli. The enzyme showed the highest activity at pH 9.0 and 65 °C, with remarkable thermostability and tolerance toward organic solvents. Its K M and v max values for taurine were 5.3 mM and 0.28 µmol s(-1) mg(-1), respectively. Determination of substrate tolerance indicated its broad donor and acceptor ranges for unnatural substrates. Notably, the enzyme showed relatively good activity toward ketoses, suggesting its potential for catalyzing the asymmetric synthesis of chiral amino alcohols. The active site of TPTAgth was identified by performing protein sequence alignment, three-dimensional structure simulation, and coenzyme pyridoxamine phosphate docking. The protein sequence and structure of TPTAgth were similar to those of TAs belonging to the 3N5M subfamily. Its active site was found to be its special large pocket and substrate tunnel. In addition, TPTAgth showed a unique mechanism of sulfonate/α-carboxylate recognition contributed by Arg163 and Gln160. We also determined the protein sequence fingerprint of TPTAs in the 3N5M subfamily, which involved Arg163 and Gln160 and seven additional residues from 413 to 419 and lacked Phe/Tyr22, Phe85, and Arg409.

A computational strategy for altering an enzyme in its cofactor preference to NAD(H) and/or NADP(H).

Coenzyme engineering, especially for altered coenzyme specificity, has been a research hotspot for more than a decade. In the present study, a novel computational strategy that enhances the hydrogen-bond interaction between an enzyme and a coenzyme was developed and utilized to alter the coenzyme preference. This novel computational strategy only required the structure of the target enzyme. No other homologous enzymes were needed to achieve alteration in the coenzyme preference of a certain enzyme. Using our novel strategy, Gox2181 was reconstructed from exhibiting complete NADPH preference to exhibiting dual cofactor specificity for NADH and NADPH. Structure-guided Gox2181 mutants were designed in silico and molecular dynamics simulations were performed to evaluate the strength of hydrogen-bond interactions between the enzyme and the coenzyme NADPH. Three Gox2181 mutants displaying high structure stability and structural compatibility to NADH/NADPH were chosen for experimental confirmation. Among the three Gox2181 mutants, Gox2181-Q20R&D43S showed the highest enzymatic activity by utilizing NADPH as its coenzyme, which was even better than the wild-type enzyme. In addition, isothermal titration calorimetry analysis further verified that Gox2181-Q20R&D43S was able to interact with NADPH but the wild-type enzyme could not. This novel computational strategy represents an insightful approach for altering the cofactor preference of target enzymes.

Structural insights into enzymatic activity and substrate specificity determination by a single amino acid in nitrilase from Syechocystis sp. PCC6803.

Nitrilases are enzymes widely expressed in prokaryotes and eukaryotes that utilize a Cys­Glu­Lys catalytic triad to hydrolyze non-peptide carbon­nitrogen bonds. Nitrilase from Syechocystis sp. Strain PCC6803 (Nit6803) shows hydrolysis activity towards a broad substrate spectrum, ranging from mononitriles to dinitriles and from aromatic nitriles to aliphatic nitriles. Yet, the structural principle of the substrate specificity of this nitrilase is still unknown. We report the crystal structure of Nit6803 at 3.1 Å resolution and propose a structural mechanism of substrate selection. Our mutagenesis data exhibited that the aromaticity of the amino acid at position 146 of Nit6803 is absolutely required for its nitrilase activity towards any substrates tested. Moreover, molecular docking and dynamic simulation analysis indicated that the distance between the sulfhydryl group of the catalytic cysteine residue and the cyano carbon of the substrate plays a crucial role in determining the nitrilase catalytic activity of Nit6803 and its mutants towards different nitrile substrates.

Structural insights into substrate and coenzyme preference by SDR family protein Gox2253 from Gluconobater oxydans.

Gox2253 from Gluconobacter oxydans belongs to the short-chain dehydrogenases/reductases family, and catalyzes the reduction of heptanal, octanal, nonanal, and decanal with NADPH. To develop a robust working platform to engineer novel G. oxydans oxidoreductases with designed coenzyme preference, we adopted a structure based rational design strategy using computational predictions that considers the number of hydrogen bonds formed between enzyme and docked coenzyme. We report the crystal structure of Gox2253 at 2.6 Å resolution, ternary models of Gox2253 mutants in complex with NADH/short-chain aldehydes, and propose a structural mechanism of substrate selection. Molecular dynamics simulation shows that hydrogen bonds could form between 2'-hydroxyl group in the adenosine moiety of NADH and the side chain of Gox2253 mutant after arginine at position 42 is replaced with tyrosine or lysine. Consistent with the molecular dynamics prediction, Gox2253-R42Y/K mutants can use both NADH and NADPH as a coenzyme. Hence, the strategies here could provide a practical platform to engineer coenzyme selectivity for any given oxidoreductase and could serve as an additional consideration to engineer substrate-binding pockets.

A method to rationally increase protein stability based on the charge-charge interaction, with application to lipase LipK107.

We report a suite of enzyme redesign protocol based on the surface charge-charge interaction calculation, which is potentially applied to improve the stability of an enzyme without compromising its catalytic activity. Together with the experimental validation, we have released a suite of enzyme redesign algorithm Enzyme Thermal Stability System, written based on our model, for open access to meet the needs in wet labs. Lipk107, a lipase of a versatile industrial use, was chosen to test our software. Our calculation determined that four residues, D113, D149, D213, and D253, located on the surface of LipK107 were critical to the stability of the enzyme. The model was validated with mutagenesis at these four residues followed by stability and activity tests. LipK107 mutants D113A and D149K were more resistant to thermal inactivation with ∼10°C higher half-inactivation temperature than wild-type LipK107. Moreover, mutant D149K exhibited significant retention in residual activity under constant heat, showing a 14-fold increase in the half-inactivation time at 50°C. Activity tests showed that these mutants retained the equal or higher specific activity, among which noteworthy was the mutant D253A with as much as 20% higher activity. We suggest that our protocol could be used as a general guideline to redesign protein enzymes with increased stabilities and enhanced activities.