Search

CYP105AS1 is a cytochrome P450 from Amycolatopsis orientalis that catalyzes monooxygenation of compactin to 6-epi-pravastatin. For fermentative production of the cholesterol-lowering drug pravastatin, the stereoselectivity of the enzyme needs to be inverted, which has been partially achieved by error-prone PCR mutagenesis and screening. In the current study, we report further optimization of the stereoselectivity by a computationally aided approach. Using the CoupledMoves protocol of Rosetta, a virtual library of mutants was designed to bind compactin in a pro-pravastatin orientation. By examining the frequency of occurrence of beneficial substitutions and rational inspection of their interactions, a small set of eight mutants was predicted to show the desired selectivity and these variants were tested experimentally. The best CYP105AS1 variant gave >99% stereoselective hydroxylation of compactin to pravastatin, with complete elimination of the unwanted 6-epi-pravastatin diastereomer. The enzyme-substrate complexes were also examined by ultrashort molecular dynamics simulations of 50 × 100 ps and 5 × 22 ns, which revealed that the frequency of occurrence of near-attack conformations agreed with the experimentally observed stereoselectivity. These results show that a combination of computational methods and rational inspection could improve CYP105AS1 stereoselectivity beyond what was obtained by directed evolution. Moreover, the work lays out a general in silico framework for specificity engineering of enzymes of known structure.

Whereas directed evolution and rational design by structural inspection are established tools for enzyme redesign, computational methods are less mature but have the potential to predict small sets of mutants with desired properties without laboratory screening of large libraries. We have explored the use of computational enzyme redesign to change the enantioselectivity of a highly thermostable alcohol dehydrogenase from Thermus thermophilus in the asymmetric reduction of ketones. The enzyme reduces acetophenone to (S)-1-phenylethanol. To invert the enantioselectivity, we used an adapted CASCO workflow which included Rosetta for enzyme design and molecular dynamics simulations for ranking. To correct for unrealistic binding modes, we used Boltzmann weighing of binding energies computed by a linear interaction energy approach. This computationally cheap method predicted four variants with inverted enantioselectivity, each with 6–8 mutations around the substrate-binding site, causing only modest reduction (2- to 7-fold) of kcat/KM values. Laboratory testing showed that three variants indeed had inverted enantioselectivity, producing (R)-alcohols with up to 99 % enantiomeric excess. The broad substrate range allowed reduction of acetophenone derivatives with full conversion to highly enantioenriched alcohols. The results demonstrate the use of computational methods to control ketoreductase stereoselectivity in asymmetric transformations with minimal experimental screening.

Context: Bronchial asthma is a chronic respiratory disorder which affects over 300 million people worldwide. Inhalation pharmacotherapy is the cornerstone in treatment of asthma, which is administered using inhaler devices. Studies show high prevalence of incorrect technique while inhaler usage, which renders to compromised disease control and increased healthcare cost. Aims: This study was aimed to monitor errors in use of inhalers and explore their relationship with patient characteristics and training given by healthcare providers, in patients suffering from mild to moderate bronchial asthma. Settings and Design: This was an observational cross-sectional study conducted after approval of Institutional ethics committee in a tertiary care hospital. Methods and Material: A total of 207 patients were recruited after they met the inclusion criteria and their informed consent was taken. Data regarding the patients’ socio-demographic information, education status and history of illness were logged on a case record form. Their inhalation techniques were assessed according to the checklist, errors were noted and patients were educated regarding correct technique using ‘teach-back training’ method. Statistical analysis used: Chi-square test, SPSS software. Results: Among the inhalers used, pMDI was most commonly prescribed (58%), followed by DPI (37.7%) and pMDI with spacer (4.3%). Irrespective of the type of inhaler used, overall 75.36% patients included in the study, showed errors in use of inhaler. Prevalence of errors in DPI, pMDI and pMDI with spacers was 78.2 %,77.8% and 22.2% respectively. Our study showed that education status of patient, training by healthcare provider and duration of inhaler use have statistically significant association (p< 0.05) with the prevalence of errors in inhaler usage. Conclusion: It is emphasized that dedicated and trained staff should be available for instructing patients and reinforcing by follow up checks should be considered.

ω-Transaminases (ω-TA) are attractive biocatalysts for the production of chiral amines from prochiral ketones via asymmetric synthesis. However, the substrate scope of ω-TAs is usually limited due to steric hindrance at the active site pockets. We explored a protein engineering strategy using computational design to expand the substrate scope of an (S)-selective ω-TA from Pseudomonas jessenii (PjTA-R6) toward the production of bulky amines. PjTA-R6 is attractive for use in applied biocatalysis due to its thermostability, tolerance to organic solvents, and acceptance of high concentrations of isopropylamine as amino donor. PjTA-R6 showed no detectable activity for the synthesis of six bicyclic or bulky amines targeted in this study. Six small libraries composed of 7-18 variants each were separately designed via computational methods and tested in the laboratory for ketone to amine conversion. In each library, the vast majority of the variants displayed the desired activity, and of the 40 different designs, 38 produced the target amine in good yield with >99% enantiomeric excess. This shows that the substrate scope and enantioselectivity of PjTA mutants could be predicted in silico with high accuracy. The single mutant W58G showed the best performance in the synthesis of five structurally similar bulky amines containing the indan and tetralin moieties. The best variant for the other bulky amine, 1-phenylbutylamine, was the triple mutant W58M + F86L + R417L, indicating that Trp58 is a key residue in the large binding pocket for PjTA-R6 redesign. Crystal structures of the two best variants confirmed the computationally predicted structures. The results show that computational design can be an efficient approach to rapidly expand the substrate scope of ω-TAs to produce enantiopure bulky amines.

Caprolactamase is the first enzyme in the caprolactam degradation pathway of Pseudomonas jessenii. It is composed of two subunits (CapA and CapB) and sequence-related to other ATP-dependent enzymes involved in lactam hydrolysis, like 5-oxoprolinases and hydantoinases. Low sequence similarity also exists with ATP-dependent acetone- and acetophenone carboxylases. The caprolactamase was produced in E. coli, isolated by His-tag affinity chromatography, and subjected to functional and structural studies. Activity towards caprolactam required ATP and was dependent on the presence of bicarbonate in the assay buffer. The hydrolysis product was identified as 6-aminocaproic acid (6-ACA). Quantum mechanical modeling indicated that the hydrolysis of caprolactam was highly disfavored (ΔG0 ' = 23 kJ/mol), which explained the ATP dependence. A crystal structure showed that the enzyme exists as an (αβ)2 tetramer and revealed an ATP-binding site in CapA and a Zn-coordinating site in CapB. Mutations in the ATP-binding site of CapA (D11A and D295A) significantly reduced product formation. Mutants with substitutions in the metal binding site of CapB (D41A, H99A, D101A and H124A) were inactive and less thermostable than the wild-type enzyme. These residues proved to be essential for activity and on basis of the experimental findings we propose possible mechanisms for ATP-dependent lactam hydrolysis. This article is protected by copyright. All rights reserved.

Protein engineering has shown widespread use in improving the industrial application of enzymes and broadening the conditions they are able to operate under by increasing their thermostability and solvent tolerance. Here, we show that protein engineering can be used to increase the thermostability of an artificial metalloenzyme. Thermostable variants of the human steroid carrier protein 2L, modified to bind a metal catalyst, were created by rational design using structural data and a 3DM database. These variants were tested to identify mutations that enhanced the stability of the protein scaffold, and a significant increase in melting temperature was observed with a number of modified metalloenzymes. The ability to withstand higher reaction temperatures resulted in an increased activity in the hydroformylation of 1-octene, with more than fivefold improvement in turnover number, whereas the selectivity for linear aldehyde remained high up to 80%.

Graphical abstract, Omniligase-1 is a broadly applicable enzyme for peptide bond formation between an activated acyl donor peptide and a non-protected acyl acceptor peptide. The enzyme is derived from an earlier subtilisin variant called peptiligase by several rounds of protein engineering aimed at increasing synthetic yields and substrate range. To examine the contribution of individual mutations on S/H ratio and substrate scope in peptide synthesis, we selected peptiligase variant M222P/L217H as a starting enzyme and introduced successive mutations. Mutation A225N in the S1′ pocket and F189W of the S2′ pocket increased the synthesis to hydrolysis (S/H) ratio and overall coupling efficiency, whereas the I107V mutation was added to S4 pocket to increase the reaction rate. The final omniligase variants appeared to have a very broad substrate range, coupling more than 250 peptides in a 400-member library of acyl acceptors, as indicated by a high-throughput FRET assay. Crystal structures and computational modelling could rationalize the exceptional properties of omniligase-1 in peptide synthesis

The mycobacterial PQS dioxygenase AqdC, a cofactor‐less protein with an α/β‐hydrolase fold, inactivates the virulence‐associated quorum‐sensing signal molecule 2‐heptyl‐3‐hydroxy‐4(1H)‐quinolone (PQS) produced by the opportunistic pathogen Pseudomonas aeruginosa and is therefore a potential anti‐virulence tool. We have used computational library design to predict stabilizing amino acid replacements in AqdC. While 57 out of 91 tested single substitutions throughout the protein led to stabilization, as judged by increases in Tappm of >2 °C, they all impaired catalytic activity. Combining substitutions, the proteins AqdC‐G40K‐A134L‐G220D‐Y238W and AqdC‐G40K‐G220D‐Y238W showed extended half‐lives and the best trade‐off between stability and activity, with increases in Tappm of 11.8 and 6.1 °C and relative activities of 22 and 72 %, respectively, compared to AqdC. Molecular dynamics simulations and principal component analysis suggested that stabilized proteins are less flexible than AqdC, and the loss of catalytic activity likely correlates with an inability to effectively open the entrance to the active site., Swings and roundabouts: The dioxygenase AqdC, an α/β‐hydrolase fold protein, inactivates a virulence‐associated signal molecule of the bacterial pathogen Pseudomonas aeruginosa. Using computational approaches to predict and engineer thermostable AqdC variants, we found that gains in thermostability (up to +11.8 °C) were traded against losses in activity, likely due to difficulties in opening the entrance to the active site.