A Photoclick-Based High-Throughput Screening for the Directed Evolution of Decarboxylase OleT.

Enzymatic oxidative decarboxylation is an up-and-coming reaction yet lacking efficient screening methods for the directed evolution of decarboxylases. Here, we describe a simple photoclick assay for the detection of decarboxylation products and its application in a proof-of-principle directed evolution study on the decarboxylase OleT. The assay was compatible with two frequently used OleT operation modes (directly using hydrogen peroxide as the enzyme's co-substrate or using a reductase partner) and the screening of saturation mutagenesis libraries identified two enzyme variants shifting the enzyme's substrate preference from long chain fatty acids toward styrene derivatives. Overall, this photoclick assay holds promise to speed-up the directed evolution of OleT and other decarboxylases.

Reversal of Regioselectivity in Zinc-Dependent Medium-Chain Alcohol Dehydrogenase from Rhodococcus erythropolis toward Octanone Derivatives.

The zinc-dependent medium-chain alcohol dehydrogenase from Rhodococcus erythropolis (ReADH) is one of the most versatile biocatalysts for the stereoselective reduction of ketones to chiral alcohols. Despite its known broad substrate scope, ReADH only accepts carbonyl substrates with either a methyl or an ethyl group adjacent to the carbonyl moiety; this limits its use in the synthesis of the chiral alcohols that serve as a building blocks for pharmaceuticals. Protein engineering to expand the substrate scope of ReADH toward bulky substitutions next to carbonyl group (ethyl 2-oxo-4-phenylbutyrate) opens up new routes in the synthesis of ethyl-2-hydroxy-4-phenylbutanoate, an important intermediate for anti-hypertension drugs like enalaprilat and lisinopril. We have performed computer-aided engineering of ReADH toward ethyl 2-oxo-4-phenylbutyrate and octanone derivatives. W296, which is located in the small binding pocket of ReADH, sterically restricts the access of ethyl 2-oxo-4-phenylbutyrate, octan-3-one or octan-4-one toward the catalytic zinc ion and thereby limits ReADH activity. Computational analysis was used to identify position W296 and site-saturation mutagenesis (SSM) yielded an improved variant W296A with a 3.6-fold improved activity toward ethyl 2-oxo-4-phenylbutyrate when compared to WT ReADH (ReADH W296A: 17.10 U/mg and ReADH WT: 4.7 U/mg). In addition, the regioselectivity of ReADH W296A is shifted toward octanone substrates. ReADH W296A has a more than 16-fold increased activity toward octan-4-one (ReADH W296A: 0.97 U/mg and ReADH WT: 0.06 U/mg) and a more than 30-fold decreased activity toward octan-2-one (ReADH W296A: 0.23 U/mg and ReADH WT: 7.69 U/mg). Computational and experimental results revealed the role of position W296 in controlling the substrate scope and regiopreference of ReADH for a variety of carbonyl substrates.

Engineering of Laccase CueO for Improved Electron Transfer in Bioelectrocatalysis by Semi-Rational Design.

Invited for the cover of this issue is the group of Ulrich Schwaneberg at the Institute of Biotechnology, RWTH-Aachen University and DWI Lebniz Institute of Interactive Materials. The picture calls for special attention to be paid to the extra Cu binding site of Copper efflux Oxidase (CueO), due to its predominant function in tuning the electrocatalytic kinetics towards oxygen reduction. Read the full text of the article at 10.1002/chem.201905598.

Engineering of Laccase CueO for Improved Electron Transfer in Bioelectrocatalysis by Semi-Rational Design.

Copper efflux oxidase (CueO) from Escherichia coli is a special bacterial laccase due to its fifth copper binding site. Herein, it is discovered that the fifth Cu occupancy plays a crucial and favorable role of electron relay in bioelectrocatalytic oxygen reduction. By substituting the residues at the four coordinated positions of the fifth Cu, 11 beneficial variants are identified with ≥2.5-fold increased currents at -250 mV (up to 6.13 mA cm-2 ). Detailed electrocatalytic characterization suggests the microenvironment of the fifth Cu binding site governs the electrocatalytic current of CueO. Additionally, further electron transfer analysis assisted by molecular dynamics (MD) simulation demonstrates that an increase in localized structural stability and a decrease of distance between the fifth Cu and the T1 Cu are two main factors contributing to the improved kinetics of CueO variants. It may guide a novel way to tailor laccases and perhaps other oxidoreductases for bioelectrocatalytic applications.

Deletion and Randomization of Structurally Variable Regions in B. subtilis Lipase A (BSLA) Alter Its Stability and Hydrolytic Performance Against Long Chain Fatty Acid Esters.

The continuous search for novel enzyme backbones and the engineering of already well studied enzymes for biotechnological applications has become an increasing challenge, especially by the increasing potential diversity space provided by directed enzyme evolution approaches and the demands of experimental data generated by rational design of enzymes. In this work, we propose a semi-rational mutational strategy focused on introducing diversity in structurally variable regions in enzymes. The identified sequences are subjected to a progressive deletion of two amino acids and the joining residues are subjected to saturation mutagenesis using NNK degenerate codons. This strategy offers a novel library diversity approach while simultaneously decreasing enzyme size in the variable regions. In this way, we intend to identify and reduce variable regions found in enzymes, probably resulting from neutral drift evolution, and simultaneously studying the functional effect of said regions. This strategy was applied to Bacillus. subtilis lipase A (BSLA), by selecting and deleting six variable enzyme regions (named regions 1 to 6) by the deletion of two amino acids and additionally randomizing the joining amino acid residues. After screening, no active variants were found in libraries 1% and 4%, 15% active variants were found in libraries 2% and 3%, and 25% for libraries 5 and 6 (n = 3000 per library, activity detected using tributyrin agar plates). Active variants were assessed for activity in microtiter plate assay (pNP-butyrate), thermal stability, substrate preference (pNP-butyrate, -palmitate), and compared to wildtype BSLA. From these analyses, variant P5F3 (F41L-ΔW42-ΔD43-K44P), from library 3 was identified, showing increased activity towards longer chain p-nitrophenyl fatty acid esters, when compared to BSLA. This study allowed to propose the targeted region 3 (positions 40-46) as a potential modulator for substrate specificity (fatty acid chain length) in BSLA, which can be further studied to increase its substrate spectrum and selectivity. Additionally, this variant showed a decreased thermal resistance but interestingly, higher isopropanol and Triton X-100 resistance. This deletion-randomization strategy could help to expand and explore sequence diversity, even in already well studied and characterized enzyme backbones such as BSLA. In addition, this strategy can contribute to investigate and identify important non-conserved regions in classic and novel enzymes, as well as generating novel biocatalysts with increased performance in specific processes, such as enzyme immobilization.

Mechanistic elucidation of amphetamine metabolism by tyramine oxidase from human gut microbiota using molecular dynamics simulations.

The human gut harbors diverse bacterial species in the gut, which play an important role in the metabolism of food and host health. Recent studies have also revealed their role in altering the pharmacological properties and efficacy of oral drugs through promiscuous metabolism. However, the atomistic details of the enzyme-drug interactions of gut bacterial enzymes which can potentially carry out the metabolism of drug molecules are still scarce. A well-known example is the FDA drug amphetamine (a central nervous system stimulant), which has been predicted to undergo promiscuous metabolism by gut bacteria. Therefore, to understand the atomistic details and energy landscape of the gut microbial enzyme-mediated metabolism of this drug, molecular dynamics studies were performed. It was observed that amphetamine binds to tyramine oxidase from the Escherichia coli strain present in the human gut microbiota at the binding site harboring polar and nonpolar amino acids. The stability analysis of amphetamine at the binding site showed that the binding is stable and the free energy for the binding of amphetamine was found to be ~ -51.71 kJ/mol. The insights provided by this study on promiscuous metabolism of amphetamine by a gut enzyme will be very useful to improve the efficacy of the drug.

KnowVolution of a Fungal Laccase toward Alkaline pH.

To date, commercial laccase preparations are used in the food, textile, and paper and pulp industries (mild pH). Laccases are attractive in the synthesis of dye molecules or oxidative lignin treatment, which take place at high pH (≥8.0). So far, one fungal laccase has been reported to be active at alkaline pH. Herein, engineering of the fungal laccase from Melanocarpus albomyces (MaL) for increased activity toward the substrate 2,6-dimethoxyphenol at pH (≥9.0) is reported. Through a knowledge-gaining directed evolution (KnowVolution) campaign, the key positions Leu365 and Leu513 were identified to increase alkaline tolerance. Both positions are located in close proximity of the T1Cu site. Molecular docking and simulations studies reveal that both substitutions act in a synergic way to stabilize and improve laccase activity at higher pH. Kinetic characterization of the final variant MaL-M1 (L365E/L513M) revealed at pH 9.8 a threefold improved kcat (kcat =(6.0±0.2) s-1 ) compared with that of wild-type M. albomyces laccase (kcat =(2.11±0.07) s-1 ).

A thermostable flavin-based fluorescent protein from Chloroflexus aggregans: a framework for ultra-high resolution structural studies.

Light-Oxygen-Voltage (LOV) domains are conserved parts of photoreceptors in plants, bacteria and fungi that bind flavins as chromophores and detect blue light. In the past, LOV domain variants have been developed as fluorescent reporter proteins (called flavin-based fluorescent proteins; FbFPs), which due to their ability to fluoresce under anaerobic conditions, fast folding kinetics and a small size of ∼12-16 kDa are a promising reporter system for quantitative real-time analysis of biological processes. Here, we present a small thermostable flavin-based fluorescent protein CagFbFP derived from a soluble LOV domain-containing histidine kinase from the thermophilic bacterium Chloroflexus aggregans. CagFbFP is composed of 107 amino acids with a molecular weight of 11.6 kDa and consists only of the conserved LOV core domain. The protein is thermostable with a melting point of about 68 °C. It crystallizes easily and its crystals diffract to 1.07 Å. Both the crystal structure and small angle scattering data show that the protein is a dimer. Unexpectedly, glutamine 148, which in LOV photoreceptor proteins is the key residue responsible for signal transduction, occupies two conformations. Molecular dynamics simulations show that the two conformations interconvert rapidly. The crystal structure of the wild-type Chloroflexus aggregans LOV domain determined at 1.22 Å resolution confirmed the presence of two alternative conformations of the glutamine 148 side chain. Overall, this protein, due to its stability and ease of crystallization, appears to be a promising model for ultra-high resolution structural studies of LOV domains and for application as a fluorescent reporter.

Directed aryl sulfotransferase evolution toward improved sulfation stoichiometry on the example of catechols.

Sulfation is an important way for detoxifying xenobiotics and endobiotics including catechols. Enzymatic sulfation occurs usually with high chemo- and/or regioselectivity under mild reaction conditions. In this study, a two-step p-NPS-4-AAP screening system for laboratory evolution of aryl sulfotransferase B (ASTB) was developed in 96-well microtiter plates to improve the sulfate transfer efficiency toward catechols. Increased transfer efficiency and improved sulfation stoichiometry are achieved through the two-step screening procedure in a one-pot reaction. In the first step, the p-NPS assay is used (detection of the colorimetric by-product, p-nitrophenol) to determine the apparent ASTB activity. The sulfated product, 3-chlorocatechol-1-monosulfate, is quantified by the 4-aminoantipyrine (4-AAP) assay in the second step. Comparison of product formation to p-NPS consumption ensures successful directed evolution campaigns of ASTB. Optimization yielded a coefficient of variation below 15% for the two-step screening system (p-NPS-4-AAP). In total, 1760 clones from an ASTB-SeSaM library were screened toward the improved sulfation activity of 3-chlorocatechol. The turnover number (kcat = 41 ± 2 s-1) and catalytic efficiency (kcat/KM = 0.41 µM-1 s-1) of the final variant ASTB-M5 were improved 2.4- and 2.3-fold compared with ASTB-WT. HPLC analysis confirmed the improved sulfate stoichiometry of ASTB-M5 with a conversion of 58% (ASTB-WT 29%; two-fold improvement). Mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR) confirmed the chemo- and regioselectivity, which yielded exclusively 3-chlorocatechol-1-monosulfate. For all five additionally investigated catechols, the variant ASTB-M5 achieved an improved kcat value of up to 4.5-fold and sulfate transfer efficiency was also increased (up to 2.3-fold).

Directed Evolution of P450 BM3 towards Functionalization of Aromatic O-Heterocycles.

The O-heterocycles, benzo-1,4-dioxane, phthalan, isochroman, 2,3-dihydrobenzofuran, benzofuran, and dibenzofuran are important building blocks with considerable medical application for the production of pharmaceuticals. Cytochrome P450 monooxygenase (P450) Bacillus megaterium 3 (BM3) wild type (WT) from Bacillus megaterium has low to no conversion of the six O-heterocycles. Screening of in-house libraries for active variants yielded P450 BM3 CM1 (R255P/P329H), which was subjected to directed evolution and site saturation mutagenesis of four positions. The latter led to the identification of position R255, which when introduced in the P450 BM3 WT, outperformed all other variants. The initial oxidation rate of nicotinamide adenine dinucleotide phosphate (NADPH) consumption increased ≈140-fold (WT: 8.3 ± 1.3 min-1; R255L: 1168 ± 163 min-1), total turnover number (TTN) increased ≈21-fold (WT: 40 ± 3; R255L: 860 ± 15), and coupling efficiency, ≈2.9-fold (WT: 8.8 ± 0.1%; R255L: 25.7 ± 1.0%). Computational analysis showed that substitution R255L (distant from the heme-cofactor) does not have the salt bridge formed with D217 in WT, which introduces flexibility into the I-helix and leads to a heme rearrangement allowing for efficient hydroxylation.

Mechanistic and structural insight into promiscuity based metabolism of cardiac drug digoxin by gut microbial enzyme.

The recent advances in microbiome studies have revealed the role of gut microbiota in altering the pharmacological properties of oral drugs, which contributes to patient-response variation and undesired effect of the drug molecule. These studies are essential to guide us for achieving the desired efficacy and pharmacological activity of the existing drug molecule or for discovering novel and more effective therapeutics. However, one of the main limitations is the lack of atomistic details on the binding and metabolism of these drug molecules by gut-microbial enzymes. Therefore, in this study, for a well-known and important FDA-approved cardiac glycoside drug, digoxin, we report the atomistic details and energy economics for its binding and metabolism by the Cgr2 protein of Eggerthella lenta DSM 2243. It was observed that the binding pocket of digoxin to Cgr2 primarily involved the negatively charged polar amino acids and a few non-polar hydrophobic residues. The drug digoxin was found to bind Cgr2 at the same binding site as that of fumarate, which is the proposed natural substrate. However, digoxin showed a much lower binding energy (17.75 ± 2 Kcal mol-1 ) than the binding energy (42.17 ± 2 Kcal mol-1 ) of fumarate. This study provides mechanistic insights into the structural and promiscuity-based metabolism of widely used cardiac drug digoxin and presents a methodology, which could be useful to confirm the promiscuity-based metabolism of other orally administrated drugs by gut microbial enzymes and also help in designing strategies for improving the efficacy of the drugs.

A Comparative Reengineering Study of cpADH5 through Iterative and Simultaneous Multisite Saturation Mutagenesis.

Positions identified in directed evolution campaigns or by (semi)rational design can be recombined iteratively or simultaneously. Iterative recombination has yielded many success stories and is beneficially used if screening capabilities are limited (four iterative SSMs generate 20×4=80 different enzyme variants). Simultaneous site saturation mutagenesis offers significantly higher diversity (204 =160 000 variants) and enables greater improvements to be found, especially if the selected positions are in close proximity to each other (cooperative effects). Here we report a first comprehensive comparison of iterative and simultaneous saturation of four residues in Candida parapsilosis alcohol dehydrogenase 5 (cpADH5) with methyl 3-hydroxyhexanoate as substrate. Screening of 7200 clones from 33 site saturation mutagenesis libraries (exploring 17 recombination paths) yielded the cpADH5 W286A variant, with a 82-fold improved initial activity toward methyl 3-hydroxyhexanoate. Screening 3500 clones from a single OmniChange library with four positions (C57, W116, L119, and W286; 1.8 % of the generated sequence space) yielded the cpADH5 C57V/W286S variant, with a 108-fold improvement in initial activity toward methyl 3-hydroxyhexanoate. A 1.8 % coverage of the sequence space of the simultaneous multisite saturation library was, in comparison to the investigated 17 recombination paths, sufficient to identify a cpADH5 variant with improved activity.

A hydroquinone-specific screening system for directed P450 evolution.

The direct hydroxylation of benzene to hydroquinone (HQ) under mild reaction conditions is a challenging task for chemical catalysts. Cytochrome P450 (CYP) monooxygenases are known to catalyze the oxidation of a variety of aromatic compounds with atmospheric dioxygen. Protein engineering campaigns led to the identification of novel P450 variants, which yielded improvements in respect to activity, specificity, and stability. An effective screening strategy is crucial for the identification of improved enzymes with desired characteristics in large mutant libraries. Here, we report a first screening system designed for screening of P450 variants capable to produce hydroquinones. The hydroquinone quantification assay is based on the interaction of 4-nitrophenylacetonitrile (NpCN) with hydroquinones under alkaline conditions. In the 96-well plate format, a low detection limit (5 µM) and a broad linear detection range (5 to 250 µM) were obtained. The NpCN assay can be used for the quantification of dihydroxylated aromatic compounds such as hydroquinones, catechols, and benzoquinones. We chose the hydroxylation of pseudocumene by P450 BM3 as a target reaction and screened for improved trimethylhydroquinone (TMHQ) formation. The new P450 BM3 variant AW2 (R47Q, Y51F, I401M, A330P) was identified by screening a saturation mutagenesis library of amino acid position A330 with the NpCN assay. In summary, a 70-fold improved TMHQ formation was achieved with P450 BM3 AW2 when compared to the wild type (WT) and a 1.8-fold improved TMHQ formation compared to the recently reported P450 BM3 M3 (R47S, Y51W, A330F, I401M).

Inversion of cpADH5 Enantiopreference and Altered Chain Length Specificity for Methyl 3-Hydroxyalkanoates.

Expanding the substrate scope of enzymes opens up new routes for synthesis of valuable chemicals. Ketone-functionalized fatty acid derivatives and corresponding chiral alcohols are valuable building blocks for the synthesis of a variety of chemicals including pharmaceuticals. The alcohol dehydrogenase from Candida parapsilosis (cpADH5) catalyzes the reversible oxidations of chiral alcohols and has a broad substrate range; a challenge for cpADH5 is to convert alcohols with small substituents (methyl or ethyl) next to the oxidized alcohol moiety. Molecular docking studies revealed that W286 is located in the small binding pocket and limits the access to substrates that contain aliphatic chains longer than ethyl substituent. In the current manuscript, we report that positions L119 and W286 are key residues to boost oxidation of medium chain methyl 3-hydroxy fatty acids; interestingly the enantiopreference toward methyl 3-hydroxybutyrate was inverted. Kinetic characterization of W286A showed a 5.5 fold increase of Vmax and a 9.6 fold decrease of Km values toward methyl 3-hydroxyhexanoate (Vmax : 2.48 U mg- and Km : 4.76 mm). Simultaneous saturation at positions 119 and 286 library yielded a double mutant (L119M/W286S) with more than 30-fold improved activity toward methyl 3-hydroxyoctanoate (WT: no conversion; L119M/W286S: 30 %) and inverted enantiopreference (S-enantiomer ≥99 % activity decrease and R-enantiomer >20-fold activity improvement) toward methyl 3-hydroxybutyrate.

What's My Substrate? Computational Function Assignment of Candida parapsilosis ADH5 by Genome Database Search, Virtual Screening, and QM/MM Calculations.

Zinc-dependent medium chain reductase from Candida parapsilosis can be used in the reduction of carbonyl compounds to pharmacologically important chiral secondary alcohols. To date, the nomenclature of cpADH5 is differing (CPCR2/RCR/SADH) in the literature, and its natural substrate is not known. In this study, we utilized a substrate docking based virtual screening method combined with KEGG, MetaCyc pathway, and Candida genome databases search for the discovery of natural substrates of cpADH5. The virtual screening of 7834 carbonyl compounds from the ZINC database provided 94 aldehydes or methyl/ethyl ketones as putative carbonyl substrates. Out of which, 52 carbonyl substrates of cpADH5 with catalytically active docking pose were identified by employing mechanism based substrate docking protocol. Comparison of the virtual screening results with KEGG, MetaCyc database search, and Candida genome pathway analysis suggest that cpADH5 might be involved in the Ehrlich pathway (reduction of fusel aldehydes in leucine, isoleucine, and valine degradation). Our QM/MM calculations and experimental activity measurements affirmed that butyraldehyde substrates are the potential natural substrates of cpADH5, suggesting a carbonyl reductase role for this enzyme in butyraldehyde reduction in aliphatic amino acid degradation pathways. Phylogenetic tree analysis of known ADHs from Candida albicans shows that cpADH5 is close to caADH5. We therefore propose, according to the experimental substrate identification and sequence similarity, the common name butyraldehyde dehydrogenase cpADH5 for Candida parapsilosis CPCR2/RCR/SADH.

Investigation of Structural Determinants for the Substrate Specificity in the Zinc-Dependent Alcohol Dehydrogenase CPCR2 from Candida parapsilosis.

Zinc-dependent alcohol dehydrogenases (ADHs) are a class of enzymes applied in different biocatalytic processes ranging from lab to industrial scale. However, one drawback is the limited substrate range, necessitating a whole array of different ADHs for the relevant substrate classes. In this study, we investigated structural determinants of the substrate spectrum in the zinc-dependent ADH carbonyl reductase 2 from Candida parapsilosis (CPCR2), combining methods of mutational analysis with in silico substrate docking. Assigned active site residues were genetically randomized, and the resulting mutant libraries were screened with a selection of challenging carbonyl substrates. Three variants (C57A, W116K, and L119M) with improved activities toward different substrates were detected at neighboring positions in the active site. Thus, all possible combinations of the mutations were generated and characterized for their substrate specificity, yielding several improved variants. The most interesting were a C57A variant, with a 27-fold increase in specific activity for 4'-acetamidoacetophenone, and the double mutant CPCR2 B16-(C57A, L119M), with a 45-fold improvement in the kcat ⋅KM (-1) value. The obtained variants were further investigated by in silico docking experiments. The results indicate that the mentioned residues are structural determinants of the substrate specificity of CPCR2, being major players in the definition of the active site. Comparison of these results with closely related enzymes suggests that these might even be transferred to other ADHs.

Activity prediction of substrates in NADH-dependent carbonyl reductase by docking requires catalytic constraints and charge parameterization of catalytic zinc environment.

Molecular docking of substrates is more challenging compared to inhibitors as the reaction mechanism has to be considered. This becomes more pronounced for zinc-dependent enzymes since the coordination state of the catalytic zinc ion is of greater importance. In order to develop a predictive substrate docking protocol, we have performed molecular docking studies of diketone substrates using the catalytic state of carbonyl reductase 2 from Candida parapsilosis (CPCR2). Different docking protocols using two docking methods (AutoDock Vina and AutoDock4.2) with two different sets of atomic charges (AM1-BCC and HF-RESP) for catalytic zinc environment and substrates as well as two sets of vdW parameters for zinc ion were examined. We have selected the catalytic binding pose of each substrate by applying mechanism based distance criteria. To compare the performance of the docking protocols, the correlation plots for the binding energies of these catalytic poses were obtained against experimental Vmax values of the 11 diketone substrates for CPCR2. The best correlation of 0.73 was achieved with AutoDock4.2 while treating catalytic zinc ion in optimized non-bonded (NBopt) state with +1.01 charge on the zinc ion, compared to 0.36 in non-bonded (+2.00 charge on the zinc ion) state. These results indicate the importance of catalytic constraints and charge parameterization of catalytic zinc environment for the prediction of substrate activity in zinc-dependent enzymes by molecular docking. The developed predictive docking protocol described here is in principle generally applicable for the efficient in silico substrate spectra characterization of zinc-dependent ADH.

Identification of dual Acetyl-CoA carboxylases 1 and 2 inhibitors by pharmacophore based virtual screening and molecular docking approach.

Acetyl-CoA carboxylase (ACC) is a crucial metabolic enzyme that plays a vital role in obesity-induced type 2 diabetes and fatty acid metabolism. To identify dual inhibitors of Acetyl-CoA carboxylase1 and Acetyl-CoA carboxylase2, a pharmacophore modelling approach has been employed. The best HypoGen pharmacophore model for ACC2 inhibitors (Hypo1_ACC2) consists of one hydrogen bond acceptor, one hydrophobic aliphatic and one hydrophobic aromatic feature, whereas the best pharmacophore (Hypo1_ACC1) for ACC1 consists of one additional hydrogen-bond donor (HBD) features. The best pharmacophore hypotheses were validated by various methods such as test set, decoy set and Cat-Scramble methodology. The validated pharmacophore models were used to screen several small-molecule databases, including Specs, NCI, ChemDiv and Natural product databases to identify the potential dual ACC inhibitors. The virtual hits were then subjected to several filters such as estimated [Formula: see text] value, quantitative estimation of drug-likeness and molecular docking analysis. Finally, three novel compounds with diverse scaffolds were selected as potential starting points for the design of novel dual ACC inhibitors.

QM/MM Simulations for the Broken-Symmetry Catalytic Reaction Mechanism of Human Arginase I.

Human arginase I (HARGI) is a metalloprotein highly expressed in the liver cytosol and catalyzes the hydrolysis of l-arginine to form l-ornithine and urea. Understanding the reaction mechanism would be highly helpful to design new inhibitor molecules for HARGI as it is a target for heart- and blood-related diseases. In this study, we explored the hydrolysis reaction mechanism of HARGI with antiferromagnetic and ferromagnetic coupling between two Mn(II) ions at the catalytic site by employing molecular dynamics simulations coupled with quantum mechanics and molecular mechanics (QM/MM). The spin states, high-spin ferromagnetic couple (S Mn1 = 5/2, S Mn2 = 5/2), low-spin ferromagnetic couple (S Mn1 = 1/2, S Mn2 = 1/2), high-spin antiferromagnetic couple (S Mn1 = 5/2, S Mn2 = -5/2), and low-spin antiferromagnetic couple (S Mn1 = 1/2, S Mn2 = -1/2) are considered, and the calculated energetics for the complex of the substrate and HARGI are compared. The results show that the high-spin antiferromagnetic couple (S Mn1 = 5/2, S Mn2 = -5/2) is more stable than other spin states. The low-spin ferromagnetic and antiferromagnetic coupled states are highly unstable compared with the corresponding high-spin states. The high-spin antiferromagnetic couple (S Mn1 = 5/2, S Mn2 = -5/2) is stabilized by 0.39 kcal/mol compared with the ferromagnetic couple (S Mn1 = 5/2, S Mn2 = 5/2). The reaction mechanism is independent of spin states; however, the energetics of transition states and intermediates are more stable in the case of the high-spin antiferromagnetic couple (S Mn1 = 5/2, S Mn2 = -5/2) than the corresponding ferromagnetic state. It is evident that the calculated coupling constants are higher for antiferromagnetic states and, interestingly, superexchange coupling is found to occur between Mn(II) ions via hydroxide ions in a reactant. The hydroxide ion enhances the coupling interaction and initiates the catalytic reaction. It is also noted that the first intermediate structure where there is no superexchange coupling is similar to the known inhibitor 2(S)-amino-6-boronohexanoic acid.

The molecular basis and enzyme engineering strategies for improvement of coupling efficiency in cytochrome P450s.

Cytochrome P450s are heme-thiolate enzymes that have been broadly applied in pharmaceutical and biosynthesis because of their efficient oxidation at inert carbons. Extensive engineering campaigns are applied to P450s to explore new non-natural substrates and reactions; however, achieving high coupling efficiency is one of the main challenges. The undesirable uncoupling reactions result in the extra consumption of expensive cofactor NAD(P)H, and lead to the accumulation of reactive oxygen species and the inactivation of enzymes and organisms. Using protein engineering methods, these limitations can be overcome by engineering and fine-tuning P450s. A systemic perspective of the enzyme structure and the catalytic mechanism is essential for P450 engineering campaigns for higher coupling efficiency. This review provide an overview on factors contributing to uncoupling and protein engineering approaches to minimize uncoupling and thereby generating efficient and robust P450s for industrials use. Contributing uncoupling factors are classified into three main groups: i) substrate binding pocket; ii) ligand access tunnel(s); and iii) electron transfer pathway(s). Finally, we draw future directions for combinations of effective state-of-the-art technologies and available software/online tools for P450s engineering campaigns.