Gaurao V. Dhoke

PHARMCADD INDIA PRIVATE LTD

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 u...

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 h...

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...

Alcohol dehydrogenase from Rhodococcus erythropolis (ReADH) is a versatile biocatalyst for stereoselective reduction of ketones to chiral alcohols. Despite the known broad substrate scope, ReADH accepts only carbonyl substrates with methyl/ethyl group adjacent to carbonyl moiety, which limits its use in chiral alcohol synthesis for pharmaceuticals....

Copper efflux oxidase (CueO) from Escherichia coli is a special bacterial laccase due to its fifth copper binding site. In contrast with previous reports, this work illustrates the relationship between the fifth copper region and electrocatalytic properties. Through a semi‐rational design, an increase in localized structural stability and a decreas...

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 electrocata...

Catechol sulfates act in our body as important antioxidants and often have anti-inflammatory properties. Enzymatic sulfation of catechol as well as substituted catechols by aryl sulfotransferases opens up an environmentally-friendly route for chemo- and/or regioselective sulfation sulfation. This study focuses on reengineering of loop12 and loop13...

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 enzyme...

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...

Antibody–drug conjugates (ADCs) combine the high specificity of antibodies with cytotoxic payloads. However, the present strategies for the synthesis of ADCs either yield unstable or heterogeneous products or involve complex processes. Here, we report a computational approach that leverages molecular docking and molecular dynamics simulations to de...

Antibody–drug conjugates (ADCs) combine the high specificity of antibodies with cytotoxic payloads. However, the present strategies for the synthesis of ADCs either yield unstable or heterogeneous products or involve complex processes. Here, we report a computational approach that leverages molecular docking and molecular dynamics simulations to de...

Chemoenzymatic cascades enable reactions with the high productivity of chemocatalysts and high selectivity of enzymes. Nevertheless, the combination of these different fields of catalysis is prone to mutual deactivation of metal‐ and biocatalysts. In this study, a one‐pot sequential two‐step catalytic cascade reaction was successfully implemented f...

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...

Ionic liquids (ILs) are widely recognized as highly attractive solvents for biocatalysis due to their high stabilities and diverse tunable chemical properties. A major challenge for biocatalysis in ILs is the reduction of enzyme activity. Thus, molecular understanding of enzyme-ILs interactions is crucial to design enzymes for improved ILs resistan...

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 microtit...

Light-Oxygen-Voltage (LOV) domains are conserved parts of photoreceptors in plants, bacteria and fungi that bind flavins as chromophore 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 anaerobi...

Downstream processing to obtain enantiopure compounds from racemic mixture relies mainly on crystallization. Nature built transporters that specifically translocate enantiomers through membranes. Here the β-barrel transmembrane protein FhuA was reengineered into a chiral channel protein (FhuAF4) to resolve racemic mixtures of D-/L-arginine. The eng...

To date, commercial laccase preparations are used in food, textile, and paper and pulp industry (mild pH). Laccases are attractive in synthesis of dye molecules or oxidative lignin treatment, which take place at high pH (pH ≥ 8.0). So far one fungal laccase has been reported to be active at alkaline pH. Herein, we report the engineering of the fung...

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 b...

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, w...

Positions identified in directed evolution campaigns or by (semi‐) rational design can be recombined iteratively or simultaneously. Iterative recombination (CAST method) yielded many success stories and is beneficially used if screening capabilities are limited (four iterative SSMs generated 20x4= 80 different enzyme variants). Simultaneous site sa...

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 existin...

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 th...

Candida parapsilosis zinc-dependent medium chain reductase enzyme can be used in the production of industrially important chiral secondary alcohols by reduction of carbonyl compounds. In literature, the nomenclature of cpADH5 is unclear (CPCR2/RCR/SADH), and its natural substrate is still unknown. In this study a substrate docking based virtual scr...

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, 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 vir...

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 d...

Details of all correlation plots between experimentally determined Vmax and calculated binding energies, binding orientations for all diketone substrates, geometry optimized structure of catalytic zinc environment, plots for the important distance criteria applied for selecting the catalytic binding orientation can be found in the supporting inform...

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 su...

Oxido-reductases from medium-chain dehydrogenase/reductase (MDR) family are excellent biocatalysts for the generation of optically pure alcohols from prochiral ketones. The mechanism of hydride and proton transfer steps in zinc-catalyzed carbonyl reduction has been investigated by quantum mechanical/molecular mechanical (QM/MM) calculations. The re...

Phosphodiesterase 4 (PDE4), is a hydrolytic enzyme, is proposed as a promising target in asthma and chronic obstructive pulmonary disease. PDE4B selective inhibitors are desirable to reduce the dose limiting adverse effect associated with non-selective PDE4B inhibitors. To achieve this goal, ligand based pharmacophore modeling and molecular docking...

The p38α mitogen-activated protein (MAP) kinase plays a vital role in treating many inflammatory diseases. In the present study, a combined ligand and structure based pharmacophore model was developed to identify potential DFG-in selective p38 MAP kinase inhibitors. Conformations of co-crystallised inhibitors were used in the development and valida...

PknB is an essential serine/threonine kinase of Mycobacterium tuberculosis, which plays an important role in a number of signalling pathways involved in cell division and metabolism. This finding supports the development of protein kinase inhibitors as new potential anti-tubercular agents. The reported PknB inhibitors were found to be less potent a...

p38 mitogen-activated protein (MAP) kinases are the serine/threonine protein kinases, which play a vital role in cellular responses to external stress signals. p38 MAP kinase inhibitors have shown anti-inflammatory effects in the preclinical disease models, primarily through inhibition of the expression of inflammatory mediators. A number of struct...

The cytochrome bc1 complex (EC 1.10.2.2, bc1) is a vital component of the cellular respiratory chain and the photosynthetic apparatus in photosynthetic bacteria  It found in the plasma membrane of bacteria and in the inner mitochondrial membrane of eukaryotes  Inhibitors of cytochrome bc1 are grouped into three classes: class P inhibitors bind to...

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_ACC...

Cytochrome bc1 (EC 1.10.2.2, bc1) is an essential component of the cellular respiratory chain, which catalyzes electron transfer from quinol to cytochrome c, and concomitantly the translocation of protons across the membrane. It has been identified as a promising target in malaria parasites. The structure based pharmacophore modelling and molecular...

Matrix metalloproteinase 12 (MMP-12) is a potential therapeutic target for the treatment of chronic obstructive pulmonary disorder and other inflammatory disorders. Ligand-based 3D QSAR pharmacophore modelling approach was employed to reveal structural and chemical features necessary for the inhibition of MMP-12. The best HypoGen pharmacophore mode...

Acetyl-CoAcarboxylase (ACC) is a crucial metabolic enzyme, which plays a vital role in fatty acid metabolism and obesity induced type 2 diabetes. Herein, we have performed 3D-QSAR and moleculardockinganalysis on novel series of (4-piperidinyl)-piperazines to design potent ACC inhibitors. This study correlates the ACC inhibitory activities of 68 (4-...

A combined ligand and structure based pharmacophore modeling approach was employed to reveal structural and chemical features necessary for PPAR-alpha agonistic activity. The best HypoGen pharmacophore model Hypo1 for PPAR-alpha agonists contains two hydrogen-bond acceptor (HBA), two general hydrophobic (H), and one negative ionizable (NI) feature....

Tuberculosis is one of the leading contagious diseases, caused by Mycobacterium tuberculosis. Despite improvements in anti-tubercular agents, it remains one of the most prevalent infectious diseases worldwide, responsible for a total of 1.6 million deaths annually. The emergence of multidrug resistant strains highlighted the need of discovering nov...

Tuberculosis is one of the leading contagious diseases, caused by Mycobacterium tuberculosis. Despite improvements in anti-tubercular agents, it remains one of the most prevalent infectious diseases worldwide, responsible for a total of 1.6 million deaths annually. The emergence of multidrug resistant strains highlighted the need of discovering nov...

> MAP kinases are serine threonine specific protein kinase that responds to the extracellular stimuli > The extracellular stimuli are mitogen, osmotic stress, cell stress, DNA damage, heat, environmental stimuli, cigarette smoke, and allergens > Four isoforms of p38 MAP kinase a) p38-α (MAPK14, p38 MAP kinase) c) p38-γ (MAPK12, ERK6, SAPK3) b) p38-...