Phylogeny and Structure of Fatty Acid Photodecarboxylases and Glucose-Methanol-Choline Oxidoreductases

Glucose-methanol-choline (GMC) oxidoreductases are a large and diverse family of flavin-binding enzymes found in all kingdoms of life. Recently, a new related family of proteins has been discovered in algae named fatty acid photodecarboxylases (FAPs). These enzymes use the energy of light to convert fatty acids to the corresponding Cn-1 alkanes or alkenes, and hold great potential for biotechnological application. In this work, we aimed at uncovering the natural diversity of FAPs and their relations with other GMC oxidoreductases. We reviewed the available GMC structures, assembled a large dataset of GMC sequences, and found that one active site amino acid, a histidine, is extremely well conserved among the GMC proteins but not among FAPs, where it is replaced with alanine. Using this criterion, we found several new potential FAP genes, both in genomic and metagenomic databases, and showed that related bacterial, archaeal and fungal genes are unlikely to be FAPs. We also identified several uncharacterized clusters of GMC-like proteins as well as subfamilies of proteins that lack the conserved histidine but are not FAPs. Finally, the analysis of the collected dataset of potential photodecarboxylase sequences revealed the key active site residues that are strictly conserved, whereas other residues in the vicinity of the flavin adenine dinucleotide (FAD) cofactor and in the fatty acid-binding pocket are more variable. The identified variants may have different FAP activity and selectivity and consequently may prove useful for new biotechnological applications, thereby fostering the transition from a fossil carbon-based economy to a bio-economy by enabling the sustainable production of hydrocarbon fuels.

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Activation and selectivity of OTUB-1 and OTUB-2 deubiquitinylases

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013073 ◽

2020 ◽

Vol 295 (20) ◽

pp. 6972-6982

Author(s):

Dakshinamurthy Sivakumar ◽

Vikash Kumar ◽

Michael Naumann ◽

Matthias Stein

Keyword(s):

Active Site ◽

Electrostatic Interactions ◽

Active Form ◽

Cysteine Proteases ◽

Binding Pocket ◽

Catalytic Triad ◽

Catalytic Site ◽

Dynamics Simulation ◽

Active Site Residues ◽

Catalytically Active

The ovarian tumor domain (OTU) deubiquitinylating cysteine proteases OTUB1 and OTUB2 (OTU ubiquitin aldehyde binding 1 and 2) are representative members of the OTU subfamily of deubiquitinylases. Deubiquitinylation critically regulates a multitude of important cellular processes, such as apoptosis, cell signaling, and growth. Moreover, elevated OTUB expression has been observed in various cancers, including glioma, endometrial cancer, ovarian cancer, and breast cancer. Here, using molecular dynamics simulation approaches, we found that both OTUB1 and OTUB2 display a catalytic triad characteristic of proteases but differ in their configuration and protonation states. The OTUB1 protein had a prearranged catalytic site, with strong electrostatic interactions between the active-site residues His265 and Asp267. In OTUB2, however, the arrangement of the catalytic triad was different. In the absence of ubiquitin, the neutral states of the catalytic-site residues in OTUB2 were more stable, resulting in larger distances between these residues. Only upon ubiquitin binding did the catalytic triad in OTUB2 rearrange and bring the active site into a catalytically feasible state. An analysis of water access channels revealed only a few diffusion trajectories for the catalytically active form of OTUB1, whereas in OTUB2 the catalytic site was solvent-accessible, and a larger number of water molecules reached and left the binding pocket. Interestingly, in OTUB2, the catalytic residues His224 and Asn226 formed a stable hydrogen bond. We propose that the observed differences in activation kinetics, protonation states, water channels, and active-site accessibility between OTUB1 and OTUB2 may be relevant for the selective design of OTU inhibitors.

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Roles of key active-site residues in flavocytochrome P450 BM3

Biochemical Journal ◽

10.1042/bj3390371 ◽

1999 ◽

Vol 339 (2) ◽

pp. 371-379 ◽

Cited By ~ 118

Author(s):

Michael A. NOBLE ◽

Caroline S. MILES ◽

Stephen K. CHAPMAN ◽

Dominikus A. LYSEK ◽

Angela C. MACKAY ◽

...

Keyword(s):

Electron Transfer ◽

Fatty Acid ◽

Fatty Acid Oxidation ◽

Active Site ◽

Side Chain ◽

Acid Oxidation ◽

Dodecanoic Acid ◽

Wild Type ◽

Active Site Residues ◽

P450 Bm3

The effects of mutation of key active-site residues (Arg-47, Tyr-51, Phe-42 and Phe-87) in Bacillus megaterium flavocytochrome P450 BM3 were investigated. Kinetic studies on the oxidation of laurate and arachidonate showed that the side chain of Arg-47 contributes more significantly to stabilization of the fatty acid carboxylate than does that of Tyr-51 (kinetic parameters for oxidation of laurate: R47A mutant, Km 859 µM, kcat 3960 min-1; Y51F mutant, Km 432 µM, kcat 6140 min-1; wild-type, Km 288 µM, kcat 5140 min-1). A slightly increased kcat for the Y51F-catalysed oxidation of laurate is probably due to decreased activation energy (ΔG‡) resulting from a smaller ΔG of substrate binding. The side chain of Phe-42 acts as a phenyl ‘cap ’ over the mouth of the substrate-binding channel. With mutant F42A, Km is massively increased and kcat is decreased for oxidation of both laurate (Km 2.08 mM, kcat 2450 min-1) and arachidonate (Km 34.9 µM, kcat 14620 min-1; compared with values of 4.7 µM and 17100 min-1 respectively for wild-type). Amino acid Phe-87 is critical for efficient catalysis. Mutants F87G and F87Y not only exhibit increased Km and decreased kcat values for fatty acid oxidation, but also undergo an irreversible conversion process from a ‘fast ’ to a ‘slow ’ rate of substrate turnover [for F87G (F87Y)-catalysed laurate oxidation: kcat ‘fast ’, 760 (1620) min-1; kcat ‘slow ’, 48.0 (44.6) min-1; kconv (rate of conversion from fast to slow form), 4.9 (23.8) min-1]. All mutants showed less than 10% uncoupling of NADPH oxidation from fatty acid oxidation. The rate of FMN-to-haem electron transfer was shown to become rate-limiting in all mutants analysed. For wild-type P450 BM3, the rate of FMN-to-haem electron transfer (8340 min-1) is twice the steady-state rate of oxidation (4100 min-1), indicating that other steps contribute to rate limitation. Active-site structures of the mutants were probed with the inhibitors 12-(imidazolyl)dodecanoic acid and 1-phenylimidazole. Mutant F87G binds 1-phenylimidazole > 10-fold more tightly than does the wild-type, whereas mutant Y51F binds the haem-co-ordinating fatty acid analogue 12-(imidazolyl)dodecanoic acid > 30-fold more tightly than wild-type.

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Amine Oxidation Mediated by N-Methyltryptophan Oxidase: Computational Insights into the Mechanism, Role of Active-Site Residues, and Covalent Flavin Binding

ACS Catalysis ◽

10.1021/cs501694q ◽

2015 ◽

Vol 5 (2) ◽

pp. 1227-1239 ◽

Cited By ~ 13

Author(s):

Bora Karasulu ◽

Walter Thiel

Keyword(s):

Active Site ◽

Active Site Residues ◽

Amine Oxidation ◽

Flavin Binding

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PickPocket : Pocket binding prediction for specific ligands family using neural networks.

10.1101/2020.04.15.042655 ◽

2020 ◽

Author(s):

Benjamin Thomas VIART ◽

Claudio Lorenzi ◽

María Moriel-Carretero ◽

Sofia Kossida

Keyword(s):

Neural Networks ◽

Fatty Acid ◽

Ligand Binding ◽

Binding Sites ◽

Structural Data ◽

Binding Pocket ◽

Fatty Acid Binding Proteins ◽

Binding Prediction ◽

Fatty Acid Binding ◽

Binding Pockets

Most of the protein biological functions occur through contacts with other proteins or ligands. The residues that constitute the contact surface of a ligand-binding pocket are usually located far away within its sequence. Therefore, the identification of such motifs is more challenging than the linear protein domains. To discover new binding sites, we developed a tool called PickPocket that focuses on a small set of user-defined ligands and uses neural networks to train a ligand-binding prediction model. We tested PickPocket on fatty acid-like ligands due to their structural similarities and their under-representation in the ligand-pocket binding literature. Our results show that for fatty acid-like molecules, pocket descriptors and secondary structures are enough to obtain predictions with accuracy >90% using a dataset of 1740 manually curated ligand-binding pockets. The trained model could also successfully predict the ligand-binding pockets using unseen structural data of two recently reported fatty acid-binding proteins. We think that the PickPocket tool can help to discover new protein functions by investigating the binding sites of specific ligand families. The source code and all datasets contained in this work are freely available at https://github.com/benjaminviart/PickPocket .

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Structural basis for UCN-01 (7-hydroxystaurosporine) specificity and PDK1 (3-phosphoinositide-dependent protein kinase-1) inhibition

Biochemical Journal ◽

10.1042/bj20031119 ◽

2003 ◽

Vol 375 (2) ◽

pp. 255-262 ◽

Cited By ~ 73

Author(s):

David KOMANDER ◽

Gursant S. KULAR ◽

Jennifer BAIN ◽

Matthew ELLIOTT ◽

Dario R. ALESSI ◽

...

Keyword(s):

Protein Kinase ◽

Hydroxy Group ◽

Active Site ◽

Binding Pocket ◽

Kinase Domain ◽

Structural Basis ◽

Active Site Residues ◽

Dependent Protein Kinase ◽

Growth Factor Signalling ◽

Dependent Protein

PDK1 (3-phosphoinositide-dependent protein kinase-1) is a member of the AGC (cAMP-dependent, cGMP-dependent, protein kinase C) family of protein kinases, and has a key role in insulin and growth-factor signalling through phosphorylation and subsequent activation of a number of other AGC kinase family members, such as protein kinase B. The staurosporine derivative UCN-01 (7-hydroxystaurosporine) has been reported to be a potent inhibitor for PDK1, and is currently undergoing clinical trials for the treatment of cancer. Here, we report the crystal structures of staurosporine and UCN-01 in complex with the kinase domain of PDK1. We show that, although staurosporine and UCN-01 interact with the PDK1 active site in an overall similar manner, the UCN-01 7-hydroxy group, which is not present in staurosporine, generates direct and water-mediated hydrogen bonds with active-site residues. Inhibition data from UCN-01 tested against a panel of 29 different kinases show a different pattern of inhibition compared with staurosporine. We discuss how these differences in inhibition could be attributed to specific interactions with the additional 7-hydroxy group, as well as the size of the 7-hydroxy-group-binding pocket. This information could lead to opportunities for structure-based optimization of PDK1 inhibitors.

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Active-site residues of a plant membrane-bound fatty acid elongase β-ketoacyl-CoA synthase, FAE1 KCS

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids ◽

10.1016/s1388-1981(00)00168-2 ◽

2001 ◽

Vol 1530 (1) ◽

pp. 77-85 ◽

Cited By ~ 45

Author(s):

Mahin Ghanevati ◽

Jan G. Jaworski

Keyword(s):

Fatty Acid ◽

Active Site ◽

Active Site Residues ◽

Fatty Acid Elongase ◽

Membrane Bound

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UHR PX and NPC studies of H-FABP water network with tiny perdeuterated crystals

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314087993 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C1200-C1200

Author(s):

Alberto Podjarny ◽

Matthew Blakeley ◽

Michael Haertlein ◽

Andre Mitschler ◽

Alexandra Cousido-Siah ◽

...

Keyword(s):

Fatty Acid ◽

Charge Distribution ◽

Binding Pocket ◽

Internal Cavity ◽

Water Molecules ◽

Fatty Acid Binding ◽

X Ray ◽

X Ray Crystallography ◽

Internal Water ◽

Dynamics Simulations

We have obtained very detailed information about the internal water molecules in the large internal cavity inside fatty acid binding (FABP) proteins , in the presence of bound fatty acids (FA), by Ultra High Resolution X-Ray Crystallography (UHR) to 0.7 Å and Neutron Protein Crystallography (NPC) to 1.9 Å using a "radically small" (V=0.05 mm3) crystal. These waters form a very well ordered dense cluster of 12 molecules, positioned between the hydrophilic internal wall of the cavity and the fatty acid molecule. This information has been used for a detailed electrostatic analysis based on the charge distribution description modeled in the multipole formalism and on the Atoms in Molecules theory. This information is also being used in molecular dynamics simulations of H-FABP and its complex with FA in order to quantify the energetic contribution of these internal waters to the binding energy. The experiment has been done with oleic acid, coming with the protein expressed in E. Coli. The results have been analyzed in order to understand the interactions between the FA, the internal water and the protein, and in particular the role played by the water molecules in determining the potency and specificity of FA binding to FABPs. The major tool for visualizing the water molecules inside the H-FABP cavity is UHR X-Ray Crystallography combined with NPC. UHR crystallographic structures give the positions of hydrogen and oxygen atoms for well-ordered water molecules. NPC determines hydrogen atom positions, particularly of water molecules which have multiple conformations, leading to the best possible crystallographic model. This model was then complemented by a transferred charge distribution to accurately determine the electrostatic and topological properties in the binding pocket, providing a description of the way water molecules in hydration layer contribute to the binding of ligand, which is essential to understand and model ligand binding.

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Genome Mining, Phylogenetic and Structural Analysis of Bacterial Nitrilases for the Biodegradation of Nitrile Compounds

10.21203/rs.3.rs-561386/v1 ◽

2021 ◽

Author(s):

Richa Salwan ◽

Vivek Sharma ◽

Surajit Das

Keyword(s):

Structural Analysis ◽

Substrate Specificity ◽

Active Site ◽

Structural Information ◽

Genome Mining ◽

Binding Pocket ◽

Catalytic Triad ◽

Vital Role ◽

Active Site Residues ◽

Motif Analysis

Abstract Microbial nitrilases play vital role in biodegradation of nitrile-containing contaminants in pollutant and effluents treatments in chemical and textile industries as well as the biosynthesis of IAA from tryptophan in plants. However, the lack of structural information hinders the correlation of its activity and substrate specificity. Here, we have identified bacterial genomes for nitrilases bearing unassigned functions including hypothetical, uncharacterized, or putative role. The genomic annotations revealed four predicted nitrilases encoding genes as uncharacterized subgroup of the nitrilase superfamily. Further, the annotation of these nitrilases revealed relatedness with nitrilase hydratases and cyanoalanine hydratases. The characterization of motif analysis of these protein sequences, predicted a single motif of 20-28 aa, and glutamate (E), lysine (K) and cysteine (C) residues as a part of catalytic triad along with several active site residues. The structural analysis of the modeled nitrilases revealed geometrical and close conformation of α-helices and β-sheets arranged in a sandwich structure. The catalytic residues constituted the substrate binding pocket and exhibited the wide nitrile substrate spectra for both aromatic and aliphatic nitriles containing compounds. The aromatic amino acid residues Y159 in active site were predicted to show importance for substrate specificity. The substitution of non-aromatic alanine residue in place of Y159 completely disrupted the catalytic activity for indole-3-acetonitrile. The present study reports several uncharacterized nitrilases which have not been reported so far for their role in the biodegradation of pollutants, xenobiotics which could find applications in industries.

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