Structural investigation of myo-inositol dehydrogenase from Bacillus subtilis: implications for catalytic mechanism and inositol dehydrogenase subfamily classification

2010 ◽  
Vol 432 (2) ◽  
pp. 237-247 ◽  
Author(s):  
Karin E. van Straaten ◽  
Hongyan Zheng ◽  
David R. J. Palmer ◽  
David A. R. Sanders
Keyword(s):  
Bacillus Subtilis ◽  
Binding Site ◽  
Catalytic Mechanism ◽  
Substrate Binding ◽  
Consensus Sequence ◽  
Catalytic Triad ◽  
Ternary Complexes ◽  
Sequence Motifs ◽  
Subfamily Classification ◽  
Inositol Dehydrogenase

Inositol dehydrogenase from Bacillus subtilis (BsIDH) is a NAD+-dependent enzyme that catalyses the oxidation of the axial hydroxy group of myo-inositol to form scyllo-inosose. We have determined the crystal structures of wild-type BsIDH and of the inactive K97V mutant in apo-, holo- and ternary complexes with inositol and inosose. BsIDH is a tetramer, with a novel arrangement consisting of two long continuous β-sheets, formed from all four monomers, in which the two central strands are crossed over to form the core of the tetramer. Each subunit in the tetramer consists of two domains: an N-terminal Rossmann fold domain containing the cofactor-binding site, and a C-terminal domain containing the inositol-binding site. Structural analysis allowed us to determine residues important in cofactor and substrate binding. Lys97, Asp172 and His176 are the catalytic triad involved in the catalytic mechanism of BsIDH, similar to what has been proposed for related enzymes and short-chain dehydrogenases. Furthermore, a conformational change in the nicotinamide ring was observed in some ternary complexes, suggesting hydride transfer to the si-face of NAD+. Finally, comparison of the structure and sequence of BsIDH with other putative inositol dehydrogenases allowed us to differentiate these enzymes into four subfamilies based on six consensus sequence motifs defining the cofactor- and substrate-binding sites.

Crystal structures of Cg1458 reveal a catalytic lid domain and a common catalytic mechanism for the FAH family

2012 ◽  
Vol 449 (1) ◽  
pp. 51-60 ◽  
Author(s):  
Tingting Ran ◽  
Yanyan Gao ◽  
May Marsh ◽  
Wenjun Zhu ◽  
Meitian Wang ◽  
...  
Keyword(s):  
Glutamic Acid ◽  
Crystal Structures ◽  
Sequence Alignment ◽  
Large Scale ◽  
Catalytic Mechanism ◽  
Enzymatic Catalysis ◽  
Catalytic Triad ◽  
Sequence Motifs ◽  
Fumarylacetoacetate Hydrolase ◽  
Lid Domain

Cg1458 was recently characterized as a novel soluble oxaloacetate decarboxylase. However, sequence alignment identified that Cg1458 has no similarity with other oxaloacetate decarboxylases and instead belongs to the FAH (fumarylacetoacetate hydrolase) family. Differences in the function of Cg1458 and other FAH proteins may suggest a different catalytic mechanism. To help elucidate the catalytic mechanism of Cg1458, crystal structures of Cg1458 in both the open and closed conformations have been determined for the first time up to a resolution of 1.9 Å (1 Å=0.1 nm) and 2.0 Å respectively. Comparison of both structures and detailed biochemical studies confirmed the presence of a catalytic lid domain which is missing in the native enzyme structure. In this lid domain, a glutamic acid–histidine dyad was found to be critical in mediating enzymatic catalysis. On the basis of structural modelling and comparison, as well as large-scale sequence alignment studies, we further determined that the catalytic mechanism of Cg1458 is actually through a glutamic acid–histidine–water triad, and this catalytic triad is common among FAH family proteins that catalyse the cleavage of the C–C bond of the substrate. Two sequence motifs, HxxE and Hxx…xxE have been identified as the basis for this mechanism.

In silicoEvaluation of Substrate Binding Site and Rare Codons in the Structure of CYP152A1

2020 ◽  
Vol 17 (1) ◽  
pp. 10-22
Author(s):  
Mojtaba Mortazavi ◽  
Navid Nezafat ◽  
Manica Negahdaripour ◽  
Mohammad J. Raee ◽  
Masoud Torkzadeh-Mahani ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Binding Site ◽  
Rational Design ◽  
Substrate Binding ◽  
Enzyme Reaction ◽  
Docking Study ◽  
Industrial Applications ◽  
Biological Information ◽  
Substrate Binding Site ◽  
Rare Codons

Background:The Cytochromes P450 (CYPs) have an essential role in the oxidation of endogenous and exogenous molecules. The CYPs are identified in all domains of life, but the CYP152A1 from Bacillus subtilis is specially considered for clinical and industrial applications. The molecular cloning of a new type of CYP from Bacillus subtilis was reported, previously. Here, we describe the hidden layer of biological information of the CYP152A1 enzyme, which can help researchers for better understanding of enzyme application. In this study, four rare codons of enzyme, including Arg63, Arg187, Arg276, and Arg338 were identified and evaluated using the bioinformatics web servers.Methods:Through in silico modeling of CYP152A1 via the I-TASSER server, the above-mentioned rare codons were studied in the structure of enzyme that may have an important role in the proper folding of CYP152A1. In the following, the substrate binding site of CYP152A1 was studied by AutoDock Vina, and the heme and palmitic acid were considered as the substrates.Results:The results of docking study elucidated the Arg242 in the active site is closely related to the substrate binding site of CYP152A1, which help us to further clarify the mechanism of the enzyme reaction.Conclusion:Studies of these hidden information’s can enhance our understanding of CYP152A1 folding and protein expression challenges. Moreover, identification of rare codons can help in the rational design of new and effective drugs.

scholarly journals Bacillus subtilis LmrA Is a Repressor of the lmrAB and yxaGH Operons: Identification of Its Binding Site and Functional Analysis of lmrB and yxaGH

2004 ◽  
Vol 186 (17) ◽  
pp. 5640-5648 ◽  
Author(s):  
Ken-ichi Yoshida ◽  
Yo-hei Ohki ◽  
Makiko Murata ◽  
Masaki Kinehara ◽  
Hiroshi Matsuoka ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Multidrug Resistance ◽  
Binding Site ◽  
Binding Sites ◽  
Promoter Region ◽  
Consensus Sequence ◽  
Efflux Transporter ◽  
Multidrug Efflux ◽  
Genome Wide

ABSTRACT The Bacillus subtilis lmrAB operon is involved in multidrug resistance. LmrA is a repressor of its own operon, while LmrB acts as a multidrug efflux transporter. LmrA was produced in Escherichia coli cells and was shown to bind to the lmr promoter region, in which an LmrA-binding site was identified. Genome-wide screening involving DNA microarray analysis allowed us to conclude that LmrA also repressed yxaGH, which was not likely to contribute to the multidrug resistance. LmrA bound to a putative yxaGH promoter region, in which two tandem LmrA-binding sites were identified. The LmrA regulon was thus determined to comprise lmrAB and yxaGH. All three LmrA-binding sites contained an 18-bp consensus sequence, TAGACCRKTCWMTATAWT, which could play an important role in LmrA binding.

The 5-ketofructose reductase of Gluconobacter sp. strain CHM43 is a novel class in the shikimate dehydrogenase family

2021 ◽  
Author(s):  
Thuy Minh Nguyen ◽  
Masaru Goto ◽  
Shohei Noda ◽  
Minenosuke Matsutani ◽  
Yuki Hodoya ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Amino Acid ◽  
Binding Site ◽  
Catalytic Mechanism ◽  
Substrate Binding ◽  
High Yield ◽  
Strong Increase ◽  
Amino Acid Residues ◽  
Shikimate Dehydrogenase ◽  
Substrate Binding Site

Gluconobacter sp. CHM43 oxidizes mannitol to fructose and then does fructose to 5-keto-D-fructose (5KF) in the periplasmic space. Since NADPH-dependent 5KF reductase was found in the soluble fraction of Gluconobacter spp., 5KF might be transported into the cytoplasm and metabolized. Here we identified the GLF_2050 gene as the kfr gene encoding 5KF reductase (KFR). A mutant strain devoid of the kfr gene showed lower KFR activity and no 5KF consumption. The crystal structure revealed that KFR is similar to NADP + -dependent shikimate dehydrogenase (SDH), which catalyzes the reversible NADP + -dependent oxidation of shikimate to 3-dehydroshikimate. We found that several amino acid residues in the putative substrate-binding site of KFR were different from those of SDH. Phylogenetic analyses revealed that only a subclass in the SDH family containing KFR conserved such a unique substrate-binding site. We constructed KFR derivatives with amino acid substitutions, including replacement of Asn21 in the substrate-binding site with Ser that is found in SDH. The KFR-N21S derivative showed a strong increase in the K M value for 5KF, but a higher shikimate oxidation activity than wild-type KFR, suggesting that Asn21 is important for 5KF binding. In addition, the conserved catalytic dyad Lys72 and Asp108 were individually substituted for Asn. The K72N and D108N derivatives showed only negligible activities without a dramatic change in the K M value for 5KF, suggesting a similar catalytic mechanism to that of SDH. Taken together, we suggest that KFR is a new member of the SDH family. Importance A limited number of species of acetic acid bacteria, such as Gluconobacter sp. strain CHM43, produce 5-ketofructose at a high yield, a potential low calorie sweetener. Here we show that an NADPH-dependent 5-ketofructose reductase (KFR) is involved in 5-ketofructose degradation and we characterize this enzyme with respect to its structure, phylogeny, and function. The crystal structure of KFR was similar to that of shikimate dehydrogenase, which is functionally crucial in the shikimate pathway in bacteria and plants. Phylogenetic analysis suggested that KFR is positioned in a small sub-group of the shikimate dehydrogenase family. Catalytically important amino acid residues were also conserved and their relevance was experimentally validated. Thus, we propose KFR as a new member of shikimate dehydrogenase family.

Crystal Structures of Creatininase Reveal the Substrate Binding Site and Provide an Insight into the Catalytic Mechanism

2004 ◽  
Vol 337 (2) ◽  
pp. 399-416 ◽  
Author(s):  
Tadashi Yoshimoto ◽  
Nobutada Tanaka ◽  
Naota Kanada ◽  
Takahiko Inoue ◽  
Yoshitaka Nakajima ◽  
...  
Keyword(s):  
Binding Site ◽  
Crystal Structures ◽  
Catalytic Mechanism ◽  
Substrate Binding ◽  
Substrate Binding Site ◽  
Insight Into

scholarly journals Crystal structures of phosphatidyl serine synthase PSS reveal the catalytic mechanism of CDP-DAG alcohol O-phosphatidyl transferases

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Martin Centola ◽  
Katharina van Pee ◽  
Heidi Betz ◽  
Özkan Yildiz
Keyword(s):  
Binding Site ◽  
Mammalian Cells ◽  
Catalytic Mechanism ◽  
De Novo ◽  
Cell Metabolism ◽  
Substrate Binding ◽  
High Energy ◽  
Phosphatidyl Serine ◽  
Substrate Binding Site ◽  
Cell Functions

AbstractPhospholipids are the major components of the membrane in all type of cells and organelles. They also are critical for cell metabolism, signal transduction, the immune system and other critical cell functions. The biosynthesis of phospholipids is a complex multi-step process with high-energy intermediates. Several enzymes in different metabolic pathways are involved in the initial phospholipid synthesis and its subsequent conversion. While the “Kennedy pathway” is the main pathway in mammalian cells, in bacteria and lower eukaryotes the precursor CDP-DAG is used in the de novo pathway by CDP-DAG alcohol O-phosphatidyl transferases to synthetize the basic lipids. Here we present the high-resolution structures of phosphatidyl serine synthase from Methanocaldococcus jannaschii crystallized in four different states. Detailed structural and functional analysis of the different structures allowed us to identify the substrate binding site and show how CDP-DAG, serine and two essential metal ions are bound and oriented relative to each other. In close proximity to the substrate binding site, two anions were identified that appear to be highly important for the reaction. The structural findings were confirmed by functional activity assays and suggest a model for the catalytic mechanism of CDP-DAG alcohol O-phosphatidyl transferases, which synthetize the phospholipids essential for the cells.

Characterization of the extracellular poly(3-hydroxybutyrate) depolymerase ofComamonassp. and of its structural gene

1995 ◽  
Vol 41 (13) ◽  
pp. 160-169 ◽  
Author(s):  
Dieter Jendrossek ◽  
Martina Backhaus ◽  
Meike Andermann
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Binding Site ◽  
Catalytic Domain ◽  
Substrate Binding ◽  
Catalytic Triad ◽  
Reading Frame ◽  
Phb Depolymerase ◽  
Substrate Binding Site

The poly(3-hydroxybutyrate) (PHB) depolymerase structural gene of Comamonas sp. (phaZCsp) was cloned in Escherichia coli and identified by halo formation on PHB-containing solid medium. The nucleotide sequence of a 1719 base pair MboI fragment was determined and contained one large open reading frame (ORF1, 1542 base pairs). This open reading frame encoded the precursor of the PHB depolymerase (514 amino acids; Mr, 53 095), and the deduced amino acid sequence was in agreement with the N-terminal amino acid sequence of the purified PHB depolymerase from amino acid 26 onwards. Analysis of the deduced amino acid sequence revealed a domain structure of the protein: a signal peptide that was 25 amino acids long was followed by a catalytic domain of about 300 amino acids, a fibronectin type III (Fn3) modul sequence, and a putative PHB-specific substrate-binding site. By comparison of the primary structure with that of other polyhydroxyalkanoate (PHA) depolymerases, the catalytic domain apparently contained a catalytic triad of serine, histidine, and aspartate. In addition, a conserved region resembling the oxyanion hole of lipases was present. The catalytic domain was linked to a C-terminal putative substrate-binding site by a sequence about 90 amino acids long resembling the Fn3 modul of fibronectin and other eukaryotic extracellular matrix proteins. A threonine-rich region, which was found in four of five PHA depolymerases of Pseudomonas lemoignei, was not present in the Comamonas sp. depolymerase. The similarities with and differences from other PHA depolymerases are discussed.Key words: biodegradable polymer, poly(3-hydroxybutyrate) depolymerase, serine hydrolase, catalytic triad, Comamonas sp., fibronectin type III modul, substrate-binding site.

Crystal structures and kinetic studies of human Kappa class glutathione transferase provide insights into the catalytic mechanism

2011 ◽  
Vol 439 (2) ◽  
pp. 215-225 ◽  
Author(s):  
Bing Wang ◽  
Yingjie Peng ◽  
Tianlong Zhang ◽  
Jianping Ding
Keyword(s):  
Binding Site ◽  
Crystal Structures ◽  
Cellular Localization ◽  
Catalytic Mechanism ◽  
Glutathione Transferase ◽  
Substrate Binding ◽  
Kinetic Studies ◽  
Thiol Group ◽  
Kinetic Mechanism ◽  
Cellular Detoxification

GSTs (glutathione transferases) are a family of enzymes that primarily catalyse nucleophilic addition of the thiol of GSH (reduced glutathione) to a variety of hydrophobic electrophiles in the cellular detoxification of cytotoxic and genotoxic compounds. GSTks (Kappa class GSTs) are a distinct class because of their unique cellular localization, function and structure. In the present paper we report the crystal structures of hGSTk (human GSTk) in apo-form and in complex with GTX (S-hexylglutathione) and steady-state kinetic studies, revealing insights into the catalytic mechanism of hGSTk and other GSTks. Substrate binding induces a conformational change of the active site from an ‘open’ conformation in the apo-form to a ‘closed’ conformation in the GTX-bound complex, facilitating formations of the G site (GSH-binding site) and the H site (hydrophobic substrate-binding site). The conserved Ser16 at the G site functions as the catalytic residue in the deprotonation of the thiol group and the conserved Asp69, Ser200, Asp201 and Arg202 form a network of interactions with γ-glutamyl carboxylate to stabilize the thiolate anion. The H site is a large hydrophobic pocket with conformational flexibility to allow the binding of different hydrophobic substrates. The kinetic mechanism of hGSTk conforms to a rapid equilibrium random sequential Bi Bi model.

scholarly journals The Bacillus subtilis DinR Binding Site: Redefinition of the Consensus Sequence

1998 ◽  
Vol 180 (8) ◽  
pp. 2201-2211 ◽  
Author(s):  
Kevin W. Winterling ◽  
David Chafin ◽  
Jeffery J. Hayes ◽  
Ji Sun ◽  
Arthur S. Levine ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Binding Site ◽  
Mutational Analysis ◽  
Consensus Sequence ◽  
Nucleotide Substitutions ◽  
Mobility Shift ◽  
Radical Cleavage ◽  
Computational Analyses ◽  
Dyad Symmetry ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT Recently, the DinR protein was established as the cellular repressor of the SOS response in the bacterium Bacillus subtilis. It is believed that DinR functions as the repressor by binding to a consensus sequence located in the promoter region of each SOS gene. The binding site for DinR is believed to be synonymous with the formerly identified Cheo box, a region of 12 bp displaying dyad symmetry (GAAC-N4-GTTC). Electrophoretic mobility shift assays revealed that highly purified DinR does bind to such sites located upstream of the dinA, dinB,dinC, and dinR genes. Furthermore, detailed mutational analysis of the B. subtilis recA operator indicates that some nucleotides are more important than others for maintaining efficient DinR binding. For example, nucleotide substitutions immediately 5′ and 3′ of the Cheo box as well as those in the N4 region appear to affect DinR binding. This data, combined with computational analyses of potential binding sites in other gram-positive organisms, yields a new consensus (DinR box) of 5′-CGAACRNRYGTTYC-3′. DNA footprint analysis of the B. subtilis dinR and recA DinR boxes revealed that the DinR box is centrally located within a DNA region of 31 bp that is protected from hydroxyl radical cleavage in the presence of DinR. Furthermore, while DinR is predominantly monomeric in solution, it apparently binds to the DinR box in a dimeric state.

Copper–oxygen Dynamics in Tyrosinase

2019 ◽  
Author(s):  
Nobutaka Fujieda ◽  
Sachiko Yanagisawa ◽  
Minoru Kubo ◽  
Genji Kurisu ◽  
Shinobu Itoh
Keyword(s):  
Catalytic Mechanism ◽  
Substrate Binding ◽  
Active Form ◽  
Copper Ion ◽  
Atomic Resolution ◽  
Oxygen Species ◽  
X Ray ◽  
Oxygen Dynamics ◽  
Insight Into ◽  
Copper Centre

To unveil the activation of dioxygen on the copper centre (Cu<sub>2</sub>O<sub>2</sub>core) of tyrosinase, we performed X-ray crystallograpy with active-form tyrosinase at near atomic resolution. This study provided a novel insight into the catalytic mechanism of the tyrosinase, including the rearrangement of copper-oxygen species as well as the intramolecular migration of copper ion induced by substrate-binding.<br>

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