Crystal structure of isoamyl acetate-hydrolyzing esterase from Saccharomyces cerevisiae reveals a novel active site architecture and the basis of substrate specificity

2010 ◽  
Vol 79 (2) ◽  
pp. 662-668 ◽  
Author(s):  
Jinming Ma ◽  
Qianda Lu ◽  
Ye Yuan ◽  
Honghua Ge ◽  
Kuai Li ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Saccharomyces Cerevisiae ◽  
Substrate Specificity ◽  
Active Site ◽  
Isoamyl Acetate

Structural insights into dihydroxylation of terephthalate, a product of polyethylene terephthalate degradation

2022 ◽  
Author(s):  
Jai Krishna Mahto ◽  
Neetu Neetu ◽  
Monica Sharma ◽  
Monika Dubey ◽  
Bhanu Prakash Vellanki ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Substrate Specificity ◽  
Polyethylene Terephthalate ◽  
Active Site ◽  
Catalytic Domain ◽  
Structural Features ◽  
Comamonas Testosteroni ◽  
Plastic Pollution ◽  
Microbial Utilization ◽  
Steady State Kinetics

Biodegradation of terephthalate (TPA) is a highly desired catabolic process for the bacterial utilization of this Polyethylene terephthalate (PET) depolymerization product, but to date, the structure of terephthalate dioxygenase (TPDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of TPA to a cis -diol is unavailable. In this study, we characterized the steady-state kinetics and first crystal structure of TPDO from Comamonas testosteroni KF1 (TPDO KF1 ). The TPDO KF1 exhibited the substrate specificity for TPA ( k cat / K m = 57 ± 9 mM −1 s −1 ). The TPDO KF1 structure harbors characteristics RO features as well as a unique catalytic domain that rationalizes the enzyme’s function. The docking and mutagenesis studies reveal that its substrate specificity to TPA is mediated by Arg309 and Arg390 residues, two residues positioned on opposite faces of the active site. Additionally, residue Gln300 is also proven to be crucial for the activity, its substitution to alanine decreases the activity ( k cat ) by 80%. Together, this study delineates the structural features that dictate the substrate recognition and specificity of TPDO. Importance The global plastic pollution has become the most pressing environmental issue. Recent studies on enzymes depolymerizing polyethylene terephthalate plastic into terephthalate (TPA) show some potential in tackling this. Microbial utilization of this released product, TPA is an emerging and promising strategy for waste-to-value creation. Research from the last decade has discovered terephthalate dioxygenase (TPDO), as being responsible for initiating the enzymatic degradation of TPA in a few Gram-negative and Gram-positive bacteria. Here, we have determined the crystal structure of TPDO from Comamonas testosteroni KF1 and revealed that it possesses a unique catalytic domain featuring two basic residues in the active site to recognize TPA. Biochemical and mutagenesis studies demonstrated the crucial residues responsible for the substrate specificity of this enzyme.

scholarly journals Crystal structure of yeast monothiol glutaredoxin Grx6 in complex with a glutathione-coordinated [2Fe–2S] cluster

2016 ◽  
Vol 72 (10) ◽  
pp. 732-737 ◽  
Author(s):  
Mohnad Abdalla ◽  
Ya-Nan Dai ◽  
Chang-Biao Chi ◽  
Wang Cheng ◽  
Dong-Dong Cao ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Saccharomyces Cerevisiae ◽  
Sequence Alignment ◽  
Multiple Sequence Alignment ◽  
Active Site ◽  
Binding Pattern ◽  
Biological Functions ◽  
Structural Comparison ◽  
Multiple Sequence ◽  
Dimeric Structure

Glutaredoxins (Grxs) constitute a superfamily of proteins that perform diverse biological functions. TheSaccharomyces cerevisiaeglutaredoxin Grx6 not only serves as a glutathione (GSH)-dependent oxidoreductase and as a GSH transferase, but also as an essential [2Fe–2S]-binding protein. Here, the dimeric structure of the C-terminal domain of Grx6 (holo Grx6C), bridged by one [2Fe–2S] cluster coordinated by the active-site Cys136 and two external GSH molecules, is reported. Structural comparison combined with multiple-sequence alignment demonstrated that holo Grx6C is similar to the [2Fe–2S] cluster-incorporated dithiol Grxs, which share a highly conserved [2Fe–2S] cluster-binding pattern and dimeric conformation that is distinct from the previously identified [2Fe–2S] cluster-ligated monothiol Grxs.

scholarly journals Structure and Mechanism of Ferulic Acid Decarboxylase (FDC1) from Saccharomyces cerevisiae

2015 ◽  
Vol 81 (12) ◽  
pp. 4216-4223 ◽  
Author(s):  
Mohammad Wadud Bhuiya ◽  
Soon Goo Lee ◽  
Joseph M. Jez ◽  
Oliver Yu
Keyword(s):  
Crystal Structure ◽  
Saccharomyces Cerevisiae ◽  
Ferulic Acid ◽  
Active Site ◽  
Three Dimensional ◽  
Structural Similarity ◽  
Dimensional Structure ◽  
Acid Decarboxylase ◽  
Content Type ◽  
Ferulic Acid Decarboxylase

ABSTRACTThe nonoxidative decarboxylation of aromatic acids occurs in a range of microbes and is of interest for bioprocessing and metabolic engineering. Although phenolic acid decarboxylases provide useful tools for bioindustrial applications, the molecular bases for how these enzymes function are only beginning to be examined. Here we present the 2.35-Å-resolution X-ray crystal structure of the ferulic acid decarboxylase (FDC1; UbiD) fromSaccharomyces cerevisiae. FDC1 shares structural similarity with the UbiD family of enzymes that are involved in ubiquinone biosynthesis. The position of 4-vinylphenol, the product ofp-coumaric acid decarboxylation, in the structure identifies a large hydrophobic cavity as the active site. Differences in the β2e-α5 loop of chains in the crystal structure suggest that the conformational flexibility of this loop allows access to the active site. The structure also implicates Glu285 as the general base in the nonoxidative decarboxylation reaction catalyzed by FDC1. Biochemical analysis showed a loss of enzymatic activity in the E285A mutant. Modeling of 3-methoxy-4-hydroxy-5-decaprenylbenzoate, a partial structure of the physiological UbiD substrate, in the binding site suggests that an ∼30-Å-long pocket adjacent to the catalytic site may accommodate the isoprenoid tail of the substrate needed for ubiquinone biosynthesis in yeast. The three-dimensional structure of yeast FDC1 provides a template for guiding protein engineering studies aimed at optimizing the efficiency of aromatic acid decarboxylation reactions in bioindustrial applications.

scholarly journals Determinants of specificity for aflatoxin B1-8,9-epoxide in Alpha-class glutathione S-transferases

1999 ◽  
Vol 339 (1) ◽  
pp. 95-101 ◽  
Author(s):  
Paul D. McDONAGH ◽  
David J. JUDAH ◽  
John D. HAYES ◽  
Lu-Yun LIAN ◽  
Gordon E. NEAL ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Substrate Specificity ◽  
High Activity ◽  
Aflatoxin B1 ◽  
Active Site ◽  
Homology Modelling ◽  
Three Dimensional ◽  
Glutathione S Transferase ◽  
Glutathione S Transferases ◽  
Aspartate Residue

We have used homology modelling, based on the crystal structure of the human glutathione S-transferase (GST) A1-1, to obtain the three-dimensional structures of rat GSTA3 and rat GSTA5 subunits bound to S-aflatoxinyl–glutathione. The resulting models highlight two residues, at positions 208 and 108, that could be important for determining, either directly or indirectly, substrate specificity for aflatoxin-exo-8,9-epoxide among the Alpha-class GSTs. Residues at these positions were mutated in human GSTA1-1 (Met-208, Leu-108), rat GSTA3-3 (Glu-208, His-108) and rat GSTA5-5 (Asp-208, Tyr-108): in the active rat GSTA5-5 to those in the inactive GSTA1-1; and in the inactive human GSTA1-1 and rat GSTA3-3 to those in the active rat GSTA5-5. These studies show clearly that, in all three GSTs, an aspartate residue at position 208 is a prerequisite for high activity in aflatoxin-exo-8,9-epoxide conjugation, although this alone is not sufficient; other residues in the vicinity, particularly residues 103–112, are important, perhaps for the optimal orientation of the aflatoxin-exo-8,9-epoxide in the active site for catalysis to occur.

scholarly journals Crystal Structure of Butyrate Kinase 2 from Thermotoga maritima, a Member of the ASKHA Superfamily of Phosphotransferases

2009 ◽  
Vol 191 (8) ◽  
pp. 2521-2529 ◽  
Author(s):  
Jiasheng Diao ◽  
Miriam S. Hasson
Keyword(s):  
Crystal Structure ◽  
Substrate Specificity ◽  
Active Site ◽  
Thermotoga Maritima ◽  
Structural Basis ◽  
Acetate Kinase ◽  
Phosphoryl Transfer ◽  
Atp Binding ◽  
Binding Motif ◽  
Butyrate Kinase

ABSTRACT The enzymatic transfer of phosphoryl groups is central to the control of many cellular processes. One of the phosphoryl transfer mechanisms, that of acetate kinase, is not completely understood. Besides better understanding of the mechanism of acetate kinase, knowledge of the structure of butyrate kinase 2 (Buk2) will aid in the interpretation of active-site structure and provide information on the structural basis of substrate specificity. The gene buk2 from Thermotoga maritima encodes a member of the ASKHA (acetate and sugar kinases/heat shock cognate/actin) superfamily of phosphotransferases. The encoded protein Buk2 catalyzes the phosphorylation of butyrate and isobutyrate. We have determined the 2.5-Å crystal structure of Buk2 complexed with (β,γ-methylene) adenosine 5′-triphosphate. Buk2 folds like an open-shelled clam, with each of the two domains representing one of the two shells. In the open active-site cleft between the N- and C-terminal domains, the active-site residues consist of two histidines, two arginines, and a cluster of hydrophobic residues. The ATP binding region of Buk2 in the C-terminal domain consists of abundant glycines for nucleotide binding, and the ATP binding motif is similar to those of other members of the ASKHA superfamily. The enzyme exists as an octamer, in which four disulfide bonds form between intermolecular cysteines. Sequence alignment and structure superposition identify the simplicity of the monomeric Buk2 structure, a probable substrate binding site, the key residues in catalyzing phosphoryl transfer, and the substrate specificity differences among Buk2, acetate, and propionate kinases. The possible enzyme mechanisms are discussed.

The roles of histidine and tyrosine residues in the active site of collagenase in Grimontia hollisae

2020 ◽  
Vol 168 (4) ◽  
pp. 385-392
Author(s):  
Kaichi Hayashi ◽  
Takeaki Ikeuchi ◽  
Ryo Morishita ◽  
Jun Qian ◽  
Kenji Kojima ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Substrate Specificity ◽  
Active Site ◽  
Zinc Binding ◽  
Binding Motif ◽  
Hydrolyze Collagen ◽  
Tyrosine Residues ◽  
Active Variant ◽  
Zinc Binding Motif ◽  
Ionizable Residue

Abstract Collagenase from the Grimontia hollisae strain 1706B (Ghcol) is a zinc metalloproteinase with the zinc-binding motif H492EXXH496. It exhibits higher collagen-degrading activity than the collagenase from Clostridium histolyticum, which is widely used in industry. We previously examined the pH and temperature dependencies of Ghcol activity; Glu493 was thought to contribute acidic pKa (pKe1), while no residue was assigned to contribute alkaline pKa (pKe2). In this study, we introduced nine single mutations at the His or Tyr residues in and near the active site. Our results showed that H412A, H485A, Y497A, H578A and H737A retained the activities to hydrolyze collagen and gelatin, while H426A, H492A, H496A and Y568A lacked them. Purification of active variants H412A, H485A, H578A and H737A, along with inactive variants H492A and H496A, were successful. H412A preferred (7-methoxycoumarin-4-yl)acetyl-L-Lys-L-Pro-L-Leu-Gly-L-Leu-[N3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH2 to collagen, while H485A preferred collagen to the peptide, suggesting that His412 and His485 are important for substrate specificity. Purification of the active variant Y497A and inactive variants H426A and Y568A were unsuccessful, suggesting that these three residues were important for stability. Based on the reported crystal structure of clostridial collagenase, Tyr568 of Ghcol is suggested to be involved in catalysis and may be the ionizable residue for pKe2.

scholarly journals Docking Sulochrin and Its Derivative as α-Glucosidase Inhibitors of Saccharomyces cerevisiae

2017 ◽  
Vol 17 (1) ◽  
pp. 144 ◽  
Author(s):  
Wening Lestari ◽  
Rizna Triana Dewi ◽  
Leonardus Broto Sugeng Kardono ◽  
Arry Yanuar
Keyword(s):  
Crystal Structure ◽  
Saccharomyces Cerevisiae ◽  
Hydrogen Bonds ◽  
Active Site ◽  
Iodine Atom ◽  
Glucosidase Inhibitors ◽  
Docking Method ◽  
Better Than

Sulochrin is known to have an activity as inhibitors of the α-glucosidase enzyme. In this report interaction of sulochrin to the active site of the α-glucosidase enzyme from Saccharomyces cerevisiae was studied by docking method. The crystal structure of α-glucosidase from S. cerevisiae obtained from the homology method using α-glucosidase from S. cerevisiae (Swiss-Prot code P53341) as a target and crystal structure of isomaltase from S. cerevisiae (PDB code 3A4A) as a template. These studies show that sulochrin and sulochrin-I could be bound in the active site of α-glucosidase from S. cerevisiae through the formation of hydrogen bonds with Arg213, Asp215, Glu277, Asp352. Sulochrin-I has stability and inhibition of the α-glucosidase enzyme better than sulochrin. The iodine atom in the structure of sulochrin can increase the activity as an inhibitor of the α-glucosidase enzyme.

scholarly journals Complexed Crystal Structure of Saccharomyces cerevisiae Dihydroorotase with Inhibitor 5-Fluoroorotate Reveals a New Binding Mode

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Hong-Hsiang Guan ◽  
Yen-Hua Huang ◽  
En-Shyh Lin ◽  
Chun-Jung Chen ◽  
Cheng-Yang Huang
Keyword(s):  
Crystal Structure ◽  
Saccharomyces Cerevisiae ◽  
Active Site ◽  
Structural Similarity ◽  
Binding Mode ◽  
Site Directed Mutagenesis ◽  
Molecular Evidence ◽  
Binding Modes ◽  
Pyrimidine Nucleotides ◽  
Quenching Method

Dihydroorotase (DHOase) possesses a binuclear metal center in which two Zn ions are bridged by a posttranslationally carbamylated lysine. DHOase catalyzes the reversible cyclization of N-carbamoyl aspartate (CA-asp) to dihydroorotate (DHO) in the third step of the pathway for the biosynthesis of pyrimidine nucleotides and is an attractive target for potential anticancer and antimalarial chemotherapy. Crystal structures of ligand-bound DHOase show that the flexible loop extends toward the active site when CA-asp is bound (loop-in mode) or moves away from the active site, facilitating the product DHO release (loop-out mode). DHOase binds the product-like inhibitor 5-fluoroorotate (5-FOA) in a similar mode to DHO. In the present study, we report the crystal structure of DHOase from Saccharomyces cerevisiae (ScDHOase) complexed with 5-FOA at 2.5 Å resolution (PDB entry 7CA0). ScDHOase shares structural similarity with Escherichia coli DHOase (EcDHOase). However, our complexed structure revealed that ScDHOase bound 5-FOA differently from EcDHOase. 5-FOA ligated the Zn atoms in the active site of ScDHOase. In addition, 5-FOA bound to ScDHOase through the loop-in mode. We also characterized the binding of 5-FOA to ScDHOase by using the site-directed mutagenesis and fluorescence quenching method. Based on these lines of molecular evidence, we discussed whether these different binding modes are species- or crystallography-dependent.

The Crystal Structure of the Cytosolic Exopolyphosphatase from Saccharomyces cerevisiae Reveals the Basis for Substrate Specificity

2007 ◽  
Vol 371 (4) ◽  
pp. 1007-1021 ◽  
Author(s):  
Emilie Ugochukwu ◽  
Andrew L. Lovering ◽  
Owen C. Mather ◽  
Thomas W. Young ◽  
Scott A. White
Keyword(s):  
Crystal Structure ◽  
Saccharomyces Cerevisiae ◽  
Substrate Specificity
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