scholarly journals High-resolution crystal structure of a polyextreme GH43 glycosidase fromHalothermothrix oreniiwith α-L-arabinofuranosidase activity

2015 ◽  
Vol 71 (3) ◽  
pp. 338-345 ◽  
Author(s):  
Noor Hassan ◽  
Lokesh D. Kori ◽  
Rosaria Gandini ◽  
Bharat K. C. Patel ◽  
Christina Divne ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Thermal Stability ◽  
Active Site ◽  
Metal Binding ◽  
Marine Bacterium ◽  
Specific Activity ◽  
Divalent Cations ◽  
Molecular Replacement ◽  
Gene Encoding ◽  
Hydrophobic Amino Acids

A gene from the heterotrophic, halothermophilic marine bacteriumHalothermothrix oreniihas been cloned and overexpressed inEscherichia coli. This gene encodes the only glycoside hydrolase of family 43 (GH43) produced byH. orenii. The crystal structure of theH. oreniiglycosidase was determined by molecular replacement and refined at 1.10 Å resolution. As for other GH43 members, the enzyme folds as a five-bladed β-propeller. The structure features a metal-binding site on the propeller axis, near the active site. Based on thermal denaturation data, theH. oreniiglycosidase depends on divalent cations in combination with high salt for optimal thermal stability against unfolding. A maximum melting temperature of 76°C was observed in the presence of 4 MNaCl and Mn2+at pH 6.5. The gene encoding theH. oreniiGH43 enzyme has previously been annotated as a putative α-L-arabinofuranosidase. Activity was detected withp-nitrophenyl-α-L-arabinofuranoside as a substrate, and therefore the nameHoAraf43 was suggested for the enzyme. In agreement with the conditions for optimal thermal stability against unfolding, the highest arabinofuranosidase activity was obtained in the presence of 4 MNaCl and Mn2+at pH 6.5, giving a specific activity of 20–36 µmol min−1 mg−1. The active site is structurally distinct from those of other GH43 members, including arabinanases, arabinofuranosidases and xylanases. This probably reflects the special requirements for degrading the unique biomass available in highly saline aqueous ecosystems, such as halophilic algae and halophytes. The amino-acid distribution ofHoAraf43 has similarities to those of mesophiles, thermophiles and halophiles, but also has unique features, for example more hydrophobic amino acids on the surface and fewer buried charged residues.

scholarly journals Crystal structure of a pyridoxal 5′-phosphate-dependent aspartate racemase derived from the bivalve mollusc Scapharca broughtonii

2017 ◽  
Vol 73 (12) ◽  
pp. 651-656 ◽  
Author(s):  
Taichi Mizobuchi ◽  
Risako Nonaka ◽  
Motoki Yoshimura ◽  
Katsumasa Abe ◽  
Shouji Takahashi ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Active Site ◽  
Metal Binding ◽  
Bivalve Mollusc ◽  
Active Site Residues ◽  
X Ray ◽  
Aspartate Racemase ◽  
Scapharca Broughtonii

Aspartate racemase (AspR) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that is responsible for D-aspartate biosynthesis in vivo. To the best of our knowledge, this is the first study to report an X-ray crystal structure of a PLP-dependent AspR, which was resolved at 1.90 Å resolution. The AspR derived from the bivalve mollusc Scapharca broughtonii (SbAspR) is a type II PLP-dependent enzyme that is similar to serine racemase (SR) in that SbAspR catalyzes both racemization and dehydration. Structural comparison of SbAspR and SR shows a similar arrangement of the active-site residues and nucleotide-binding site, but a different orientation of the metal-binding site. Superposition of the structures of SbAspR and of rat SR bound to the inhibitor malonate reveals that Arg140 recognizes the β-carboxyl group of the substrate aspartate in SbAspR. It is hypothesized that the aromatic proline interaction between the domains, which favours the closed form of SbAspR, influences the arrangement of Arg140 at the active site.

scholarly journals A trapped human PPM1A–phosphopeptide complex reveals structural features critical for regulation of PPM protein phosphatase activity

2018 ◽  
Vol 293 (21) ◽  
pp. 7993-8008 ◽  
Author(s):  
Subrata Debnath ◽  
Dalibor Kosek ◽  
Harichandra D. Tagad ◽  
Stewart R. Durell ◽  
Daniel H. Appella ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Metal Ions ◽  
Active Site ◽  
Metal Binding ◽  
Metal Ion ◽  
Catalytic Domain ◽  
Protein Phosphatases ◽  
Random Order ◽  
Structural Features

Metal-dependent protein phosphatases (PPM) are evolutionarily unrelated to other serine/threonine protein phosphatases and are characterized by their requirement for supplementation with millimolar concentrations of Mg2+ or Mn2+ ions for activity in vitro. The crystal structure of human PPM1A (also known as PP2Cα), the first PPM structure determined, displays two tightly bound Mn2+ ions in the active site and a small subdomain, termed the Flap, located adjacent to the active site. Some recent crystal structures of bacterial or plant PPM phosphatases have disclosed two tightly bound metal ions and an additional third metal ion in the active site. Here, the crystal structure of the catalytic domain of human PPM1A, PPM1Acat, complexed with a cyclic phosphopeptide, c(MpSIpYVA), a cyclized variant of the activation loop of p38 MAPK (a physiological substrate of PPM1A), revealed three metal ions in the active site. The PPM1Acat D146E–c(MpSIpYVA) complex confirmed the presence of the anticipated third metal ion in the active site of metazoan PPM phosphatases. Biophysical and computational methods suggested that complex formation results in a slightly more compact solution conformation through reduced conformational flexibility of the Flap subdomain. We also observed that the position of the substrate in the active site allows solvent access to the labile third metal-binding site. Enzyme kinetics of PPM1Acat toward a phosphopeptide substrate supported a random-order, bi-substrate mechanism, with substantial interaction between the bound substrate and the labile metal ion. This work illuminates the structural and thermodynamic basis of an innate mechanism regulating the activity of PPM phosphatases.

scholarly journals Crystal structure of human dehydroepiandrosterone sulphotransferase in complex with substrate

2002 ◽  
Vol 364 (1) ◽  
pp. 165-171 ◽  
Author(s):  
Peter H. REHSE ◽  
Ming ZHOU ◽  
Sheng-Xiang LIN
Keyword(s):  
Crystal Structure ◽  
Hydrogen Bond ◽  
Active Site ◽  
Substrate Binding ◽  
Substrate Inhibition ◽  
Molecular Replacement ◽  
Alternative Substrate ◽  
Hydrophobic Residues ◽  
Sequence Identity ◽  
Binding Orientations

Dehydroepiandrosterone sulphotransferase (DHEA-ST) is an enzyme that converts dehydroepiandrosterone (DHEA), and some other steroids, into their sulphonated forms. The enzyme catalyses the sulphonation of DHEA on the 3α-oxygen, with 3′-phosphoadenosine-5′-phosphosulphate contributing the sulphate. The structure of human DHEA-ST in complex with its preferred substrate DHEA has been solved here to 1.99Å using molecular replacement with oestradiol sulphotransferase (37% sequence identity) as a model. Two alternative substrate-binding orientations have been identified. The primary, catalytic, orientation has the DHEA 3α-oxygen and the highly conserved catalytic histidine in nearly identical positions as are seen for the related oestradiol sulphotransferase. The substrate, however, shows rotations of up to 30°, and there is a corresponding rearrangement of the protein loops contributing to the active site. This may also reflect the low identity between the two enzymes. The second orientation penetrates further into the active site and can form a potential hydrogen bond with the desulphonated cofactor 3′,5′-phosphoadenosine (PAP). This second site contains more van der Waal interactions with hydrophobic residues than the catalytic site and may also reflect the substrate-inhibition site. The PAP position was obtained from the previously solved structure of DHEA-ST co-crystallized with PAP. This latter structure, due to the arrangement of loops within the active site and monomer interactions, cannot bind substrate. The results presented here describe details of substrate binding to DHEA-ST and the potential relationship to substrate inhibition.

scholarly journals Elucidating the regulation of glucose tolerance through the interaction between the reaction product and active site pocket residues of a β-glucosidase from Halothermothrix orenii

2019 ◽  
Author(s):  
Sushant K Sinha ◽  
Shibashis Das ◽  
Sukanya Konar ◽  
Pradip Kr. Ghorai ◽  
Rahul Das ◽  
...  
Keyword(s):  
Glucose Tolerance ◽  
Active Site ◽  
Specific Activity ◽  
Gene Encoding ◽  
Conserved Residues ◽  
Active Site Pocket ◽  
Glucose Inhibition ◽  
Glucose Tolerant ◽  
Dynamics Simulations ◽  
Hydrolysis Of

Abstractβ-glucosidase catalyzes the hydrolysis of β-1,4 linkage between two glucose molecules in cello-oligosaccharides and is prone to inhibition by the reaction product glucose. Relieving the glucose inhibition of β-glucosidase is a significant challenge. Towards the goal of understanding how glucose interacts with β-glucosidase, we expressed in Escherichia coli, the Hore_15280 gene encoding a β-glucosidase in Halothermothrix orenii. Our results show that the enzyme is glucose tolerant, and its activity stimulated in the presence of up to 0.5 M glucose. NMR analyses show the unexpected interactions between glucose and the β-glucosidase at lower concentrations of glucose that however does not lead to enzyme inhibition. We identified non-conserved residues at the aglycone-binding and the gatekeeper site and show that increased hydrophobicity at the pocket entrance and a reduction in steric hindrances are critical towards enhanced substrate accessibility and significant improvement in activity. Analysis of structures and in combination with molecular dynamics simulations show that glucose increases the accessibility of the substrate by enhancing the structural flexibility of the active site pocket and may explain the stimulation in specific activity up to 0.5 M glucose. Such novel regulation of β-glucosidase activity by its reaction product may offer novel ways of engineering glucose tolerance.

scholarly journals Crystal Structure of the Terminal Oxygenase Component of Cumene Dioxygenase from Pseudomonas fluorescens IP01

2005 ◽  
Vol 187 (7) ◽  
pp. 2483-2490 ◽  
Author(s):  
Xuesong Dong ◽  
Shinya Fushinobu ◽  
Eriko Fukuda ◽  
Tohru Terada ◽  
Shugo Nakamura ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Pseudomonas Fluorescens ◽  
Active Site ◽  
Complex Structure ◽  
Binding Pocket ◽  
Nonheme Iron ◽  
Single Amino Acid ◽  
Molecular Replacement ◽  
Catalytic Subunits ◽  
Single Amino Acid Residue

ABSTRACT The crystal structure of the terminal component of the cumene dioxygenase multicomponent enzyme system of Pseudomonas fluorescens IP01 (CumDO) was determined at a resolution of 2.2 Å by means of molecular replacement by using the crystal structure of the terminal oxygenase component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4 (NphDO). The ligation of the two catalytic centers of CumDO (i.e., the nonheme iron and Rieske [2Fe-2S] centers) and the bridging between them in neighboring catalytic subunits by hydrogen bonds through a single amino acid residue, Asp231, are similar to those of NphDO. An unidentified external ligand, possibly dioxygen, was bound at the active site nonheme iron. The entrance to the active site of CumDO is different from the entrance to the active site of NphDO, as the two loops forming the lid exhibit great deviation. On the basis of the complex structure of NphDO, a biphenyl substrate was modeled in the substrate-binding pocket of CumDO. The residues surrounding the modeled biphenyl molecule include residues that have already been shown to be important for its substrate specificity by a number of engineering studies of biphenyl dioxygenases.

scholarly journals The crystal structure of a class II fructose-1,6-bisphosphate aldolase shows a novel binuclear metal-binding active site embedded in a familiar fold

Structure ◽  
1996 ◽  
Vol 4 (11) ◽  
pp. 1303-1315 ◽  
Author(s):  
Serena J Cooper ◽  
Gordon A Leonard ◽  
Sean M McSweeney ◽  
Andrew W Thompson ◽  
James H Naismith ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Metal Binding ◽  
Class Ii ◽  
Binuclear Metal ◽  
Fructose 1,6 Bisphosphate

Crystal structure of dUTPase from equine infectious anaemia virus; active site metal binding in a substrate analogue complex 1 1Edited by K. Nagai

1999 ◽  
Vol 285 (2) ◽  
pp. 655-673 ◽  
Author(s):  
Zbigniew Dauter ◽  
Rebecca Persson ◽  
Anna Maria Rosengren ◽  
Per Olof Nyman ◽  
Keith S Wilson ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Metal Binding ◽  
Substrate Analogue ◽  
Equine Infectious Anaemia Virus

scholarly journals Overexpression of celB Gene Coding for β-Glucosidase from Pyrococcus furiosus Using a Baculovirus Expression Vector System in Silkworm, Bombyx mori

2006 ◽  
Vol 61 (7-8) ◽  
pp. 595-600 ◽  
Author(s):  
Xu’Ai Lin ◽  
Wei Zhang ◽  
Yin Chen ◽  
Bin Yao ◽  
Zhi Fang Zhang
Keyword(s):  
Bombyx Mori ◽  
Specific Activity ◽  
Pyrococcus Furiosus ◽  
Divalent Cations ◽  
Gene Promoter ◽  
Protein Quantification ◽  
Vector System ◽  
Glycosyl Hydrolases ◽  
Gene Encoding ◽  
Baculovirus Expression Vector

β-Glucosidase is a member of the glycosyl hydrolases that specifically catalyze the hydrolysis of terminal nonreducing β-ᴅ-glucose residues from the end of various oligosaccharides with the release of β-ᴅ-glucose. CelB gene, encoding the thermostable β-glucosidase, was amplified from the Pyrococcus furiosus genome and then cloned into the baculoviral transfer vector under the control of the polyhedrin gene promoter. After co-transfection with the genetically modified parental Bombyx mori nucleopolyhedrovirus (BmNPV), the recombinant virus containing celB gene was used to express β-glucosidase in silkworm. The recombinant β-glucosidase was purified to about 81% homogeneity in a single heat-treatment step. The optimal activity of the expressed β-glucosidase was obtained at pH 5.0 and about 105 °C; divalent cations and high ionic strength did not affect the activity remarkably. This suggested that the enzymatic characteristics of recombinant β-glucosidase were similar to the native counterpart. The expressed β-glucosidase accounted for more than 10% of silkworm total haemolymph proteins according to the protein quantification and densimeter scanning. The expression level reached 10,199.5 U per ml haemolymph and 19,797.4 U per silkworm larva, and the specific activity of the one-step purified crude enzyme was 885 U per mg. It was demonstrated to be an attractive approach for mass production of thermostable β-glucosidase using this system.

scholarly journals Crystal Structure of the Mycobacterium fortuitum Class A β-Lactamase: Structural Basis for Broad Substrate Specificity

2006 ◽  
Vol 50 (7) ◽  
pp. 2516-2521 ◽  
Author(s):  
Eric Sauvage ◽  
Eveline Fonzé ◽  
Birgit Quinting ◽  
Moreno Galleni ◽  
Jean-Marie Frère ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Broad Spectrum ◽  
Active Site ◽  
Bacterial Resistance ◽  
Specific Activity ◽  
Structural Basis ◽  
Mycobacterium Fortuitum ◽  
Bacterial Strains ◽  
Class A ◽  
Extended Spectrum

ABSTRACT β-Lactamases are the main cause of bacterial resistance to penicillins and cephalosporins. Class A β-lactamases, the largest group of β-lactamases, have been found in many bacterial strains, including mycobacteria, for which no β-lactamase structure has been previously reported. The crystal structure of the class A β-lactamase from Mycobacterium fortuitum (MFO) has been solved at 2.13-Å resolution. The enzyme is a chromosomally encoded broad-spectrum β-lactamase with low specific activity on cefotaxime. Specific features of the active site of the class A β-lactamase from M. fortuitum are consistent with its specificity profile. Arg278 and Ser237 favor cephalosporinase activity and could explain its broad substrate activity. The MFO active site presents similarities with the CTX-M type extended-spectrum β-lactamases but lacks a specific feature of these enzymes, the VNYN motif (residues 103 to 106), which confers on CTX-M-type extended-spectrum β-lactamases a more efficient cefotaximase activity.

scholarly journals Evaluation of the role of two conserved active-site residues in Beta class glutathione S-transferases

2000 ◽  
Vol 351 (2) ◽  
pp. 341-346 ◽  
Author(s):  
Nerino ALLOCATI ◽  
Enrico CASALONE ◽  
Michele MASULLI ◽  
Galina POLEKHINA ◽  
Jamie ROSSJOHN ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Thermal Stability ◽  
Catalytic Activity ◽  
Proteus Mirabilis ◽  
Active Site ◽  
Binding Capacity ◽  
Active Site Residues ◽  
Terminal Domain ◽  
Glutathione S Transferases

Glutathione S-transferases (GSTs) normally use hydroxy-group-containing residues in the N-terminal domain of the enzyme for stabilizing the activated form of the co-substrate, glutathione. However, previous mutagenesis studies have shown that this is not true for Beta class GSTs and thus the origin of the stabilization remains a mystery. The recently determined crystal structure of Proteus mirabilis GST B1-1 (PmGST B1-1) suggested that the stabilizing role might be fulfilled in Beta class GSTs by one or more residues in the C-terminal domain of the enzyme. To test this hypothesis we mutated His106 and Lys107 of PmGST B1-1 to investigate their possible role in the enzyme's catalytic activity. His106 was mutated to Ala, Asn and Phe, and Lys107 to Ala and Arg. The effects of the replacement on the activity, thermal stability and antibiotic-binding capacity of the enzyme were examined. The results are consistent with the involvement of His106 and Lys107 in interacting with glutathione at the active site but these residues do not contribute significantly to catalysis, folding or antibiotic binding.

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