Structural contributions of Delta class glutathione transferase active-site residues to catalysis

2010 ◽  
Vol 428 (1) ◽  
pp. 25-32 ◽  
Author(s):  
Jantana Wongsantichon ◽  
Robert C. Robinson ◽  
 Albert J. Ketterman
Keyword(s):  
Crystal Structures ◽  
Glutathione Transferase ◽  
Substrate Binding ◽  
Conformational Stability ◽  
Site Directed Mutagenesis ◽  
Active Site Residues ◽  
Molecular Rearrangement ◽  
Hydrophobic Substrate ◽  
Substrate Processing ◽  
First Time

GST (glutathione transferase) is a dimeric enzyme recognized for biotransformation of xenobiotics and endogenous toxic compounds. In the present study, residues forming the hydrophobic substrate-binding site (H-site) of a Delta class enzyme were investigated in detail for the first time by site-directed mutagenesis and crystallographic studies. Enzyme kinetics reveal that Tyr111 indirectly stabilizes GSH binding, Tyr119 modulates hydrophobic substrate binding and Phe123 indirectly modulates catalysis. Mutations at Tyr111 and Phe123 also showed evidence for positive co-operativity for GSH and 1-chloro-2,4-dinitrobenzene respectively, strongly suggesting a role for these residues in manipulating subunit–subunit communication. In the present paper we report crystal structures of the wild-type enzyme, and two mutants, in complex with S-hexylglutathione. This study has identified an aromatic ‘zipper’ in the H-site contributing a network of aromatic π–π interactions. Several residues of the cluster directly interact with the hydrophobic substrate, whereas others indirectly maintain conformational stability of the dimeric structure through the C-terminal domain (domain II). The Y119E mutant structure shows major main-chain rearrangement of domain II. This reorganization is moderated through the ‘zipper’ that contributes to the H-site remodelling, thus illustrating a role in co-substrate binding modulation. The F123A structure shows molecular rearrangement of the H-site in one subunit, but not the other, explaining weakened hydrophobic substrate binding and kinetic co-operativity effects of Phe123 mutations. The three crystal structures provide comprehensive evidence of the aromatic ‘zipper’ residues having an impact upon protein stability, catalysis and specificity. Consequently, ‘zipper’ residues appear to modulate and co-ordinate substrate processing through permissive flexing.

scholarly journals Domain sliding of two Staphylococcus aureus N-acetylglucosaminidases enables their substrate-binding prior to its catalysis

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Sara Pintar ◽  
Jure Borišek ◽  
Aleksandra Usenik ◽  
Andrej Perdih ◽  
Dušan Turk
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Drug Targets ◽  
Substrate Binding ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Human Pathogen ◽  
Active Site Cleft ◽  
Novel Drug ◽  
Novel Drug Targets

AbstractTo achieve productive binding, enzymes and substrates must align their geometries to complement each other along an entire substrate binding site, which may require enzyme flexibility. In pursuit of novel drug targets for the human pathogen S. aureus, we studied peptidoglycan N-acetylglucosaminidases, whose structures are composed of two domains forming a V-shaped active site cleft. Combined insights from crystal structures supported by site-directed mutagenesis, modeling, and molecular dynamics enabled us to elucidate the substrate binding mechanism of SagB and AtlA-gl. This mechanism requires domain sliding from the open form observed in their crystal structures, leading to polysaccharide substrate binding in the closed form, which can enzymatically process the bound substrate. We suggest that these two hydrolases must exhibit unusual extents of flexibility to cleave the rigid structure of a bacterial cell wall.

scholarly journals Crystal Structures of Yeast β-Alanine Synthase Complexes Reveal the Mode of Substrate Binding and Large Scale Domain Closure Movements

2007 ◽  
Vol 282 (49) ◽  
pp. 36037-36047 ◽  
Author(s):  
Stina Lundgren ◽  
Birgit Andersen ◽  
Jure Piškur ◽  
Doreen Dobritzsch
Keyword(s):  
Crystal Structures ◽  
Large Scale ◽  
Catalytic Domain ◽  
Substrate Binding ◽  
Salt Bridge ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Dimerization Domain ◽  
Binding Residues ◽  
Present Site

β-Alanine synthase is the final enzyme of the reductive pyrimidine catabolic pathway, which is responsible for the breakdown of uracil and thymine in higher organisms. The fold of the homodimeric enzyme from the yeast Saccharomyces kluyveri identifies it as a member of the AcyI/M20 family of metallopeptidases. Its subunit consists of a catalytic domain harboring a di-zinc center and a smaller dimerization domain. The present site-directed mutagenesis studies identify Glu159 and Arg322 as crucial for catalysis and His262 and His397 as functionally important but not essential. We determined the crystal structures of wild-type β-alanine synthase in complex with the reaction product β-alanine, and of the mutant E159A with the substrate N-carbamyl-β-alanine, revealing the closed state of a dimeric AcyI/M20 metallopeptidase-like enzyme. Subunit closure is achieved by a ∼30° rigid body domain rotation, which completes the active site by integration of substrate binding residues that belong to the dimerization domain of the same or the partner subunit. Substrate binding is achieved via a salt bridge, a number of hydrogen bonds, and coordination to one of the zinc ions of the di-metal center.

scholarly journals Cloning, Expression, and Site-Directed Mutagenesis of the Propene Monooxygenase Genes from Mycobacterium sp. Strain M156

2005 ◽  
Vol 71 (4) ◽  
pp. 1909-1914 ◽  
Author(s):  
Chan K. Chan Kwo Chion ◽  
Sarah E. Askew ◽  
David J. Leak
Keyword(s):  
Mutational Analysis ◽  
Substrate Binding ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Directed Mutagenesis ◽  
Active Site Residues ◽  
Styrene Oxidation ◽  
Overlapping Reading Frames ◽  
Reading Frames

ABSTRACT Propene monooxygenase has been cloned from Mycobacterium sp. strain M156, based on hybridization with the amoABCD genes of Rhodococcus corallinus B276. Sequencing indicated that the mycobacterial enzyme is a member of the binuclear nonheme iron monooxygenase family and, in gene order and sequence, is most similar to that from R. corallinus B-276. Attempts were made to express the pmoABCD operon in Escherichia coli and Mycobacterium smegmatis mc2155. In the former, there appeared to be a problem resolving overlapping reading frames between pmoA and -B and between pmoC and -D, while in the latter, problems were encountered with plasmid instability when the pmoABCD genes were placed under the control of the hsp60 heat shock promoter in the pNBV1 vector. Fortuitously, constructs with the opposite orientation were constitutively expressed at a level sufficient to allow preliminary mutational analysis. Two PMO active-site residues (A94 and V188) were targeted by site-directed mutagenesis to alter their stereoselectivity. The results suggest that changing the volume occupied by the side chain at V188 leads to a systematic alteration in the stereoselectivity of styrene oxidation, presumably by producing different orientations for substrate binding during catalysis. Changing the volume occupied by the side chain at A94 produced a nonsystematic change in stereoselectivity, which may be attributable to the role of this residue in expansion of the binding site during substrate binding. Neither set of mutations changed the enzyme's specificity for epoxidation.

scholarly journals Evidence that Asn542 of neprilysin (EC 3.4.24.11) is involved in binding of the P2′ residue of substrates and inhibitors

1995 ◽  
Vol 311 (2) ◽  
pp. 623-627 ◽  
Author(s):  
N Dion ◽  
H Le Moual ◽  
M C Fournié-Zaluski ◽  
B P Roques ◽  
P Crine ◽  
...  
Keyword(s):  
Active Site ◽  
Substrate Binding ◽  
Biologically Active ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Active Site Residues ◽  
Hydrophobic Cluster Analysis ◽  
Active Peptides ◽  
Ic50 Values

Neprilysin (EC 3.4.24.11) is a Zn2+ metallopeptidase involved in the degradation of biologically active peptides, e.g. enkephalins and atrial natriuretic peptide. The substrate specificity and catalytic activity of neprilysin resemble those of thermolysin, a crystallized bacterial Zn2+ metalloprotease. Despite little overall homology between the primary structures of thermolysin and neprilysin, many of the amino acid residues involved in catalysis, as well as Zn2+ and substrate binding, are highly conserved. Most of the active-site residues of neprilysin have their homologues in thermolysin and have been characterized by site-directed mutagenesis. Furthermore, hydrophobic cluster analysis has revealed some other analogies between the neprilysin and thermolysin sequences [Benchetrit, Bissery, Mornon, Devault, Crine and Roques (1988) Biochemistry 27, 592-596]. According to this analysis the role of Asn542 in the neprilysin active site is analogous to that of Asn112 of thermolysin, which is to bind the substrate. Site-directed mutagenesis was used to change Asn542 to Gly or Gln residues. The effect of these mutations on substrate catalysis and inhibitor binding was examined with a series of thiorphan-like compounds containing various degrees of methylation at the P2′ residue. For both mutated enzymes, determination of kinetic parameters with [D-Ala2,Leu5]enkephalin as substrate showed that the large decrease in activity was attributable to an increase in Km (14-16-fold) whereas kcat values were only slightly affected (2-3-fold decrease). This is in agreement with Asn542 being involved in substrate binding rather than directly in catalysis. Finally, the IC50 values for thiorphan and substituted thiorphans strongly suggest that Asn542 of neprilysin binds the substrate on the amino side of the P2′ residue by formation of a unique hydrogen bond.

scholarly journals Structural basis for the differential regulatory roles of the PDZ domain in C-terminal processing proteases

2019 ◽  
Author(s):  
Chuang-Kai Chueh ◽  
Nilanjan Som ◽  
Lu-Chu Ke ◽  
Meng-Ru Ho ◽  
Manjula Reddy ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Active Site ◽  
Resting State ◽  
Substrate Binding ◽  
Pdz Domain ◽  
Structural Basis ◽  
Active Site Residues ◽  
Structural Remodeling ◽  
Hydrophobic Substrate ◽  
Specific Protease

AbstractCarboxyl (C)-terminal processing proteases (CTPs) participate in protective and regulatory proteolysis in bacteria. The PDZ domain is central to the activity of CTPs but plays inherently different regulatory roles. For example, the PDZ domain inhibits the activity of the signaling protease CtpB by blocking the active site but is required for the activation of Prc (or Tsp), a tail-specific protease that degrades the ssrA-tagged proteins. Here, by structural and functional analysis we show that in the unliganded resting state of Prc, the PDZ domain is docked inside the bowl-shaped scaffold without contacting the active site, which is kept in a default misaligned conformation. In Prc, a hydrophobic substrate sensor distinct from CtpB engages substrate binding to the PDZ domain and triggers a structural remodeling to align the active site residues. Therefore, this work reveals the structural basis for understanding the contrasting roles of the PDZ domain in the regulation of CTPs.

scholarly journals The molecular basis of the nonprocessive elongation mechanism in levansucrases

2020 ◽  
pp. jbc.RA120.015853
Author(s):  
Enrique Raga-Carbajal ◽  
Adelaida Díaz-Vilchis ◽  
Sonia P. Rojas-Trejo ◽  
Enrique Rudiño-Piñera ◽  
Clarita Olvera
Keyword(s):  
Molecular Weight ◽  
Molecular Basis ◽  
Substrate Binding ◽  
Site Directed Mutagenesis ◽  
Crystallographic Structure ◽  
Substrate Affinity ◽  
Km Value ◽  
Pharmaceutical Industries ◽  
First Time ◽  
Binding Subsites

Levansucrases (LSs) synthesize levan, a β2-6-linked fructose polymer, by successively transferring the fructosyl moiety from sucrose to a growing acceptor molecule. Elucidation of the levan polymerization mechanism is important for using LSs in the production of size-defined products for application in the food and pharmaceutical industries. For a deeper understanding of the levan synthesis reaction, we determined the crystallographic structure of Bacillus subtilis LS (SacB) in complex with a levan-type fructooligosaccharide (FOS) and utilized site-directed mutagenesis to identify residues involved in substrate binding. The presence of a levanhexaose molecule in the central catalytic cavity allowed us to identify five substrate-binding subsites (-1, +1, +2, +3, and +4). Mutants affecting residues belonging to the identified acceptor subsites showed similar substrate affinity (Km) values to the wild type (WT) Km value but had a lower turnover number and transfructosylation/hydrolysis ratio. Most importantly, compared to the WT, the variants progressively yielded smaller-sized low-molecular weight (LMW) levans, as the affected subsites that were closer to the catalytic site, but without affecting their ability to synthesized high-molecular weight (HMW) levans. Furthermore, an additional oligosaccharide-binding (OB) site 20 Å away from the catalytic pocket was identified,and its potential participation in the elongation mechanism is discussed. Our results clarify, for the first time, the interaction of the enzyme with an acceptor/product oligosaccharide and elucidate the molecular basis of the nonprocessive levan elongation mechanism of LSs.

scholarly journals Complexes ofThermotoga maritimaS-adenosylmethionine decarboxylase provide insights into substrate specificity

2010 ◽  
Vol 66 (2) ◽  
pp. 181-189 ◽  
Author(s):  
Shridhar Bale ◽  
Kavita Baba ◽  
Diane E. McCloskey ◽  
Anthony E. Pegg ◽  
Steven E. Ealick
Keyword(s):  
Substrate Specificity ◽  
Ligand Binding ◽  
Crystal Structures ◽  
Active Sites ◽  
Thermotoga Maritima ◽  
Binding Mode ◽  
Cellular Growth ◽  
Active Site Residues ◽  
Adenosylmethionine Decarboxylase ◽  
First Time

The polyamines putrescine, spermidine and spermine are ubiquitous aliphatic cations and are essential for cellular growth and differentiation.S-Adenosylmethionine decarboxylase (AdoMetDC) is a critical pyruvoyl-dependent enzyme in the polyamine-biosynthetic pathway. The crystal structures of AdoMetDC from humans and plants and of the AdoMetDC proenzyme fromThermotoga maritimahave been obtained previously. Here, the crystal structures of activatedT. maritimaAdoMetDC (TmAdoMetDC) and of its complexes withS-adenosylmethionine methyl ester and 5′-deoxy-5′-dimethylthioadenosine are reported. The results demonstrate for the first time that TmAdoMetDC autoprocesses without the need for additional factors and that the enzyme contains two complete active sites, both of which use residues from both chains of the homodimer. The complexes provide insights into the substrate specificity and ligand binding of AdoMetDC in prokaryotes. The conservation of the ligand-binding mode and the active-site residues between human andT. maritimaAdoMetDC provides insight into the evolution of AdoMetDC.

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