Crystal structure of the wild-type and D30A mutant thioredoxin h of Chlamydomonas reinhardtii and implications for the catalytic mechanism

2001 ◽  
Vol 359 (1) ◽  
pp. 65-75 ◽  
Author(s):  
Valeria MENCHISE ◽  
Catherine CORBIER ◽  
Claude DIDIERJEAN ◽  
Michele SAVIANO ◽  
Ettore BENEDETTI ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Proton Transfer ◽  
Chlamydomonas Reinhardtii ◽  
Catalytic Mechanism ◽  
Structural Data ◽  
Careful Analysis ◽  
Wild Type ◽  
Thioredoxin H ◽  
Dynamics Simulations

Thioredoxins are ubiquitous proteins which catalyse the reduction of disulphide bridges on target proteins. The catalytic mechanism proceeds via a mixed disulphide intermediate whose breakdown should be enhanced by the involvement of a conserved buried residue, Asp-30, as a base catalyst towards residue Cys-39. We report here the crystal structure of wild-type and D30A mutant thioredoxin h from Chlamydomonas reinhardtii, which constitutes the first crystal structure of a cytosolic thioredoxin isolated from a eukaryotic plant organism. The role of residue Asp-30 in catalysis has been revisited since the distance between the carboxylate OD1 of Asp-30 and the sulphur SG of Cys-39 is too great to support the hypothesis of direct proton transfer. A careful analysis of all available crystal structures reveals that the relative positioning of residues Asp-30 and Cys-39 as well as hydrophobic contacts in the vicinity of residue Asp-30 do not allow a conformational change sufficient to bring the two residues close enough for a direct proton transfer. This suggests that protonation/deprotonation of Cys-39 should be mediated by a water molecule. Molecular-dynamics simulations, carried out either in vacuo or in water, as well as proton-inventory experiments, support this hypothesis. The results are discussed with respect to biochemical and structural data.

scholarly journals Crystal structure of the wild-type and D30A mutant thioredoxin h of Chlamydomonas reinhardtii and implications for the catalytic mechanism

2001 ◽  
Vol 359 (1) ◽  
pp. 65 ◽  
Author(s):  
Valeria MENCHISE ◽  
Catherine CORBIER ◽  
Claude DIDIERJEAN ◽  
Michele SAVIANO ◽  
Ettore BENEDETTI ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Chlamydomonas Reinhardtii ◽  
Catalytic Mechanism ◽  
Wild Type ◽  
Thioredoxin H

scholarly journals Roles of the Conserved Aspartate and Arginine in the Catalytic Mechanism of an Archaeal β-Class Carbonic Anhydrase

2002 ◽  
Vol 184 (15) ◽  
pp. 4240-4245 ◽  
Author(s):  
Kerry S. Smith ◽  
Cheryl Ingram-Smith ◽  
James G. Ferry
Keyword(s):  
Crystal Structure ◽  
Proton Transfer ◽  
Rate Constants ◽  
Active Sites ◽  
Catalytic Mechanism ◽  
Zinc Hydroxide ◽  
Carbonic Anhydrases ◽  
Wild Type ◽  
Transfer Step ◽  
Mechanism Of Catalysis

ABSTRACT The roles of an aspartate and an arginine, which are completely conserved in the active sites of β-class carbonic anhydrases, were investigated by steady-state kinetic analyses of replacement variants of the β-class enzyme (Cab) from the archaeon Methanobacterium thermoautotrophicum. Previous kinetic analyses of wild-type Cab indicated a two-step zinc-hydroxide mechanism of catalysis in which the k cat/Km value depends only on the rate constants for the CO2 hydration step, whereas k cat also depends on rate constants from the proton transfer step (K. S. Smith, N. J. Cosper, C. Stalhandske, R. A. Scott, and J. G. Ferry, J. Bacteriol. 182:6605-6613, 2000). The recently solved crystal structure of Cab shows the presence of a buffer molecule within hydrogen bonding distance of Asp-34, implying a role for this residue in the proton transport step (P. Strop, K. S. Smith, T. M. Iverson, J. G. Ferry, and D. C. Rees, J. Biol. Chem. 276:10299-10305, 2001). The k cat/Km values of Asp-34 variants were decreased relative to those of the wild type, although not to an extent which supports an essential role for this residue in the CO2 hydration step. Parallel decreases in k cat and k cat/Km values for the variants precluded any conclusions regarding a role for Asp-34 in the proton transfer step; however, the k cat of the D34A variant was chemically rescued by replacement of 2-(N-morpholino)propanesulfonic acid buffer with imidazole at pH 7.2, supporting a role for the conserved aspartate in the proton transfer step. The crystal structure of Cab also shows Arg-36 with two hydrogen bonds to Asp-34. Arg-36 variants had both k cat and k cat/Km values that were decreased at least 250-fold relative to those of the wild type, establishing an essential function for this residue. Imidazole was unable to rescue the k cat of the R36A variant; however, partial rescue of the kinetic parameter was obtained with guanidine-HCl indicating that the guanido group of this residue is important.

scholarly journals Arabidopsis Disulfide Reductase, Trx-h2, Functions as an RNA Chaperone under Cold Stress

2021 ◽  
Vol 11 (15) ◽  
pp. 6865
Author(s):  
Eun Seon Lee ◽  
Joung Hun Park ◽  
Seong Dong Wi ◽  
Ho Byoung Chae ◽  
Seol Ki Paeng ◽  
...  
Keyword(s):  
Cold Stress ◽  
Wild Type ◽  
Rna Chaperone ◽  
E Coli ◽  
Cold Sensitive ◽  
Thioredoxin H ◽  
Functional Rnas

The thioredoxin-h (Trx-h) family of Arabidopsis thaliana comprises cytosolic disulfide reductases. However, the physiological function of Trx-h2, which contains an additional 19 amino acids at its N-terminus, remains unclear. In this study, we investigated the molecular function of Trx-h2 both in vitro and in vivo and found that Arabidopsis Trx-h2 overexpression (Trx-h2OE) lines showed significantly longer roots than wild-type plants under cold stress. Therefore, we further investigated the role of Trx-h2 under cold stress. Our results revealed that Trx-h2 functions as an RNA chaperone by melting misfolded and non-functional RNAs, and by facilitating their correct folding into active forms with native conformation. We showed that Trx-h2 binds to and efficiently melts nucleic acids (ssDNA, dsDNA, and RNA), and facilitates the export of mRNAs from the nucleus to the cytoplasm under cold stress. Moreover, overexpression of Trx-h2 increased the survival rate of the cold-sensitive E. coli BX04 cells under low temperature. Thus, our data show that Trx-h2 performs function as an RNA chaperone under cold stress, thus increasing plant cold tolerance.

WHICH IS THE PROTON-SHUTTLE IN ISOXANTHOPTERIN DEAMINASE? QM/MM MD UNDERSTANDING

2013 ◽  
Vol 12 (08) ◽  
pp. 1341002 ◽  
Author(s):  
XIN ZHANG ◽  
MING LEI
Keyword(s):  
Crystal Structure ◽  
Molecular Dynamics ◽  
Hydrogen Bonds ◽  
Proton Transfer ◽  
Glutamic Acid ◽  
Active Site ◽  
Nucleophilic Attack ◽  
Zinc Ions ◽  
Initial Model ◽  
Dynamics Simulations

The deamination process of isoxanthopterin catalyzed by isoxanthopterin deaminase was determined using the combined QM(PM3)/MM molecular dynamics simulations. In this paper, the updated PM3 parameters were employed for zinc ions and the initial model was built up based on the crystal structure. Proton transfer and following steps have been investigated in two paths: Asp336 and His285 serve as the proton shuttle, respectively. Our simulations showed that His285 is more effective than Aap336 in proton transfer for deamination of isoxanthopterin. As hydrogen bonds between the substrate and surrounding residues play a key role in nucleophilic attack, we suggested mutating Thr195 to glutamic acid, which could enhance the hydrogen bonds and help isoxanthopterin get close to the active site. The simulations which change the substrate to pterin 6-carboxylate also performed for comparison. Our results provide reference for understanding of the mechanism of deaminase and for enhancing the deamination rate of isoxanthopterin deaminase.

scholarly journals Intracellular Proton-Transfer Mutants in a CLC Cl−/H+ Exchanger

2009 ◽  
Vol 133 (2) ◽  
pp. 131-138 ◽  
Author(s):  
Hyun-Ho Lim ◽  
Christopher Miller
Keyword(s):  
Proton Transfer ◽  
Mutational Analysis ◽  
Side Chain ◽  
Side Chains ◽  
Extracellular Water ◽  
Wild Type ◽  
Cl Transport ◽  
Structural And Functional Properties ◽  
Key Residues

CLC-ec1, a bacterial homologue of the CLC family’s transporter subclass, catalyzes transmembrane exchange of Cl− and H+. Mutational analysis based on the known structure reveals several key residues required for coupling H+ to the stoichiometric countermovement of Cl−. E148 (Gluex) transfers protons between extracellular water and the protein interior, and E203 (Gluin) is thought to function analogously on the intracellular face of the protein. Mutation of either residue eliminates H+ transport while preserving Cl− transport. We tested the role of Gluin by examining structural and functional properties of mutants at this position. Certain dissociable side chains (E, D, H, K, R, but not C and Y) retain H+/Cl− exchanger activity to varying degrees, while other mutations (V, I, or C) abolish H+ coupling and severely inhibit Cl− flux. Transporters substituted with other nonprotonatable side chains (Q, S, and A) show highly impaired H+ transport with substantial Cl− transport. Influence on H+ transport of side chain length and acidity was assessed using a single-cysteine mutant to introduce non-natural side chains. Crystal structures of both coupled (E203H) and uncoupled (E203V) mutants are similar to wild type. The results support the idea that Gluin is the internal proton-transfer residue that delivers protons from intracellular solution to the protein interior, where they couple to Cl− movements to bring about Cl−/H+ exchange.

scholarly journals The role of tyrosine-9 and the C-terminal helix in the catalytic mechanism of Alpha-class glutathione S-transferases

1999 ◽  
Vol 343 (3) ◽  
pp. 525-531 ◽  
Author(s):  
Claire S. ALLARDYCE ◽  
Paul D. MCDONAGH ◽  
Lu-Yun LIAN ◽  
C. Roland WOLF ◽  
Gordon C. K. ROBERTS
Keyword(s):  
Conformational Change ◽  
Catalytic Mechanism ◽  
Ethacrynic Acid ◽  
Marked Decrease ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Turnover Number ◽  
Hydrophobic Substrate ◽  
Glutathione S Transferases

Glutathione S-transferases (GSTs) play a key role in the metabolism of drugs and xenobiotics. To investigate the catalytic mechanism, substrate binding and catalysis by the wild-type and two mutants of GST A1-1 have been studied. Substitution of the ‘essential’ Tyr9 by phenylalanine leads to a marked decrease in the kcat for 1-chloro-2,4-dinitrobenzene (CDNB), but has no affect on kcat for ethacrynic acid. Similarly, removal of the C-terminal helix by truncation of the enzyme at residue 209 leads to a decrease in kcat for CDNB, but an increase in kcat for ethacrynic acid. The binding of a GSH analogue increases the affinity of the wild-type enzyme for CDNB, and increases the rate of the enzyme-catalysed conjugation of this substrate with the small thiols 2-mercaptoethanol and dithiothreitol. This suggests that GSH binding produces a conformational change which is transmitted to the binding site for the hydrophobic substrate, where it alters both the affinity for the substrate and the catalytic-centre activity (‘turnover number‘) for conjugation, perhaps by increasing the proportion of the substrate bound productively. Neither of these two effects of GSH analogues are seen in the C-terminally truncated enzyme, indicating a role for the C-terminal helix in the GSH-induced conformational change.

scholarly journals Role of βAsn-243 in the Phosphate-binding Subdomain of Catalytic Sites ofEscherichia coliF1-ATPase

2004 ◽  
Vol 279 (44) ◽  
pp. 46057-46064 ◽  
Author(s):  
Zulfiqar Ahmad ◽  
Alan E. Senior
Keyword(s):  
Atpase Activity ◽  
Atp Synthase ◽  
Catalytic Mechanism ◽  
Wild Type ◽  
Phosphate Binding ◽  
Γ Subunit ◽  
Catalytic Sites ◽  
The Face ◽  
Extensive Involvement

In the catalytic mechanism of ATP synthase, phosphate (Pi) binding and release steps are believed to be correlated to γ-subunit rotation, and Pibinding is proposed to be prerequisite for binding ADP in the face of high cellular [ATP]/[ADP] ratios. In x-ray structures, residue βAsn-243 appears centrally located in the Pi-binding subdomain of catalytic sites. Here we studied the role of βAsn-243 inEscherichia coliATP synthase by mutagenesis to Ala and Asp. Mutation βN243A caused 30-fold impairment of F1-ATPase activity; 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole inhibited this activity less potently than in wild type and Piprotected from inhibition. ADP-fluoroaluminate was more inhibitory than in wild-type, but ADP-fluoroscandium was less inhibitory. βN243D F1-ATPase activity was impaired by 1300-fold and was not inhibited by ADP-fluoroaluminate or ADP-fluoroscandium. 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole activated βN243D F1-ATPase, and Pidid not affect activation. We conclude that residue βAsn-243 is not involved in Pibinding directly but is necessary for correct organization of the transition state complex through extensive involvement in hydrogen bonding to neighboring residues. It is also probably involved in orientation of the “attacking water” and of an associated second water.

scholarly journals Conformational effects of Lys191 in the human GnRH receptor: mutagenesis and molecular dynamics simulations studies

2009 ◽  
Vol 201 (2) ◽  
pp. 297-307 ◽  
Author(s):  
Eduardo Jardón-Valadez ◽  
Arturo Aguilar-Rojas ◽  
Guadalupe Maya-Núñez ◽  
Alfredo Leaños-Miranda ◽  
Ángel Piñeiro ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Dynamic Behavior ◽  
Gnrh Receptor ◽  
Receptor Trafficking ◽  
Wild Type ◽  
Conformational Effects ◽  
Dynamics Simulations ◽  
And Function

In the present study, we analyzed the role of Lys191 on function, structure, and dynamic behavior of the human GnRH receptor (hGnRHR) and the formation of the Cys14–Cys200 bridge, which is essential for receptor trafficking to the plasma membrane. Several mutants were studied; mutants lacked either the Cys14–Cys200 bridge, Lys191 or both. The markedly reduced expression and function of a Cys14Ser mutant lacking the 14–200 bridge, was nearly restored to wild-type/ΔLys191 levels upon deletion of Lys191. Lys191 removal resulted in changes in the dynamic behavior of the mutants as disclosed by molecular dynamics simulations: the distance between the sulfur- (or oxygen-) sulfur groups of Cys (or Ser)14 and Cys200 was shorter and more constant, and the conformation of the NH2-terminus and the exoloop 2 exhibited fewer fluctuations than when Lys191 was present. These data provide novel information on the role of Lys191 in defining an optimal configuration for the hGnRHR intracellular trafficking and function.

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