On the molecular basis of inhibition by excess substrate in wild-type and Y143F flavocytochrome b2

1996 ◽  
Vol 24 (1) ◽  
pp. 15S-15S ◽  
Author(s):  
Martine Mayer ◽  
Nathalie Rouviere-Fourmy ◽  
Mariella Tegoni ◽  
Chantal Capeillere-Blandin ◽  
Florence Lederer
Keyword(s):  
Molecular Basis ◽  
Wild Type ◽  
Flavocytochrome B2 ◽  
Excess Substrate

scholarly journals The importance of the interdomain hinge in intramolecular electron transfer in flavocytochrome b2

1993 ◽  
Vol 291 (1) ◽  
pp. 89-94 ◽  
Author(s):  
P White ◽  
F D C Manson ◽  
C E Brunt ◽  
S K Chapman ◽  
G A Reid
Keyword(s):  
Electron Transfer ◽  
Steady State ◽  
Cytochrome C ◽  
Kinetic Properties ◽  
Stopped Flow ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Cytochrome C Reductase ◽  
Flavocytochrome B2 ◽  
Cytochrome C Reduction

The two distinct domains of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase) are connected by a typical hinge peptide. The amino acid sequence of this interdomain hinge is dramatically different in flavocytochromes b2 from Saccharomyces cerevisiae and Hansenula anomala. This difference in the hinge is believed to contribute to the difference in kinetic properties between the two enzymes. To probe the importance of the hinge, an interspecies hybrid enzyme has been constructed comprising the bulk of the S. cerevisiae enzyme but containing the H. anomala flavocytochrome b2 hinge. The kinetic properties of this ‘hinge-swap’ enzyme have been investigated by steady-state and stopped-flow methods. The hinge-swap enzyme remains a good lactate dehydrogenase as is evident from steady-state experiments with ferricyanide as acceptor (only 3-fold less active than wild-type enzyme) and stopped-flow experiments monitoring flavin reduction (2.5-fold slower than in wild-type enzyme). The major effect of the hinge-swap mutation is to lower dramatically the enzyme's effectiveness as a cytochrome c reductase; kcat. for cytochrome c reduction falls by more than 100-fold, from 207 +/- 10 s-1 (25 degrees C, pH 7.5) in the wild-type enzyme to 1.62 +/- 0.41 s-1 in the mutant enzyme. This fall in cytochrome c reductase activity results from poor interdomain electron transfer between the FMN and haem groups. This can be demonstrated by the fact that the kcat. for haem reduction in the hinge-swap enzyme (measured by the stopped-flow method) has a value of 1.61 +/- 0.42 s-1, identical with the value for cytochrome c reduction and some 300-fold lower than the value for the wild-type enzyme. From these and other kinetic parameters, including kinetic isotope effects with [2-2H]lactate, we conclude that the hinge plays a crucial role in allowing efficient electron transfer between the two domains of flavocytochrome b2.

Molecular Interpretation of Inhibition by Excess Substrate in Flavocytochromeb2:  A Study with Wild-Type and Y143F Mutant Enzymes†

Biochemistry ◽  
1997 ◽  
Vol 36 (23) ◽  
pp. 7126-7135 ◽  
Author(s):  
Nathalie Rouvière ◽  
Martine Mayer ◽  
Mariella Tegoni ◽  
Chantal Capeillère-Blandin ◽  
Florence Lederer
Keyword(s):  
Wild Type ◽  
Excess Substrate ◽  
Molecular Interpretation

scholarly journals Acquisition of the Ability To Assimilate Mannitol by Saccharomyces cerevisiae through Dysfunction of the General Corepressor Tup1-Cyc8

2014 ◽  
Vol 81 (1) ◽  
pp. 9-16 ◽  
Author(s):  
Moeko Chujo ◽  
Shiori Yoshida ◽  
Anri Ota ◽  
Kousaku Murata ◽  
Shigeyuki Kawai
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Basis ◽  
Mutant Allele ◽  
Wild Type ◽  
Growth Data ◽  
Content Type ◽  
Corepressor Complex ◽  
Marine Biomass ◽  
Prolonged Culture ◽  
Genes Encoding

ABSTRACTSaccharomyces cerevisiaenormally cannot assimilate mannitol, a promising brown macroalgal carbon source for bioethanol production. The molecular basis of this inability remains unknown. We found that cells capable of assimilating mannitol arose spontaneously from wild-typeS. cerevisiaeduring prolonged culture in mannitol-containing medium. Based on microarray data, complementation analysis, and cell growth data, we demonstrated that acquisition of mannitol-assimilating ability was due to spontaneous mutations in the genes encoding Tup1 or Cyc8, which constitute a general corepressor complex that regulates many kinds of genes. We also showed that anS. cerevisiaestrain carrying a mutant allele ofCYC8exhibited superior salt tolerance relative to other ethanologenic microorganisms; this characteristic would be highly beneficial for the production of bioethanol from marine biomass. Thus, we succeeded in conferring the ability to assimilate mannitol onS. cerevisiaethrough dysfunction of Tup1-Cyc8, facilitating production of ethanol from mannitol.

scholarly journals Tyr-143 facilitates interdomain electron transfer in flavocytochrome b2

1992 ◽  
Vol 285 (1) ◽  
pp. 187-192 ◽  
Author(s):  
C S Miles ◽  
N Rouvière-Fourmy ◽  
F Lederer ◽  
F S Mathews ◽  
G A Reid ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Steady State ◽  
Isotope Effects ◽  
Mutant Enzyme ◽  
Stopped Flow ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Flavocytochrome B2 ◽  
Rate Determining Step ◽  
Kinetic Isotope

The role of Tyr-143 in the catalytic cycle of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase) has been examined by replacement of this residue with phenylalanine. The electron-transfer steps in wild-type and mutant flavocytochromes b2 have been investigated by using steady-state and stopped-flow kinetic methods. The most significant effect of the Tyr-143----Phe mutation is a change in the rate-determining step in the reduction of the enzyme. For wild-type enzyme the main rate-determining step is proton abstraction at the C-2 position of lactate, as shown by the 2H kinetic-isotope effect. However, for the mutant enzyme it is clear that the slowest step is interdomain electron transfer between the FMN and haem prosthetic groups. In fact, the rate of haem reduction by lactate, as determined by the stopped-flow method, is decreased by more than 20-fold, from 445 +/- 50 s-1 (25 degrees C, pH 7.5) in the wild-type enzyme to 21 +/- 2 s-1 in the mutant enzyme. Decreases in kinetic-isotope effects seen with [2-2H]lactate for mutant enzyme compared with wild-type, both for flavin reduction (from 8.1 +/- 1.4 to 4.3 +/- 0.8) and for haem reduction (from 6.3 +/- 1.2 to 1.6 +/- 0.5) also provide support for a change in the nature of the rate-determining step. Other kinetic parameters determined by stopped-flow methods and with two external electron acceptors (cytochrome c and ferricyanide) under steady-state conditions are all consistent with this mutation having a dramatic effect on interdomain electron transfer. We conclude that Tyr-143, an active-site residue which lies between the flavodehydrogenase and cytochrome domains of flavocytochrome b2, plays a key role in facilitating electron transfer between FMN and haem groups.

Computational analysis of molecular basis of 1:1 interactions of NRG-1β wild-type and variants with ErbB3 and ErbB4

2005 ◽  
Vol 59 (4) ◽  
pp. 742-756 ◽  
Author(s):  
Cheng Luo ◽  
Lingfei Xu ◽  
Suxin Zheng ◽  
Xiaomin Luo ◽  
Jianhua Shen ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Computational Analysis ◽  
Wild Type

scholarly journals Molecular basis of attenuation of neurovirulence of wild-type Japanese encephalitis virus strain SA14

1995 ◽  
Vol 76 (2) ◽  
pp. 409-413 ◽  
Author(s):  
H. Ni ◽  
G.-J. J. Chang ◽  
H. Xie ◽  
D. W. Trent ◽  
A. D. T. Barrett
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Virus Strain ◽  
Molecular Basis ◽  
Encephalitis Virus ◽  
Wild Type

scholarly journals Molecular Basis of Ca2+ Activation of the Mouse Cardiac Ca2+ Release Channel (Ryanodine Receptor)

2001 ◽  
Vol 118 (1) ◽  
pp. 33-44 ◽  
Author(s):  
Pin Li ◽  
S.R. Wayne Chen
Keyword(s):  
Lipid Bilayers ◽  
Ryanodine Receptor ◽  
Molecular Basis ◽  
Single Channel ◽  
Single Point ◽  
Hek293 Cells ◽  
Single Point Mutation ◽  
Single Channels ◽  
Wild Type ◽  
Structural And Functional Properties

Activation of the cardiac ryanodine receptor (RyR2) by Ca2+ is an essential step in excitation-contraction coupling in heart muscle. However, little is known about the molecular basis of activation of RyR2 by Ca2+. In this study, we investigated the role in Ca2+ sensing of the conserved glutamate 3987 located in the predicted transmembrane segment M2 of the mouse RyR2. Single point mutation of this conserved glutamate to alanine (E3987A) reduced markedly the sensitivity of the channel to activation by Ca2+, as measured by using single-channel recordings in planar lipid bilayers and by [3H]ryanodine binding assay. However, this mutation did not alter the affinity of [3H]ryanodine binding and the single-channel conductance. In addition, the E3987A mutant channel was activated by caffeine and ATP, was inhibited by Mg2+, and was modified by ryanodine in a fashion similar to that of the wild-type channel. Coexpression of the wild-type and mutant E3987A RyR2 proteins in HEK293 cells produced individual single channels with intermediate sensitivities to activating Ca2+. These results are consistent with the view that glutamate 3987 is a major determinant of Ca2+ sensitivity to activation of the mouse RyR2 channel, and that Ca2+ sensing by RyR2 involves the cooperative action between ryanodine receptor monomers. The results of this study also provide initial insights into the structural and functional properties of the mouse RyR2, which should be useful for studying RyR2 function and regulation in genetically modified mouse models.

scholarly journals Mutations at pipX Suppress Lethality of PII-Deficient Mutants of Synechococcus elongatus PCC 7942

2009 ◽  
Vol 191 (15) ◽  
pp. 4863-4869 ◽  
Author(s):  
Javier Espinosa ◽  
Miguel Angel Castells ◽  
Karim Boumediene Laichoubi ◽  
Asunción Contreras
Keyword(s):  
Molecular Basis ◽  
Synechococcus Elongatus ◽  
Wild Type ◽  
Growth Defects ◽  
Genetic Inactivation ◽  
Domains Of Life ◽  
Synechococcus Elongatus Pcc 7942 ◽  
Pcc 7942 ◽  
Severe Growth ◽  
Null Mutants

ABSTRACT The PII proteins are found in all three domains of life as key integrators of signals reflecting the balance of nitrogen and carbon. Genetic inactivation of PII proteins is typically associated with severe growth defects or death. However, the molecular basis of these defects depends on the specific functions of the proteins with which PII proteins interact to regulate nitrogen metabolism in different organisms. In Synechococcus elongatus PCC 7942, where PII forms complexes with the NtcA coactivator PipX, attempts to engineer PII-deficient strains failed in a wild-type background but were successful in pipX null mutants. Consistent with the idea that PII is essential to counteract the activity of PipX, four different spontaneous mutations in the pipX gene were found in cultures in which glnB had been genetically inactivated.

scholarly journals S19W, T27W, and N330Y mutations in ACE2 enhance SARS-CoV-2 S-RBD binding toward both wild-type and antibody-resistant viruses and its molecular basis

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Fei Ye ◽  
Xi Lin ◽  
Zimin Chen ◽  
Fanli Yang ◽  
Sheng Lin ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Structural Data ◽  
Van Der Waals Interactions ◽  
Side Chains ◽  
Receptor Binding Domain ◽  
Converting Enzyme ◽  
Wild Type ◽  
Angiotensin Converting Enzyme 2 ◽  
Domain Protein ◽  
Entry Inhibition

AbstractSARS-CoV-2 recognizes, via its spike receptor-binding domain (S-RBD), human angiotensin-converting enzyme 2 (ACE2) to initiate infection. Ecto-domain protein of ACE2 can therefore function as a decoy. Here we show that mutations of S19W, T27W, and N330Y in ACE2 could individually enhance SARS-CoV-2 S-RBD binding. Y330 could be synergistically combined with either W19 or W27, whereas W19 and W27 are mutually unbeneficial. The structures of SARS-CoV-2 S-RBD bound to the ACE2 mutants reveal that the enhanced binding is mainly contributed by the van der Waals interactions mediated by the aromatic side-chains from W19, W27, and Y330. While Y330 and W19/W27 are distantly located and devoid of any steric interference, W19 and W27 are shown to orient their side-chains toward each other and to cause steric conflicts, explaining their incompatibility. Finally, using pseudotyped SARS-CoV-2 viruses, we demonstrate that these residue substitutions are associated with dramatically improved entry-inhibition efficacy toward both wild-type and antibody-resistant viruses. Taken together, our biochemical and structural data have delineated the basis for the elevated S-RBD binding associated with S19W, T27W, and N330Y mutations in ACE2, paving the way for potential application of these mutants in clinical treatment of COVID-19.

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