Solvent Effects on Environmentally Coupled Hydrogen Tunnelling During Catalysis by Dihydrofolate Reductase from Thermotoga maritima

2008 ◽  
Vol 14 (34) ◽  
pp. 10782-10788 ◽  
Author(s):  
E. Joel Loveridge ◽  
Rhiannon M. Evans ◽  
Rudolf K. Allemann
Keyword(s):  
Dihydrofolate Reductase ◽  
Solvent Effects ◽  
Thermotoga Maritima

scholarly journals Differences in Thermal Structural Changes and Melting Between Mesophilic and Thermophilic Dihydrofolate Reductase Enzymes

2020 ◽  
Author(s):  
Irene Maffucci ◽  
Damien Laage ◽  
Guillaume Stirnemann ◽  
Fabio Sterpone
Keyword(s):  
Dihydrofolate Reductase ◽  
Conformational Changes ◽  
Structural Changes ◽  
Molecular Mechanisms ◽  
Thermotoga Maritima ◽  
Conformational Sampling ◽  
Detailed Understanding ◽  
Wide Range ◽  
The Difference ◽  
Dynamics Simulations

A key aspect of life's evolution on Earth is the adaptation of proteins to be stable and work in a very wide range of temperature conditions. A detailed understanding of the associated molecular mechanisms would also help to design enzymes optimized for biotechnological processes. Despite important advances, a comprehensive picture of how thermophilic enzymes succeed in functioning under extreme temperatures remains incomplete. Here, we examine the temperature dependence of stability and of flexibility in the mesophilic monomeric Escherichia coli (Ec) and thermophilic dimeric Thermotoga maritima (Tm) homologs of the paradigm dihydrofolate reductase (DHFR) enzyme. We use all-atom molecular dynamics simulations and a replica-exchange scheme that allows to enhance the conformational sampling while providing at the same time a detailed understanding of the enzymes' behavior at increasing temperatures. We show that this approach reproduces the stability shift between the two homologs, and provides a molecular description of the denaturation mechanism by identifying the sequence of secondary structure elements melting as temperature increases, which is not straightforwardly obtained in the experiments. By repeating our approach on the hypothetical TmDHFR monomer, we further determine the respective effects of sequence and oligomerization in the exceptional stability of TmDFHR. We show that the intuitive expectation that protein flexibility and thermal stability are correlated is not verified. Finally, our simulations reveal that significant conformational fluctuations already take place much below the melting temperature. While the difference between the TmDHFR and EcDHFR catalytic activities is often interpreted via a simplified two-state picture involving the open and closed conformations of the key M20 loop, our simulations suggest that the two homologs' markedly different activity temperature dependences are caused by changes in the ligand-cofactor distance distributions in response to these conformational changes.

scholarly journals Moritella Cold-Active Dihydrofolate Reductase: Are There Natural Limits to Optimization of Catalytic Efficiency at Low Temperature?

2003 ◽  
Vol 185 (18) ◽  
pp. 5519-5526 ◽  
Author(s):  
Ying Xu ◽  
Georges Feller ◽  
Charles Gerday ◽  
Nicolas Glansdorff
Keyword(s):  
Low Temperature ◽  
Dihydrofolate Reductase ◽  
Thermotoga Maritima ◽  
Catalytic Efficiency ◽  
Metabolic Enzyme ◽  
Psychrophilic Bacterium ◽  
E Coli ◽  
Enthalpy Changes ◽  
Maximum Stability ◽  
Order Of Magnitude

ABSTRACT Adapting metabolic enzymes of microorganisms to low temperature environments may require a difficult compromise between velocity and affinity. We have investigated catalytic efficiency in a key metabolic enzyme (dihydrofolate reductase) of Moritella profunda sp. nov., a strictly psychrophilic bacterium with a maximal growth rate at 2°C or less. The enzyme is monomeric (M r = 18,291), 55% identical to its Escherichia coli counterpart, and displays Tm and denaturation enthalpy changes much lower than E. coli and Thermotoga maritima homologues. Its stability curve indicates a maximum stability above the temperature range of the organism, and predicts cold denaturation below 0°C. At mesophilic temperatures the apparent Km value for dihydrofolate is 50- to 80-fold higher than for E. coli, Lactobacillus casei, and T. maritima dihydrofolate reductases, whereas the apparent Km value for NADPH, though higher, remains in the same order of magnitude. At 5°C these values are not significantly modified. The enzyme is also much less sensitive than its E. coli counterpart to the inhibitors methotrexate and trimethoprim. The catalytic efficiency (k cat /Km ) with respect to dihydrofolate is thus much lower than in the other three bacteria. The higher affinity for NADPH could have been maintained by selection since NADPH assists the release of the product tetrahydrofolate. Dihydrofolate reductase adaptation to low temperature thus appears to have entailed a pronounced trade-off between affinity and catalytic velocity. The kinetic features of this psychrophilic protein suggest that enzyme adaptation to low temperature may be constrained by natural limits to optimization of catalytic efficiency.

The crystal structure of dihydrofolate reductase from Thermotoga maritima: molecular features of thermostability

2000 ◽  
Vol 297 (3) ◽  
pp. 659-672 ◽  
Author(s):  
Thomas Dams ◽  
Günter Auerbach ◽  
Gerd Bader ◽  
Uwe Jacob ◽  
Tarmo Ploom ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Dihydrofolate Reductase ◽  
Thermotoga Maritima ◽  
Molecular Features

Probing coupled motions in enzymatic hydrogen tunnelling reactions

2009 ◽  
Vol 37 (2) ◽  
pp. 349-353 ◽  
Author(s):  
Rudolf K. Allemann ◽  
Rhiannon M. Evans ◽  
E. Joel Loveridge
Keyword(s):  
Protein Dynamics ◽  
Dihydrofolate Reductase ◽  
Thermotoga Maritima ◽  
Quantum Mechanical ◽  
Extreme Conditions ◽  
Chemical Transformations ◽  
Complex Reaction ◽  
Detailed Mechanism ◽  
The Relationship ◽  
Catalytic Power

Much work has gone into understanding the physical basis of the enormous catalytic power of enzymes over the last 50 years or so. Nevertheless, the detailed mechanism used by Nature's catalysts to speed chemical transformations remains elusive. DHFR (dihydrofolate reductase) has served as a paradigm to study the relationship between the structure, function and dynamics of enzymatic transformations. A complex reaction cascade, which involves rearrangements and movements of loops and domains of the enzyme, is used to orientate cofactor and substrate in a reactive configuration from which hydride is transferred by quantum mechanical tunnelling. In the present paper, we review results from experiments that probe the influence of protein dynamics on the chemical step of the reaction catalysed by TmDHFR (DHFR from Thermotoga maritima). This enzyme appears to have evolved an optimal structure that can maintain a catalytically competent conformation under extreme conditions.

scholarly journals Differences in Thermal Structural Changes and Melting Between Mesophilic and Thermophilic Dihydrofolate Reductase Enzymes

2020 ◽  
Author(s):  
Irene Maffucci ◽  
Damien Laage ◽  
Guillaume Stirnemann ◽  
Fabio Sterpone
Keyword(s):  
Dihydrofolate Reductase ◽  
Conformational Changes ◽  
Structural Changes ◽  
Molecular Mechanisms ◽  
Thermotoga Maritima ◽  
Conformational Sampling ◽  
Detailed Understanding ◽  
Wide Range ◽  
The Difference ◽  
Dynamics Simulations

A key aspect of life's evolution on Earth is the adaptation of proteins to be stable and work in a very wide range of temperature conditions. A detailed understanding of the associated molecular mechanisms would also help to design enzymes optimized for biotechnological processes. Despite important advances, a comprehensive picture of how thermophilic enzymes succeed in functioning under extreme temperatures remains incomplete. Here, we examine the temperature dependence of stability and of flexibility in the mesophilic monomeric Escherichia coli (Ec) and thermophilic dimeric Thermotoga maritima (Tm) homologs of the paradigm dihydrofolate reductase (DHFR) enzyme. We use all-atom molecular dynamics simulations and a replica-exchange scheme that allows to enhance the conformational sampling while providing at the same time a detailed understanding of the enzymes' behavior at increasing temperatures. We show that this approach reproduces the stability shift between the two homologs, and provides a molecular description of the denaturation mechanism by identifying the sequence of secondary structure elements melting as temperature increases, which is not straightforwardly obtained in the experiments. By repeating our approach on the hypothetical TmDHFR monomer, we further determine the respective effects of sequence and oligomerization in the exceptional stability of TmDFHR. We show that the intuitive expectation that protein flexibility and thermal stability are correlated is not verified. Finally, our simulations reveal that significant conformational fluctuations already take place much below the melting temperature. While the difference between the TmDHFR and EcDHFR catalytic activities is often interpreted via a simplified two-state picture involving the open and closed conformations of the key M20 loop, our simulations suggest that the two homologs' markedly different activity temperature dependences are caused by changes in the ligand-cofactor distance distributions in response to these conformational changes.

Thermodynamics and Solvent Effects on Substrate and Cofactor Binding inEscherichia coliChromosomal Dihydrofolate Reductase

Biochemistry ◽  
2011 ◽  
Vol 50 (18) ◽  
pp. 3673-3685 ◽  
Author(s):  
Jordan Grubbs ◽  
Sharghi Rahmanian ◽  
Alexa DeLuca ◽  
Chetan Padmashali ◽  
Michael Jackson ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Solvent Effects ◽  
Cofactor Binding
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