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

Dams, Thomas; Auerbach, Günter; Bader, Gerd; Jacob, Uwe; Ploom, Tarmo; Huber, Robert; Jaenicke, Rainer

doi:10.1006/jmbi.2000.3570

Cited by 98 publications

(154 citation statements)

References 33 publications

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“…DHFRs are usually monomeric enzymes. However, the thermophilic T. maritima DHFR was found to be a dimer (6,41) and crystallographic analysis confirmed that the dimeric state plays an important role in the stabilization of this enzyme (7). Accordingly, DHFR Mp was expected to be a monomeric protein, as confirmed here by gel filtration and mass spectrometry.…”

Section: Discussionsupporting

confidence: 71%

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

Xu¹,

Feller

Gerday

et al. 2003

J Bacteriol

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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 T m 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 K m value for dihydrofolate is 50-to 80-fold higher than for E. coli, Lactobacillus casei, and T. maritima dihydrofolate reductases, whereas the apparent K m 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 /K m ) 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 temperature range compatible with procaryotic life extends from well below 0°C up to at least 113°C. The comparative analysis of series of homologous enzymes spanning this range suggested the molecular strategies which achieve the necessary compromises between structural flexibility and stability. Thermophilic enzymes appear to owe their stability to a variety of extrinsic or intrinsic factors (for a recent review, see reference 13). Moreover, oligomerization is the dominant stabilizing factor in several proteins (20,22,39); a basically similar situation is obtained when an enzyme is stabilized by physical association with another one (37).At the other end of the temperature scale, psychrophilic (24) enzymes face the necessity of being efficient catalysts at a low energy cost (4, 9, 14). On the basis of a much more limited set of data than for thermophilic enzymes (see recent reviews in references 33, 35, and 45), two basic strategies for psychrophily were distinguished: either an overall increase of flexibility generally associated with a marked decrease of stability, or a more subtle strategy which keeps one domain rigid enough to provide enough affinity for the substrate while holding the catalytic domain in a more flexible state (2,21,45). In the former case thermodynamic and genetic data even suggest that some enzymes, such as...

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Section: Discussionsupporting

confidence: 71%

“…DHFR catalyzes the NADPH-linked reduction of 7,8-dihydrofolate (DHF) to 5,6,7,. It is a metabolically and clinically important enzyme since it is involved in over 20 different one-carbon transfer reactions and is the target of several antibacterial, antiprotozoal and anticancer drugs.…”

mentioning

confidence: 99%

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

Xu¹,

Feller

Gerday

et al. 2003

J Bacteriol

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“…493 Asa paradigmatic system, DHFR also has been subject to numerous experimental and theoretical investigations. 37,38,49,131,133,146,153,161,172,[180][181][182]186,188,[195][196][197]289,408,433,[494][495][496][497][498][499] Agarwal et al employed the MQCMD approach to study the reaction mechanism and KIEs in the hydride transfer reaction catalyzed by EcDHFR; recrossing transmission coefficients of 0.80 ± 0.03 and 0.85 ± 0.01 were obtained for reactions transferring a hydride and deuteride, respectively, at 300 K. 131 The same system was investigated by Garcia-Viloca et al with the EA-VTST/MT approach. 133 Their transmission coefficients are in Table 4, along with the standard deviations.…”

Section: Dihydrofolate Reductase From E Colimentioning

confidence: 99%

Multidimensional Tunneling, Recrossing, and the Transmission Coefficient for Enzymatic Reactions

2006

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“…The hydrophobic core and ion-pair network at the subunit interface are the major stabilizing factors which stabilize the intersubunit interface (50). Moreover, oligomerization can be a significant stabilization mechanism for hyperthermophilic enzymes (50)(51)(52)(53). On the basis of stability studies of dimeric globular proteins, it was calculated that quaternary interactions could provide 25% to 100% of the conformational stability in protein dimers (54).…”

Section: Oligomeric Enzymesmentioning

confidence: 99%

Extreme catalysts from low-temperature environments

Hoyoux

Blaise

Collins

et al. 2004

Journal of Bioscience and Bioengineering

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Cold-loving or psychrophilic organisms are widely distributed in nature as a large part of the earth's surface is at temperatures around 0°C. To maintain metabolic rates and to prosper in cold environments, these extremophilic organisms have developed a vast array of adaptations. One main adaptive strategy developed in order to cope with the reduction of chemical reaction rates induced by low temperatures is the synthesis of cold-adapted or psychrophilic enzymes. These enzymes are characterized by a high catalytic activity at low temperatures associated with a low thermal stability. A study of protein adaptation strategies suggests that the high activity of psychrophilic enzymes could be achieved by the destabilization of the active site, allowing the catalytic center to be more flexible at low temperatures, whereas other protein regions may be destabilized or as rigid as their mesophilic counterparts. Due to these particular properties, psychrophilic enzymes offer a high potential not only for fundamental research but also for biotechnological applications.[Key words: psychrophile, extremophiles, cold adaptation, enzyme kinetics, flexibility strategy]Life under low-temperature conditions was identified as early as 1840 by Hooker, who observed that algae were associated with sea ice. In 1887, Forster was the first who reported that microorganisms isolated from fish could grow well at 0°C (1). The term "psychrophilic" was first used in 1902 by Schmidt-Nielsen to describe such cold-adapted organisms (2). Psychrophile is defined as an organism, prokaryotic or eukaryotic, living permanently at temperatures close to the freezing point of water in thermal equilibrium with the medium. Thus psychrophiles are numerous, including a large range of species of gram-positive and gram-negative bacteria, yeast, algae, marine invertebrates, insects and polar fish, and are widely distributed (3). Psychrophiles have developed mechanisms of adaptation to temperature including a huge range of structural and physiological adjustments in order to cope with the deleterious effect of low temperatures. Indeed, they display metabolic fluxes at low temperatures that are more or less comparable to those exhibited by closely related mesophiles living at moderate temperatures (4-6). This is explained by the capability of these psychrophilic organisms to produce "cold-adapted" enzymes which are able to cope with the reduction of chemical reaction rates induced by low temperatures. However, most cellular adaptations to low temperatures and the underlying molecular mechanisms are not fully understood and are still being investigated. Moreover, a study of proteins and enzymes from cold-adapted organisms is not only useful in the understanding of some general processes related to the protein structure and function but also in protein folding investigations. In addition, cold-active and heat-labile psychrophilic enzymes possess an interesting biotechnological potential.

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

Cited by 98 publications

References 33 publications

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

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

Multidimensional Tunneling, Recrossing, and the Transmission Coefficient for Enzymatic Reactions

Extreme catalysts from low-temperature environments

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