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| | {{STRUCTURE_1zdr| PDB=1zdr | SCENE= }} | | {{STRUCTURE_1zdr| PDB=1zdr | SCENE= }} |
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| - | '''DHFR from Bacillus Stearothermophilus'''
| + | ===DHFR from Bacillus Stearothermophilus=== |
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| - | ==Overview==
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| - | Dihydrofolate reductase (DHFR) from a moderate thermophilic organism, Bacillus stearothermophilus, has been cloned and expressed. Physical characterization of the protein (BsDHFR) indicates that it is a monomeric protein with a molecular mass of 18,694.6 Da (0.8), coincident with the mass of 18 694.67 Da calculated from the primary sequence. Determination of the X-ray structure of BsDHFR provides the first structure for a monomeric DHFR from a thermophilic organism, indicating a high degree of conservation of structure in relation to all chromosomal DHFRs. Structurally based sequence alignment of DHFRs indicates the following levels of sequence identity and similarity for BsDHFR: 38 and 58% with Escherichia coli, 35 and 56% with Lactobacillus casei, and 23 and 40% with Thermotoga maritima, respectively. Steady state kinetic isotope effect studies indicate an ordered kinetic mechanism at elevated temperatures, with NADPH binding first to the enzyme. This converts to a more random mechanism at reduced temperatures, reflected in a greatly reduced K(m) for dihydrofolate at 20 degrees C in relation to that at 60 degrees C. A reduction in either temperature or pH reduces the degree to which the hydride transfer step is rate-determining for the second-order reaction of DHF with the enzyme-NADPH binary complex. Transient state kinetics have been used to study the temperature dependence of the isotope effect on hydride transfer at pH 9 between 10 and 50 degrees C. The data support rate-limiting hydride transfer with a moderate enthalpy of activation (E(a) = 5.5 kcal/mol) and a somewhat greater temperature dependence for the kinetic isotope effect than predicted from classical behavior [A(H)/A(D) = 0.57 (0.15)]. Comparison of kinetic parameters for BsDHFR to published data for DHFR from E. coli and T. maritima shows a decreasing trend in efficiency of hydride transfer with increasing thermophilicity of the protein. These results are discussed in the context of the capacity of each enzyme to optimize H-tunneling from donor (NADPH) to acceptor (DHF) substrates.
| + | The line below this paragraph, {{ABSTRACT_PUBMED_16114879}}, adds the Publication Abstract to the page |
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| | ==About this Structure== | | ==About this Structure== |
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| | [[Category: Nadp]] | | [[Category: Nadp]] |
| | [[Category: Oxidoreductase]] | | [[Category: Oxidoreductase]] |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sat May 3 17:30:18 2008'' | + | |
| | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Mon Jul 28 19:52:23 2008'' |
Revision as of 16:52, 28 July 2008
Template:STRUCTURE 1zdr
DHFR from Bacillus Stearothermophilus
Template:ABSTRACT PUBMED 16114879
About this Structure
Full crystallographic information is available from OCA.
Reference
Structure and hydride transfer mechanism of a moderate thermophilic dihydrofolate reductase from Bacillus stearothermophilus and comparison to its mesophilic and hyperthermophilic homologues., Kim HS, Damo SM, Lee SY, Wemmer D, Klinman JP, Biochemistry. 2005 Aug 30;44(34):11428-39. PMID:16114879
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