1zdr
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(New page: 200px<br /><applet load="1zdr" size="450" color="white" frame="true" align="right" spinBox="true" caption="1zdr, resolution 2.0Å" /> '''DHFR from Bacillus St...)
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Revision as of 02:42, 25 November 2007
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DHFR from Bacillus Stearothermophilus
Overview
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.
About this Structure
1ZDR is a Protein complex structure of sequences from Geobacillus stearothermophilus with SO4 and GOL as ligands. Active as Dihydrofolate reductase, with EC number 1.5.1.3 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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