1ZDR

DHFR from Bacillus Stearothermophilus

Summary for 1ZDR

• Entry DOI: 10.2210/pdb1zdr/pdb
• Descriptor: dihydrofolate reductase, SULFATE ION, GLYCEROL, ... (4 entities in total)
• Functional Keywords: dhfr, nadp, oxidoreductase
• Biological source: Geobacillus stearothermophilus
• Total number of polymer chains: 2
• Total formula weight: 38015.51

Authors

Kim, H.S.,Damo, S.M.,Lee, S.Y.,Wemmer, D.,Klinman, J.P. (deposition date: 2005-04-14, release date: 2005-08-30, Last modification date: 2023-08-23)

Primary citation

Kim, H.S.,Damo, S.M.,Lee, S.Y.,Wemmer, D.,Klinman, J.P.
Structure and hydride transfer mechanism of a moderate thermophilic dihydrofolate reductase from Bacillus stearothermophilus and comparison to its mesophilic and hyperthermophilic homologues.
Biochemistry, 44:11428-11439, 2005

PubMed Abstract: 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.

PubMed: 16114879
DOI: 10.1021/bi050630j

Experimental method

X-RAY DIFFRACTION (2 Å)