PDBsum entry 1zdr

Oxidoreductase

PDB id: 1zdr

Contents

Protein chains

161 a.a. *

Ligands

SO4 ×5

GOL

Waters ×234

* Residue conservation analysis

PDB id:

1zdr

Name:

Oxidoreductase

Title:

Dhfr from bacillus stearothermophilus

Structure:

Dihydrofolate reductase. Chain: a, b. Engineered: yes

Source:

Geobacillus stearothermophilus. Organism_taxid: 1422. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Resolution:

2.00Å R-factor: 0.210 R-free: 0.248

Authors:

H.S.Kim,S.M.Damo,S.Y.Lee,D.Wemmer,J.P.Klinman

Key ref:

H.S.Kim et al. (2005). 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. PubMed id: 16114879 DOI: 10.1021/bi050630j

Date:

14-Apr-05 Release date: 30-Aug-05

Protein chains

Q5KZ26  (Q5KZ26_GEOKA) -  Dihydrofolate reductase from Geobacillus kaustophilus (strain HTA426)

Seq: 164 a.a.

Struc: 161 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.1.5.1.3 - dihydrofolate reductase.
• Pathway: Folate Coenzymes
• Reaction: (6S)-5,6,7,8-tetrahydrofolate + NADP⁺ = 7,8-dihydrofolate + NADPH + H⁺

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1021/bi050630j

Biochemistry 44:11428-11439 (2005)

PubMed id: 16114879

Structure and hydride transfer mechanism of a moderate thermophilic dihydrofolate reductase from Bacillus stearothermophilus and comparison to its mesophilic and hyperthermophilic homologues.

H.S.Kim, S.M.Damo, S.Y.Lee, D.Wemmer, J.P.Klinman.

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.