PMID:16114879

Citation	Kim, HS, Damo, SM, Lee, SY, Wemmer, D and Klinman, JP (2005) Structure and hydride transfer mechanism of a moderate thermophilic dihydrofolate reductase from Bacillus stearothermophilus and comparison to its mesophilic and hyperthermophilic homologues. Biochemistry 44:11428-39
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.
Links	PubMed Online version:10.1021/bi050630j
Keywords	Amino Acid Sequence; Animals; Cloning, Molecular; Conserved Sequence; Crystallography, X-Ray; Geobacillus stearothermophilus/enzymology; Humans; Kinetics; Models, Molecular; Molecular Sequence Data; Protein Conformation; Recombinant Proteins/chemistry; Recombinant Proteins/metabolism; Sequence Alignment; Sequence Homology, Amino Acid; Species Specificity; Tetrahydrofolate Dehydrogenase/chemistry; Tetrahydrofolate Dehydrogenase/metabolism
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
GEOSE:A0A087LEM1		GO:0004146: dihydrofolate reductase activity	ECO:0000314:		F		The three-dimensional structure of the dihydrofoate reductase (via X-ray crystallography) in Bacillus Stearothermophilus is iterated by Figure 3. This structure of Bacillus Stearothermophilus DHFR is the first monomeric DHFR structure from a thermophilic organism; there is also comparison between E. coli and T. maritima enzymes. IDA was used because this was the actual determination of the structure; the comparison to the other 2 enzymes helps support the functional conservation.	complete CACAO 11999
GEOSE:A0A087LEM1		GO:0004146: dihydrofolate reductase activity	ECO:0000315:		F		Figure 4 shows BsDHFR activity as a function of reaction conditions through its pH dependence within MTEN buffer at different pH values and 40 °C with the rest at standard conditions. Optimum temperature for neutral pH of 7 was 75*C although enzyme stability was seen at pH 6.8 as a function of temperature due to very rapid loss of activity in the absence of substrate above 64 °C. pKa under standard assay was 7.5. The formation of THF is shown to be sensitive to pH since as pH increases, activity declines.	complete CACAO 12000

References

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