PMID:19950924

Citation	Lee, J, Yennawar, NH, Gam, J and Benkovic, SJ (2010) Kinetic and structural characterization of dihydrofolate reductase from Streptococcus pneumoniae. Biochemistry 49:195-206
Abstract	Drug resistance associated with dihydrofolate reductase (DHFR) has emerged as a critical issue in the treatment of bacterial infections. In our efforts to understand the mechanism of a drug-resistant dihydrofolate reductase (DHFR) from a pathogenic bacterial source, we report the first kinetic characterization of Streptococcus pneumoniae DHFR (spDHFR) along with its X-ray structure. This study revealed that the kinetic properties of spDHFR were significantly different from those of Escherichia coli DHFR. The product (tetrahydrofolate) dissociation step that is the rate-limiting step in E. coli DHFR is significantly accelerated in spDHFR so that hydride transfer or a preceding step is rate-limiting. Comparison of the binding parameters of this enzyme to those of a mutant spDHFR (Sp9) confirmed that the Leu100 residue in spDHFR is the critical element for the trimethoprim (TMP) resistance. Steady-state kinetics exhibited a pH dependence in k(cat), which prompted us to elucidate the role of the new catalytic residue (His33) in the active site of spDHFR. Structural data of the Sp9 mutant in complex with NADPH and methotrexate confirmed the participation of His33 in a hydrogen bonding network involving a water molecule, the hydroxyl group of Thr119, and the carboxylate ion of Glu30. Sequence analysis of the DHFR superfamily revealed that the His residue is the major amino acid component at this position and is found mostly in pathogenic bacterial DHFRs. A mutation of Val100 to Leu demonstrated a steric clash of the leucine side chain with the side chains of Ile8 and Phe34, rationalizing weaker binding of trimethoprim to Leu100 DHFR. Understanding the role of specific amino acids in the active site coupled with detailed structural analysis will inform us on how to better design inhibitors targeting drug-resistant pathogenic bacterial DHFRs.
Links	PubMed PMC3773979 Online version:10.1021/bi901614m
Keywords	Amino Acid Sequence; Binding Sites; Catalytic Domain; Crystallography, X-Ray; G-Quadruplexes; Histidine/genetics; Histidine/metabolism; Hydrogen-Ion Concentration; Kinetics; Models, Molecular; Molecular Sequence Data; Mutation; Protein Conformation; Protein Folding; Streptococcus pneumoniae/enzymology; Tetrahydrofolate Dehydrogenase/chemistry; Thermodynamics; Trimethoprim/pharmacology; Trimethoprim Resistance
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
STREE:O85255		GO:0004146: dihydrofolate reductase activity	ECO:0000315:		F		Figure 8 shows how the mutation of Val100 to Leu makes the leucine side chain clash with side chains of Ile8 and Phe34, shifting their positions and stacking interactions with the trimethoprim molecule. "Weaker binding of trimethoprim to Leu100 DHFR would lead to trimethoprim resistance". The KM values of NADPH and H2F for the wild type spDHFR and the mutant Sp9 were measured (Table 2B). The KM values for the wild type spDHFR were at least order of magnitude higher than those for the Sp9 mutant that showed full enzyme activity even at a limiting low concentration of 0.63 μM H2F consistent with a much lower KM value for this substrate.	complete CACAO 10955
STREE:O85255	enables	GO:0004146: dihydrofolate reductase activity	ECO:0000315: mutant phenotype evidence used in manual assertion		F		Seeded From UniProt	complete

References

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