Kinetic and structural characterization of dihydrofolate reductase from Streptococcus pneumoniae

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.

Figure 1

A) Sequence of spDHFR and the mutant Sp9. Note that the Sp9 mutant DHFR has Val-100 whereas the wild type S. pneumoniae DHFR has Leu at this position. A list of nine mutations from S. pneumoniae DHFR includes H26Y, Q60K, A77V, V78A, Q81H, Q91S, L100V, E133A, and A149T. B) Sequence alignment of DHFRs from S. pneumoniae (spDHFR), E. coli (ecDHFR), and human (hDHFR). Alignment was generated using ClustalW2 web server (http://www.ebi.ac.uk/Tools/clustalw2/index.html). C) The locations of the nine mutations in Sp9 DHFR. Note that most of these mutations are on the surface of the protein except the L100V mutation.

Figure 2

A) Stopped-flow time course for the binding of 0.5μM spDHFR with 5 μM NADPH at pH 7.0. Solid line represents the fit of the data to a single exponential function. B) Measurement of the time course for the hydride transfer rate catalyzed by spDHFR as monitored by stopped-flow absorbance. spDHFR was preincubated with NADPH and the reaction was initiated by mixing with H₂F. The final concentrations were 5 μM, 100 μM, and 100 μM for enzyme, NADPH, and H₂F, respectively at pH 7.0. The solid line represents the fit of the absorbance data by using Dynafit under the given experimental condition and kinetic parameters derived from this simulation are summarized in Table 6.

Figure 3

pH-rate profiles at 25 ºC in MTEN buffer. A) Both S. pneumoniae DHFRs (wild type •, Sp9 DHFR □) showed maximum activities at pH 7.0. The two pKa values of S. pneumoniae DHFR were calculated to be 6.2 and 7.9 from the curve fit equation 1 (14). B) The His33Phe mutant DHFR showed a similar pH profile to that of E. coli DHFR from reference (12) with a pKa value of 8.2 when fitted to equation 2 (15).

Figure 4

(a) Superposition of the E. coli and Sp9 DHFR monomers colored according to their temperature factors with red representing mobile regions and blue, the rigid regions. The substrate binding loops and helix in the E. coli structure seem to be more flexible compared to the Sp9 mutant. (b) Cartoon representation of the Sp9 DHFR dimer. (c) Stereo view of the dimer interface. Residues are shown in stick representation.

Figure 5

(a) Electron density shown as a stereoview for the active site region of the Sp9 DHFR mutant including His33 and Glu30 residues (sigma value of 1.5 ) and (b) Superposition of E. coli (pink) and spDHFR (cyan) – eight of the active site residues differ. The labeled amino acids are in the order S. pneumoniae DHFR Sp9 followed by the E. coli DHFR residue. Note that spDHFR has 168 amino acids while ecDHFR has 159 amino acids. This causes different numbering when comparing the two sequences.

Figure 6

Amino acid distribution in the His33 position among members of the DHFR superfamily (cd00209). Further sequence analysis of this DHFR family revealed that the histidine residue is the major occupant of this position (see text).

Figure 7

Ternary complex as seen in the E. coli crystal structure with dideazatetrahydrofolate (DDF) (A) and an energy minimized model of S. pneumoniae with DDF and NADPH (B).

Figure 8

Superposition of the Streptococcus pneumoniae DHFR Sp9 structure and a complex of Mycobacterium tuberculosis DHFR with trimethoprim (1DG5). Val100 in Streptococcus pneumoniae DHFR Sp9 and Ile94 in Mycobacterium tuberculosis DHFR showed stacking (Phe with the pyrimidine ring of trimethoprim) and hydrogen bond interactions (black dashes). No steric clashes occur. Mutation of Val100 to Leu100 as seen in the wild type protein results in a steric clash of the side-chains (red dashes). The first numbered label in the figure is the S. pneumoniae DHFR Sp9 mutant residue and the second is 1DG5.

Figure 9

S. pneumoniae DHFR activity decreased by TMP binding at pH 7.0 in MTEN buffer.

Scheme 1

Schematic representation of E. coli DHFR reaction (12) and S. pneumoniae DHFR at pH 7.0. Highlighted with bold italic are the rate constants measured from experiments for S. pneumoniae DHFR. E = DHFR; NH = NADPH; H₂F = 7, 8-dihydrofolate; H₄F = tetrahydrofolate; N⁺ = NADP⁺

Scheme 2

Schematic representation of spDHFR reaction. The data were obtained from simulated fits as described in Table 6.