Bisubstrate kinetics using SAS computer software.
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Biomedical subjects
R D Smiley
Publications and source records attributed to R D Smiley.
Role of S65, Q67, I68, and Y69 residues in homotetrameric R67 dihydrofolate reductase.
R67 dihydrofolate reductase (DHFR) shares no sequence or structural homology with chromosomal DHFRs. This enzyme arose recently in response to the clinical use of the antibacterial drug trimethoprim. R67 DHFR is a homotetramer possessing a single active site pore. A high-resolution crystal structure shows the homotetramer possesses exact 222 symmetry [Narayana, N., et al. (1995) Nat. Struct. Biol. 2, 1018-1025]. This symmetry dictates four symmetry-related binding sites must exist for each substrate as well as each cofactor. Isothermal titration calorimetry studies, however, indicate only two molecules bind: either two dihydrofolate molecules, two NADPH molecules, or one substrate and one cofactor [Bradrick, T. D., et al. (1996) Biochemistry 35, 11414-11424]. The latter is the productive ternary complex. To evaluate the role of S65, Q67, I68, and Y69 residues, located near the center of the active site pore, site-directed mutagenesis was performed. One mutation in the gene creates four mutations per active site pore which typically result in large cumulative effects. Steady state kinetic data indicate the mutants have altered K(m) values for both cofactor and substrate. For example, the Y69F R67 DHFR displays an 8-fold increase in the K(m) for dihydrofolate and a 20-fold increase in the K(m) for NADPH. Residues involved in ligand binding in R67 DHFR display very little, if any, specificity, consistent with their possessing dual roles in binding. These results support a model where R67 DHFR utilizes an unusual "hot spot" binding surface capable of binding both ligands and indicate this enzyme has adopted a novel yet simple approach to catalysis.
Amino Acid Sequence↗
One site fits both: a model for the ternary complex of folate + NADPH in R67 dihydrofolate reductase, a D2 symmetric enzyme.
R67 dihydrofolate reductase (DHFR) is a novel enzyme that confers resistance to the antibiotic trimethoprim. The crystal structure of R67 DHFR displays a toroidal structure with a central active-site pore. This homotetrameric protein exhibits 222 symmetry, with only a few residues from each chain contributing to the active site, so related sites must be used to bind both substrate (dihydrofolate) and cofactor (NADPH) in the productive R67 DHFR.NADPH.dihydrofolate complex. Whereas the site of folate binding has been partially resolved crystallographically, an interesting question remains: how can the highly symmetrical active site also bind and orient NADPH for catalysis? To model this ternary complex, we employed DOCK and SLIDE, two methods for docking flexible ligands into proteins using quite different algorithms. The bound pteridine ring of folate (Fol I) from the crystal structure of R67 DHFR was used as the basis for docking the nicotinamide-ribose-Pi (NMN) moiety of NADPH. NMN was positioned by both DOCK and SLIDE on the opposite side of the pore from Fol I, where it interacts with Fol I at the pore's center. Numerous residues serve dual roles in binding. For example, Gln 67 from both the B and D subunits has several contacts with the pteridine ring, while the same residue from the A and C subunits has several contacts with the nicotinamide ring. The residues involved in dual roles are generally amphipathic, allowing them to make both hydrophobic and hydrophilic contacts with the ligands. The result is a 'hot spot' binding surface allowing the same residues to co-optimize the binding of two ligands, and orient them for catalysis.
Algorithms↗
Preliminary evidence that degradation of aflatoxin B1 by Flavobacterium aurantiacum is enzymatic.
The ability of crude protein extracts from Flavobacterium aurantiacum to degrade aflatoxin B1 (AB1) in aqueous solution was evaluated. Crude protein extracts (800 microg of total protein per ml) degraded 74.5% of AB1 in solution. An average of 94.5% of AB1 was recovered after incubation with heat-treated crude protein extracts (800 microg of total protein per ml). DNase I-treated crude protein extracts degraded 80.5% of AB1 in solution, suggesting that removal of aflatoxin by F. aurantiacum is not due to nonspecific binding with the bacterium's genomic DNA. Proteinase K-treated crude protein extracts degraded 34.5% of AB1, providing evidence that degradation of aflatoxin is linked to a protein that is possibly an enzyme. Solution pH affected the amount of AB1 degraded by crude protein extracts after 24 h. Maximum degradation was observed at pH 7 (pH levels tested: 5, 6, 7, and 8), with some AB1 degradation occurring at pH levels as low as 5 and as high as 8. Acidic pH levels were more detrimental to the ability of crude protein extracts to degrade AB1 than was basic pH. The results of this work indicate that the degradation of AB1 by F. aurantiacum may be enzymatic.
Aflatoxin B1↗