3n0h | pdb_00003n0h

hDHFR double mutant Q35S/N64F Trimethoprim Binary Complex

Structural highlights

3n0h is a 1 chain structure with sequence from Homo sapiens. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance.
Method:	X-ray diffraction, Resolution 1.92Å
Ligands:	SO4, TOP
Resources:	FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT

Disease

DYR_HUMAN Defects in DHFR are the cause of megaloblastic anemia due to dihydrofolate reductase deficiency (DHFRD) [MIM:613839. DHFRD is an inborn error of metabolism, characterized by megaloblastic anemia and/or pancytopenia, severe cerebral folate deficiency, and cerebral tetrahydrobiopterin deficiency. Clinical features include variable neurologic symptoms, ranging from severe developmental delay and generalized seizures in infancy, to childhood absence epilepsy with learning difficulties, to lack of symptoms.^[1] ^[2]

Function

DYR_HUMAN Key enzyme in folate metabolism. Contributes to the de novo mitochondrial thymidylate biosynthesis pathway. Catalyzes an essential reaction for de novo glycine and purine synthesis, and for DNA precursor synthesis. Binds its own mRNA and that of DHFRL1.^[3] ^[4]

Publication Abstract from PubMed

In order to produce a more potent replacement for trimethoprim (TMP) used as a therapy for Pneumocystis pneumonia and targets dihydrofolate reductase from Pneumocystis jirovecii (pjDHFR), it is necessary to understand the determinants of potency and selectivity against DHFR from the mammalian host and fungal pathogen cells. To this end, active site residues in human (h) DHFR were replaced with those from pjDHFR. Structural data are reported for two complexes of TMP with the double mutants Gln35Ser/Asn64Phe (Q35S/N64F) and Gln35Lys/Asn64Phe (Q35K/N64F) of hDHFR that unexpectedly show evidence for the binding of two molecules of TMP: one molecule that binds in the normal folate binding site and the second molecule that binds in a novel subpocket site such that the mutated residue Phe64 is involved in van der Waals contacts to the trimethoxyphenyl ring of the second TMP molecule. Kinetic data for the binding of TMP to hDHFR and pjDHFR reveal an 84-fold selectivity of TMP against pjDHFR (K(i) 49nM) compared to hDHFR (K(i) 4093nM). Two mutants that contain one substitution from pj- and one from the closely related Pneumocystis carinii DHFR (pcDHFR) (Q35K/N64F and Q35S/N64F) show K(i) values of 593 and 617nM, respectively; these K(i) values are well above both the K(i) for pjDHFR and are similar to pcDHFR (Q35K/N64F and Q35S/N64F) (305nM). These results suggest that active site residues 35 and 64 play key roles in determining selectivity for pneumocystis DHFR, but that other residues contribute to the unique binding of inhibitors to these enzymes.

Crystallographic analysis reveals a novel second binding site for trimethoprim in active site double mutants of human dihydrofolate reductase.,Cody V, Pace J, Piraino J, Queener SF J Struct Biol. 2011 Oct;176(1):52-9. Epub 2011 Jun 13. PMID:21684339^[5]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

References

↑ Banka S, Blom HJ, Walter J, Aziz M, Urquhart J, Clouthier CM, Rice GI, de Brouwer AP, Hilton E, Vassallo G, Will A, Smith DE, Smulders YM, Wevers RA, Steinfeld R, Heales S, Crow YJ, Pelletier JN, Jones S, Newman WG. Identification and characterization of an inborn error of metabolism caused by dihydrofolate reductase deficiency. Am J Hum Genet. 2011 Feb 11;88(2):216-25. doi: 10.1016/j.ajhg.2011.01.004. PMID:21310276 doi:10.1016/j.ajhg.2011.01.004
↑ Cario H, Smith DE, Blom H, Blau N, Bode H, Holzmann K, Pannicke U, Hopfner KP, Rump EM, Ayric Z, Kohne E, Debatin KM, Smulders Y, Schwarz K. Dihydrofolate reductase deficiency due to a homozygous DHFR mutation causes megaloblastic anemia and cerebral folate deficiency leading to severe neurologic disease. Am J Hum Genet. 2011 Feb 11;88(2):226-31. doi: 10.1016/j.ajhg.2011.01.007. PMID:21310277 doi:10.1016/j.ajhg.2011.01.007
↑ Anderson DD, Quintero CM, Stover PJ. Identification of a de novo thymidylate biosynthesis pathway in mammalian mitochondria. Proc Natl Acad Sci U S A. 2011 Sep 13;108(37):15163-8. doi:, 10.1073/pnas.1103623108. Epub 2011 Aug 26. PMID:21876188 doi:10.1073/pnas.1103623108
↑ Klon AE, Heroux A, Ross LJ, Pathak V, Johnson CA, Piper JR, Borhani DW. Atomic structures of human dihydrofolate reductase complexed with NADPH and two lipophilic antifolates at 1.09 a and 1.05 a resolution. J Mol Biol. 2002 Jul 12;320(3):677-93. PMID:12096917
↑ Cody V, Pace J, Piraino J, Queener SF. Crystallographic analysis reveals a novel second binding site for trimethoprim in active site double mutants of human dihydrofolate reductase. J Struct Biol. 2011 Oct;176(1):52-9. Epub 2011 Jun 13. PMID:21684339 doi:10.1016/j.jsb.2011.06.001

Proteopedia Page Contributors and Editors (what is this?)

OCA

[1] Banka S, Blom HJ, Walter J, Aziz M, Urquhart J, Clouthier CM, Rice GI, de Brouwer AP, Hilton E, Vassallo G, Will A, Smith DE, Smulders YM, Wevers RA, Steinfeld R, Heales S, Crow YJ, Pelletier JN, Jones S, Newman WG. Identification and characterization of an inborn error of metabolism caused by dihydrofolate reductase deficiency. Am J Hum Genet. 2011 Feb 11;88(2):216-25. doi: 10.1016/j.ajhg.2011.01.004. PMID:21310276 doi:10.1016/j.ajhg.2011.01.004

[2] Cario H, Smith DE, Blom H, Blau N, Bode H, Holzmann K, Pannicke U, Hopfner KP, Rump EM, Ayric Z, Kohne E, Debatin KM, Smulders Y, Schwarz K. Dihydrofolate reductase deficiency due to a homozygous DHFR mutation causes megaloblastic anemia and cerebral folate deficiency leading to severe neurologic disease. Am J Hum Genet. 2011 Feb 11;88(2):226-31. doi: 10.1016/j.ajhg.2011.01.007. PMID:21310277 doi:10.1016/j.ajhg.2011.01.007

[3] Anderson DD, Quintero CM, Stover PJ. Identification of a de novo thymidylate biosynthesis pathway in mammalian mitochondria. Proc Natl Acad Sci U S A. 2011 Sep 13;108(37):15163-8. doi:, 10.1073/pnas.1103623108. Epub 2011 Aug 26. PMID:21876188 doi:10.1073/pnas.1103623108

[4] Klon AE, Heroux A, Ross LJ, Pathak V, Johnson CA, Piper JR, Borhani DW. Atomic structures of human dihydrofolate reductase complexed with NADPH and two lipophilic antifolates at 1.09 a and 1.05 a resolution. J Mol Biol. 2002 Jul 12;320(3):677-93. PMID:12096917

[5] Cody V, Pace J, Piraino J, Queener SF. Crystallographic analysis reveals a novel second binding site for trimethoprim in active site double mutants of human dihydrofolate reductase. J Struct Biol. 2011 Oct;176(1):52-9. Epub 2011 Jun 13. PMID:21684339 doi:10.1016/j.jsb.2011.06.001

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3n0h | pdb_00003n0h

hDHFR double mutant Q35S/N64F Trimethoprim Binary Complex

Structural highlights

Disease

Function

Publication Abstract from PubMed

See Also

References

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