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Title: Nonconserved Residues Ala287 and Ser290 of the Cryptosporidium hominis Thymidylate Synthase Domain Facilitate Its Rapid Rate of Catalysis

Abstract

Cryptosporidium hominis TS-DHFR exhibits an unusually high rate of catalysis at the TS domain, at least 10-fold greater than those of other TS enzymes. Using site-directed mutagenesis, we have mutated residues Ala287 and Ser290 in the folate-binding helix to phenylalanine and glycine, respectively, the corresponding residues in human and most other TS enzymes. Our results show that the mutant A287F, the mutant S290G, and the double mutant all have reduced affinities for methylene tetrahydrofolate and reduced rates of reaction at the TS domain. Interestingly, the S290G mutant enzyme had the lowest TS activity, with a catalytic efficiency {approx}200-fold lower than that of the wild type (WT). The rate of conformational change of the S290G mutant is {approx}80 times slower than that of WT, resulting in a change in the rate-limiting step from hydride transfer to covalent ternary complex formation. We have determined the crystal structure of ligand-bound S290G mutant enzyme, which shows that the primary effect of the mutation is an increase in the distance between the TS ligands. The kinetic and crystal structure data presented here provide the first evidence explaining the unusually fast TS rate in C. hominis.

Authors:
; ; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
929919
Report Number(s):
BNL-80508-2008-JA
Journal ID: ISSN 0006-2960; TRN: US200822%%1092
DOE Contract Number:
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry; Journal Volume: 46; Journal Issue: 28
Country of Publication:
United States
Language:
English
Subject:
36 MATERIALS SCIENCE; CATALYSIS; CONFORMATIONAL CHANGES; CRYSTAL STRUCTURE; EFFICIENCY; ENZYMES; GLYCINE; HUMAN POPULATIONS; HYDRIDES; KINETICS; LIGANDS; MUTAGENESIS; MUTANTS; MUTATIONS; PARASITES; PHENYLALANINE; RESIDUES; national synchrotron light source

Citation Formats

Doan,L., Martucci, W., Vargo, M., Atreya, C., and Anderson, K.. Nonconserved Residues Ala287 and Ser290 of the Cryptosporidium hominis Thymidylate Synthase Domain Facilitate Its Rapid Rate of Catalysis. United States: N. p., 2007. Web. doi:10.1021/bi700531r.
Doan,L., Martucci, W., Vargo, M., Atreya, C., & Anderson, K.. Nonconserved Residues Ala287 and Ser290 of the Cryptosporidium hominis Thymidylate Synthase Domain Facilitate Its Rapid Rate of Catalysis. United States. doi:10.1021/bi700531r.
Doan,L., Martucci, W., Vargo, M., Atreya, C., and Anderson, K.. Mon . "Nonconserved Residues Ala287 and Ser290 of the Cryptosporidium hominis Thymidylate Synthase Domain Facilitate Its Rapid Rate of Catalysis". United States. doi:10.1021/bi700531r.
@article{osti_929919,
title = {Nonconserved Residues Ala287 and Ser290 of the Cryptosporidium hominis Thymidylate Synthase Domain Facilitate Its Rapid Rate of Catalysis},
author = {Doan,L. and Martucci, W. and Vargo, M. and Atreya, C. and Anderson, K.},
abstractNote = {Cryptosporidium hominis TS-DHFR exhibits an unusually high rate of catalysis at the TS domain, at least 10-fold greater than those of other TS enzymes. Using site-directed mutagenesis, we have mutated residues Ala287 and Ser290 in the folate-binding helix to phenylalanine and glycine, respectively, the corresponding residues in human and most other TS enzymes. Our results show that the mutant A287F, the mutant S290G, and the double mutant all have reduced affinities for methylene tetrahydrofolate and reduced rates of reaction at the TS domain. Interestingly, the S290G mutant enzyme had the lowest TS activity, with a catalytic efficiency {approx}200-fold lower than that of the wild type (WT). The rate of conformational change of the S290G mutant is {approx}80 times slower than that of WT, resulting in a change in the rate-limiting step from hydride transfer to covalent ternary complex formation. We have determined the crystal structure of ligand-bound S290G mutant enzyme, which shows that the primary effect of the mutation is an increase in the distance between the TS ligands. The kinetic and crystal structure data presented here provide the first evidence explaining the unusually fast TS rate in C. hominis.},
doi = {10.1021/bi700531r},
journal = {Biochemistry},
number = 28,
volume = 46,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}