TbSMT [Trypanosoma brucei 24-SMT (sterol C-24-methyltransferase)] synthesizes an unconventional 24-alkyl sterol product set consisting of Δ24(25)-, Δ24(28)- and Δ25(27)-olefins. The C-methylation reaction requires Si(β)-face C-24-methyl addition coupled to reversible migration of positive charge from C-24 to C-25. The hydride shifts responsible for charge migration in formation of multiple ergostane olefin isomers catalysed by TbSMT were examined by incubation of a series of sterol acceptors paired with AdoMet (S-adenosyl-L-methionine). Results obtained with zymosterol compared with the corresponding 24-2H and 27-13C derivatives revealed isotopic-sensitive branching in the hydride transfer reaction on the path to form a 24-methyl-Δ24(25)-olefin product (kinetic isotope effect, kH/kD=1.20), and stereospecific CH3→CH2 elimination at the C28 branch and C27 cis-terminal methyl to form Δ24(28) and Δ25(27) products respectively. Cholesta-5,7,22,24-tetraenol converted into ergosta-5,7,22,24(28)-tetraenol and 24β–hydroxy ergosta-5,7,23-trienol (new compound), whereas ergosta-5,24-dienol converted into 24-dimethyl ergosta-5,25(27)-dienol and cholesta-5,7,24-trienol converted into ergosta-5,7,25(27)trienol, ergosta-5,7,24(28)-trienol, ergosta-5,7,24-trienol and 24 dimethyl ergosta-5,7,25(27)-trienol. We made use of our prior research and molecular modelling of 24-SMT to identify contact amino acids that might affect catalysis. Conserved tyrosine residues at positions 66, 177 and 208 in TbSMT were replaced with phenylalanine residues. The substitutions generated variable loss of activity during the course of the first C-1-transfer reaction, which differs from the corresponding Erg6p mutants that afforded a gain in C-2-transfer activity. The results show that differences exist among 24-SMTs in control of C-1- and C-2-transfer activities by interactions of intermediate and aromatic residues in the activated complex and provide an opportunity for rational drug design of a parasite enzyme not synthesized by the human host.
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Research Article|
October 13 2011
Effect of substrate features and mutagenesis of active site tyrosine residues on the reaction course catalysed by Trypanosoma brucei sterol C-24-methyltransferase
Jialin Liu;
Jialin Liu
1
*Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409, U.S.A.
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Kulothungan Ganapathy;
Kulothungan Ganapathy
1
*Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409, U.S.A.
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Ewa Wywial;
Ewa Wywial
†The Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology in Warsaw, ul. Ks. Trojdena 4, 02-109 Warsaw, Poland
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Janusz M. Bujnicki;
Janusz M. Bujnicki
†The Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology in Warsaw, ul. Ks. Trojdena 4, 02-109 Warsaw, Poland
‡The Laboratory of Bioinformatics, Institute of Molecular Biology and the Biotechnology, Faculty of Biology, Adam Mickiewicz University, ul. Umultowska 89, 61-614 Poznan, Poland
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Chizaram A. Nwogwugwu;
Chizaram A. Nwogwugwu
*Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409, U.S.A.
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W. David Nes
W. David Nes
2
*Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409, U.S.A.
2To whom correspondence should be addressed (email wdavid.nes@ttu.edu).
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Publisher: Portland Press Ltd
Received:
May 13 2011
Revision Received:
June 30 2011
Accepted:
July 08 2011
Accepted Manuscript online:
July 08 2011
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2011 Biochemical Society
2011
Biochem J (2011) 439 (3): 413–428.
Article history
Received:
May 13 2011
Revision Received:
June 30 2011
Accepted:
July 08 2011
Accepted Manuscript online:
July 08 2011
Citation
Jialin Liu, Kulothungan Ganapathy, Ewa Wywial, Janusz M. Bujnicki, Chizaram A. Nwogwugwu, W. David Nes; Effect of substrate features and mutagenesis of active site tyrosine residues on the reaction course catalysed by Trypanosoma brucei sterol C-24-methyltransferase. Biochem J 1 November 2011; 439 (3): 413–428. doi: https://doi.org/10.1042/BJ20110865
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