The Pneumocystis carinii drug target S-adenosyl-L-methionine:sterol C-24 methyl transferase has a unique substrate preference.
Pneumocystis is an opportunistic pathogen that can cause pneumonitis in immunodeficient people such as AIDS patients. Pneumocystis remains difficult to study in the absence of culture methods for luxuriant growth. Recombinant protein technology now makes it possible to avoid some major obstacles. The P. carinii expressed sequence tag (EST) database contains 11 entries of a sequence encoding a protein homologous to S-adenosyl-L-methionine (SAM):C-24 sterol methyl transferase (SMT), suggesting high activity of this enzyme in the organism. We sequenced the erg6 cDNA, identified the putative peptide motifs for the sterol and SAM binding sites in the deduced amino acid sequence and expressed the protein in Escherichia coli. Unlike SAM:SMT from other organisms, the P. carinii enzyme had higher affinities for lanosterol and 24-methylenelanosterol than for zymosterol, the preferred substrate in other fungi. Cycloartenol was not a productive substrate. With lanosterol and 24-methylenelanosterol as substrates, the major reaction products were 24-methylenelanosterol and pneumocysterol respectively. Thus, the P. carinii SAM:SMT catalysed the transfer of both the first and the second methyl groups to the sterol C-24 position, and the substrate preference was found to be a unique property of the P. carinii SAM:SMT. These observations, together with the absence of SAM:SMT among mammals, further support the identification of sterol C-24 alkylation reactions as excellent targets for the development of drugs specifically directed against this pathogen.
Kaneshiro
Edna S
ES
Department of Biological Sciences, University of Cincinnati, Cincinnati, OH 45221-0006, USA. Edna.Kaneshiro@uc.edu
Rosenfeld
Jill A
JA
Basselin-Eiweida
Mireille
M
Stringer
James R
JR
Keely
Scott P
SP
Smulian
A George
AG
Giner
José-Luis
JL
D000478:Alkylation; D000935:Antifungal Agents / Q000494:pharmacology*; D015139:Blotting, Southern; D002784:Cholesterol; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007810:Lanosterol / Q000031:analogs & derivatives / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010840:Phytosterols / Q000737:chemistry / Q000378:metabolism; D011010:Pneumocystis / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism; D013261:Sterols / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature; D014315:Triterpenes
D000935:Antifungal Agents; D010840:Phytosterols; D011994:Recombinant Proteins; D013261:Sterols; D014315:Triterpenes; D007810:Lanosterol; D012436:S-Adenosylmethionine; D002784:Cholesterol; D008780:Methyltransferases; C022670:delta 24-sterol methyltransferase; C015582:zymosterol; C100089:cycloartenol