DIHYDRONEOPTERIN ALDOLASE, A TUBERCULOSIS DRUG TARGET

描述：这个试点研究项目的目的是调查

DESCRIPTION: The purpose of this pilot research project is to investigate the enzyme dihydroneopterin aldolase (DHNA, EC 4.1.2.25) as a target for therapeutic intervention in disease caused by Mycobacterium tuberculosis (MTB). Unlike mammalian cells, which acquire folates exogenously through active transport, MTB and many other bacteria must synthesize folates de novo. DHNA is an enzyme present early in the metabolic pathway for the synthesis of reduced folates from GTP. The absence of DHNA in mammalian cells makes this enzyme an attractive target for chemotherapy. Depletion of reduced folates through inhibition of this pathway leads to inhibition of DNA, RNA and protein synthesis. Comprehensive studies of the folate biosynthetic pathway in MTB are lacking but genes coding for enzymes in this pathway have been identified through the Sanger Centre MTB genome sequencing project. A DNA sequence in the MTB genome data base has been identified tentatively as coding for DHNA. For this pilot study, we propose to establish that the gene listed as foIX (embi locus MTCY7H7B, accession Z95557.1) and foIB (swissprot locus FOLB MYCTU, accession 006275) codes for DHNA. Our objectives will be to clone and express the foiX/foiB in Escherichia coli, and prove that the protein is functionally DHNA. We will also assess the essentiality of the gene by construction of DHNA-deficient MTB strains. This will be done in MTB by allelic exchange mutagenesis and a counter selection method based upon a mycobacterial thermosensitive origin of replication and toxicity of the sacB gene to MTB in the presence of sucrose. The results of this pilot study will enable us to better understand the biochemistry of folate metabolism in MTB. It will also provide purified DHNA for future drug discovery studies based upon structure-activity relationships, molecular modeling and crystallographic structure-based drug design.