(3R)-hydroxyacyl-ACP dehydratase enzymes involved in the FAS-II system of mycobacteria, useful for antibiotic screening

摘要

#CMT# #/CMT# (3R)-hydroxyacyl-ACP dehydratase enzymes (I) involved in the FAS-II system of mycobacteria are new. #CMT# : #/CMT# Enzymes (I) involved in the FAS-II system of mycobacteria are in the form of homodimers or homomultimers comprising two or more proteins comprising a hydrolase 2 unit of formula (II) or a a related catalytic unit of formula (III), or in the form of heterodimers or heteromultimers comprising a protein as above and a protein that lacks a hydrolase 2 unit but has a hotdog fold structure, and catalyze the hydration of (3R)-hydroxyacyl compounds of formula (IV) to 2-trans-enoyl compounds of formula (V) and/or catalyze the hydration of (V) to (IV). [YF]-x(1,2)-[LIVG]-[STGC]-G-D-x-N-P-[LIV]-H-x(5)-[AS] (II) D-x(4)-H (III) CH 3(CH 2) n-CH(OH)-CH 2-CO-S-X (IV) CH 2(CH 2) n-CH=CH-CO-S-X (V) x(n) : n amino acids; X : ACP or CoA; n : 0 or more; amino acids in square brackets : are alternatives. Independent claims are also included for: (1) polynucleotides coding for (I); (2) expression vectors containing the polynucleotides; prokaryotic or eukaryotic host cells transformed with the polynucleotides or vectors; (3) producing (I) by culturing the cells; (4) inhibiting biosynthesis of mycolata envelope compounds by inhibiting the expression or activity of (I). #CMT#USE : #/CMT# (I) are useful for screening for antibiotics active against microorganisms comprising a FAS-II system (claimed), especially pathogenic Mycobacterium, Nocardia. Rhodococcus and Gordona species. #CMT#BIOTECHNOLOGY : #/CMT# Preferred Enzymes: (I) are homodimers or homomultimers of Rv0636 with SEQ ID NO:2 (defined sequence of 142 amino acids given in the specification), heterodimers or heteromultimers of Rv0636 and Rv0635 with SEQ ID NO:4 (defined sequence of 158 amino acids given in the specification) or heterodimers or heteromultimers of Rv0636 and Rv0637 with SEQ ID NO:6 (defined sequence of 166 amino acids given in the specification). #CMT#SPECIFIC PROTEINS : #/CMT# Mycobacterium tubersulosis proteins Rv0635, Rv0636 and Rv0637 in monomeric form are specifically claimed. #CMT#EXAMPLE : #/CMT# The gene coding for protein Rv0636 was amplified from the total DNA of Mycobacterium tuberculosis H37Rv by PCR using the primers 5'-GATTTTCTGATGGCGCTGCGTGAGTTC-3' and 5'-CGGTCTTGAGCGCCATAAACTA-3'.