Identification and targeted disruption of the gene encoding the main 3-ketosteroid dehydrogenase in Mycobacterium smegmatis.
The catabolic potential for sterol degradation of fast-growing mycobacteria is well known. However, no genes or enzymes responsible for the steroid degradation process have been identified as yet in these species. One of the key enzymes required for degradation of the steroid ring structure is 3-ketosteroid Delta(1)-dehydrogenase (KsdD). The recent annotation of the Mycobacterium smegmatis genome (TIGR database) revealed six KsdD homologues. Targeted disruption of the MSMEG5898 (ksdD-1) gene, but not the MSMEG4855 (ksdD-2) gene, resulted in partial inactivation of the cholesterol degradation pathway and accumulation of the intermediate 4-androstene-3,17-dione. This effect was reversible by the introduction of the wild-type ksdD-1 gene into M. smegmatis DeltaksdD-1 or overexpression of ksdD-2. The data indicate that KsdD1 is the main KsdD in M. smegmatis, but that KsdD2 is able to perform the cholesterol degradation process when overproduced.
Medical Biology Centre, Polish Academy of Sciences, Lodowa 106, 93-232 Łodz, Poland.
Anna
A
Brzostek
Department of Biotechnology and Food Science, Technical University of Łodz, Wolczanska 171/173, 90-924 Łodz, Poland.
Tomasz
T
Śliwiński
Medical Biology Centre, Polish Academy of Sciences, Lodowa 106, 93-232 Łodz, Poland.
Anna
A
Rumijowska-Galewicz
Medical Biology Centre, Polish Academy of Sciences, Lodowa 106, 93-232 Łodz, Poland.
Małgorzata
M
Korycka-Machała
Medical Biology Centre, Polish Academy of Sciences, Lodowa 106, 93-232 Łodz, Poland.
Jarosław
J
Dziadek
D003001:Cloning, Molecular; D007664:Ketosteroids / Q000378:metabolism; D020102:Mycobacterium smegmatis / Q000201:enzymology* / Q000235:genetics; D016366:Open Reading Frames; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
D007664:Ketosteroids; D010088:Oxidoreductases; C022656:3-oxosteroid delta(1) dehydrogenase