PhD ceremony: Mr. J. Knol, 12.45 uur, Aula Academiegebouw, Broerstraat 5, Groningen
Dissertation: Natural diversity and catalytic mechanisms of 1- and 4 3 -ketosteroid dehydrogenases from actinomycetal origin
Promotor(s): prof. L. Dijkhuizen
Faculty: Mathematics and Natural Sciences
Phylogenetic analysis of known and putative KSTD amino acid sequences showed that the KSTD enzymes cluster into distinct groups. A representative enzyme of each group was further characterized. The newly identified ∆1 KSTD3 of Rhodococcus erythropolis SQ1, its orthologue Rv3537 (∆1 KSTD) of Mycobacterium tuberculosis H37Rv and the ∆4 KSTD Rv1817 of M. tuberculosis H37Rv clearly preferred saturated steroids while other KSTD enzymes showed higher activity on unsaturated steroids. The genes encoding ∆1 KSTD3 of R. erythropolis SQ1 and iorthologue Rv3537 (∆1 KSTD) of M. tuberculosis H37Rv both are located on a DNA fragment containing other genes involved in cholesterol catabolism. Rv3537 was also identified as one of several essential pathogenicity proteins. The results of Knol imply that saturated steroids are newly identified intermediates in the cholesterol catabolic pathway and important steroids with respect to pathogenicity.
At the start of this research, information about the reaction mechanism and catalytic important residues was lacking for KSTD enzymes. The obtained crystal structure of native and steroid bound ∆4 KSTD Ro05698 of Rhodococcus jostii RHA1 allowed elucidation of its reaction mechanism. Site-directed mutagenesis confirmed the identity of the catalytic residues. This structure was also used as template for ∆1 KSTD homology modelling and helped to identify candidate catalytic residues in ∆1 KSTD enzymes. In all KSTD enzymes tyrosines play a key role in the dehydrogenation reaction. The amino acid that abstract the proton from the steroid substrate is specific for the ∆1- and ∆4 KSTD enzymes. This detailed information may stimulate structure-based design of inhibitors for the virulence-associated KSTD enzymes of M. tuberculosis to combat tuberculosis, a disease that causes millions of victims worldwide every year.
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