1.3.99.4 A255F site-directed mutagenesis, inactive mutant 763180 1.3.99.4 A255G site-directed mutagenesis, inactive mutant 763180 1.3.99.4 E140V site-directed mutagenesis, no significant change in enzyme expression is caused by the mutation, the mutant shows reduced activity with 4-androstene-3,17-dione compared to wild-type enzyme 763772 1.3.99.4 F266A site-directed mutagenesis, inactive mutant -, 763180 1.3.99.4 G258A site-directed mutagenesis, inactive mutant -, 763180 1.3.99.4 G532A site-directed mutagenesis, the mutant shows over 70% reduced activity compare to wild-type enzyme 763180 1.3.99.4 L261A site-directed mutagenesis, inactive mutant 763180 1.3.99.4 additional information among the three KstD isozymes, KstD2 shows the highest enzymatic activity when expressed heterogeneously, and KstD1 performs poorly, especially in Escherichia coli. For isozyme KstD3, the KstD enzyme activities are hardly detected in either host, Escherichia coli or Bacillus subtilis -, 763441 1.3.99.4 additional information construction of a kstD1 deletion mutant. Among the three KstD isozymes, KstD2 shows the highest enzymatic activity when expressed heterogeneously, and KstD1 performs poorly, especially in Escherichia coli. For isozyme KstD3, the KstD enzyme activities are hardly detected in either host, Escherichia coli or Bacillus subtilis -, 763441 1.3.99.4 additional information development of a synthetic 3-ketosteroid DELTA1-dehydrogenase (DELTA1-KstDA) for the generation of a catabolic pathway enabling cholesterol degradation in human cells. Coexpression with cholesterol-3-hydroxy-dehydrogenase (CholD) and 3-oxosteroid-9alpha-hydroxylase (Kst-9alphaH). CholD activity catalyzes the generation of a C-3 ketone, which is needed for DELTA1-KstD activity, which introduces a double bond between the C-1 and C-2 atoms of 3-oxosteroids. Cholesterol catabolism pathway in Actinomycetes, overview 763735 1.3.99.4 additional information development of a synthetic 3-ketosteroid DELTA1-dehydrogenase (DELTA1-KstDR) for the generation of a catabolic pathway enabling cholesterol degradation in human cells. Coexpression with cholesterol-3-hydroxy-dehydrogenase (CholD) and 3-oxosteroid-9alpha-hydroxylase (Kst-9alphaH). CholD activity catalyzes the generation of a C-3 ketone, which is needed for DELTA1-KstD activity, which introduces a double bond between the C-1 and C-2 atoms of 3-oxosteroids. Cholesterol catabolism pathway in Actinomycetes, overview. The downstream enzymes need to have equal or greater activity when compared to the enzymes acting upstream to allow the pathway to flow and to prevent the accumulation of toxic intermediates, kinetic study 763735 1.3.99.4 additional information enzyme from strain ST-095 only differs in residue V366 from strain JC-12 (S366). Mutation results in almost 3fold higher catalytic efficiency in strain JC-12 -, 743031 1.3.99.4 additional information enzyme mutants are constructed based on in silico protein docking modeling using site-directed mutagenesis and exogenous expression -, 763772 1.3.99.4 additional information targeted disruption of the MSMEG5898 (ksdD-1) gene, but not the MSMEG4855 (ksdD-2) gene, results in partial inactivation of the cholesterol degradation pathway and accumulation of the intermediate 4-androstene-3,17-dione, reversible by the introduction of the wild-type ksdD-1 gene into deltaksdD-1 or overexpression of ksdD-2 -, 675829 1.3.99.4 R256A site-directed mutagenesis, almost inactive mutant -, 763180 1.3.99.4 R256H site-directed mutagenesis, the mutant shows over 70% reduced activity compare to wild-type enzyme 763180 1.3.99.4 R273A site-directed mutagenesis, inactive mutant 763180 1.3.99.4 S138A site-directed mutagenesis, no significant change in enzyme expression is caused by the mutation, the mutant shows reduced activity with 4-androstene-3,17-dione compared to wild-type enzyme -, 763772 1.3.99.4 S325F mutant enzyme has no detectable KSTD enzyme activity -, 656722 1.3.99.4 T503I mutant enzyme has no detectable KSTD enzyme activity -, 656722 1.3.99.4 V259A site-directed mutagenesis, inactive mutant -, 763180 1.3.99.4 V259F site-directed mutagenesis, inactive mutant 763180 1.3.99.4 Y118A site-directed mutagenesis, almost inactive mutant -, 763180 1.3.99.4 Y119F almost complete loss of activity -, 742850 1.3.99.4 Y122F site-directed mutagenesis, no significant change in enzyme expression is caused by the mutation, the mutant shows similar activity with 4-androstene-3,17-dione compared to wild-type enzyme -, 763772 1.3.99.4 Y125F site-directed mutagenesis, no significant change in enzyme expression is caused by the mutation, the mutant shows highly reduced activity with 4-androstene-3,17-dione compared to wild-type enzyme -, 763772 1.3.99.4 Y318F almost complete loss of activity -, 742850 1.3.99.4 Y355A site-directed mutagenesis, the mutant shows over 70% reduced activity compare to wild-type enzyme 763180 1.3.99.4 Y365F site-directed mutagenesis, no significant change in enzyme expression is caused by the mutation, the mutant shows highly reduced activity with 4-androstene-3,17-dione compared to wild-type enzyme -, 763772 1.3.99.4 Y472F site-directed mutagenesis, no significant change in enzyme expression is caused by the mutation, the mutant shows similar activity with 4-androstene-3,17-dione compared to wild-type enzyme 763772 1.3.99.4 Y487F almost complete loss of activity -, 742850 1.3.99.4 Y528A site-directed mutagenesis, inactive mutant 763180 1.3.99.4 Y541F site-directed mutagenesis, no significant change in enzyme expression is caused by the mutation, the mutant shows highly reduced activity with 4-androstene-3,17-dione compared to wild-type enzyme 763772