Microbial phytosterol degradation is accompanied by the formation of steroid pathway intermediates, which are potential precursors in the synthesis of bioactive steroids. Degradation of these steroid intermediates is initiated by ⌬ 1 -dehydrogenation of the steroid ring structure. Characterization of a 2.9-kb DNA fragment of Rhodococcus erythropolis SQ1 revealed an open reading frame (kstD) showing similarity with known 3-ketosteroid ⌬ 1 -dehydrogenase genes. Heterologous expression of kstD yielded 3-ketosteroid ⌬ 1 -dehydrogenase (KSTD) activity under the control of the lac promoter in Escherichia coli. Targeted disruption of the kstD gene in R. erythropolis SQ1 was achieved, resulting in loss of more than 99% of the KSTD activity. However, growth on the steroid substrate 4-androstene-3,17-dione or 9␣-hydroxy-4-androstene-3,17-dione was not abolished by the kstD gene disruption. Bioconversion of phytosterols was also not blocked at the level of ⌬ 1 -dehydrogenation in the kstD mutant strain, since no accumulation of steroid pathway intermediates was observed. Thus, inactivation of kstD is not sufficient for inactivation of the ⌬ 1 -dehydrogenase activity. Native polyacrylamide gel electrophoresis of cell extracts stained for KSTD activity showed that R. erythropolis SQ1 in fact harbors two activity bands, one of which is absent in the kstD mutant strain.Rhodococcus species are well known for their catabolic potential (5, 40). Several Rhodococcus species degrade natural phytosterols. Microbial phytosterol degradation proceeds via the formation of steroids as pathway intermediates (16,21,22), i.e., 4-androstene-3,17-dione, 1,4-androstadiene-3,17-dione, and 9␣-hydroxy-4-androstene-3,17-dione (Fig.
2-(Methylthio)ethanesulfonate (CH3S-CoM) is formed as an intermediate in methanogenesis from methanol by cell-free extracts of Methanosarcina barkeri. The enzyme system involved in the methyl transfer from methanol to 2-mercaptoethanesulfonate (HS-CoM) was resolved into two enzyme fractions. One enzyme (methanol:5-hydroxy-benzimidazolylcobamide methyltransferase) appears to be a cobalamin-containing protein, which is oxygen sensitive. The other enzyme (Co-methyl-5-hydroxybenzimidazolylcobamide: HS-CoM methyltransferase) was purified. It is insensitive to oxygen and it transfers also the methylgroup from Co-methyl-5,6-dimethylbenzimidazolylcobamide to HS-CoM.
Summary9a-Hydroxylation of 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) is catalysed by 3-ketosteroid 9a-hydroxylase (KSH), a key enzyme in microbial steroid catabolism. Very limited knowledge is presently available on the KSH enzyme. Here, we report for the first time the identification and molecular characterization of genes encoding KSH activity. The kshA and kshB genes, encoding KSH in Rhodococcus erythropolis strain SQ1, were cloned by functional complementation of mutant strains blocked in AD(D) 9a-hydroxylation. Analysis of the deduced amino acid sequences of kshA and kshB showed that they contain domains typically conserved in class IA terminal oxygenases and class IA oxygenase reductases respectively. By definition, class IA oxygenases are made up of two components, thus classifying the KSH enzyme system in R. erythropolis strain SQ1 as a two-component class IA monooxygenase composed of KshA and KshB. Unmarked in frame gene deletion mutants of parent strain R. erythropolis SQ1, designated strains RG2 (kshA mutant) and RG4 (kshB mutant), were unable to grow on steroid substrates AD(D), whereas growth on 9a-hydroxy-4-androstene-3,17-dione (9OHAD) was not affected. Incubation of these mutant strains with AD resulted in the accumulation of ADD (30-50% conversion), confirming the involvement of KshA and KshB in AD(D) 9a-hydroxylation. Strain RG4 was also impaired in sterol degradation, suggesting a dual role for KshB in both sterol and steroid degradation.
Methanol:5-hydroxybenzinmidazolylcobamide methyltransferase from Methanosarcina barkeri has been purified to approximately 90% homogeneity by ion-exchange chromatography on DEAE-cellulose and QAE-A50 Sephadex columns. The molecular weight, estimated by gel electrophoresis, was found to be 122,000, and the enzyme contained two different subunits with molecular weights of 34,000 and 53,000, which indicates an a213 structure. The enzyme contains three or four molecules of 5-hydroxybenzimidazolylcobamide, which could be removed by treatment of the enzyme with 2-mercaptoethanol or sodium dodecyl sulfate. In both cases the enzyme dissociated into its subunits. For stability, the enzyme required the presence of divalent cations such as Mge+, Mn2+, Sr2+, Ca2+, or Ba2+. ATP, GTP, or CTP was needed in a reductive activation process of the enzyme. This activation was brought about by a mixture of H2, ferredoxin, and hydrogenase, but also by CO, which is thought to reduce the corrinoid chemically. The CO dehydrogenase-like activity of the methyltransferase is discussed. Methanosarcina barkeri is a methanogenic bacterium that can grow on various one-carbon compounds such as C02, methylamines, methanol, and CO and on acetate (3, 10, 11, 23). Growth on methanol has been reported in both the presence and absence of H2; in the latter case the reduction equivalents needed in methanogenesis were derived from the oxidation of part of the methanol to CO2 (11). The reduction of methanol to CH4 in cell-free extracts of M. barkeri was found to depend on the presence of coenzyme M (2-mercaptoethanesulfonic acid; HS-CoM) and ATP under an atmosphere of H2 (12). First, HS-CoM is methylated to 2-(methylthio)ethanesulfonic acid (methylcoenzyme M; CH3S-CoM) (18, 21). CH3S-CoM is subsequently reduced to methane by a methylreductase system that contains an enzyme-bound coenzyme MF430 (7, 8, 13). The involvement of two distinct methyltransferases in the formation of CH3S-CoM from methanol was recently reported (21). Methanol:5-hydroxybenzimidazolylcobamide methyltransferase (MT1) binds the methyl group of methanol to a corrinoid bound to this enzyme (22). The enzyme is subject to activation and inactivation. Inactivation is brought about by 02 and other oxidizing agents, and activation is achieved in the presence of ATP and H2 (21). Activation of the partially purified MT, requires also the presence of hydrogenase and ferredoxin and leads to the formation of a Co(I) corrinoid (B12) (22b). The role of the catalytic amount of ATP in this activation has not been elucidated. The second methyltransferase, methylcobalamin:HS-CoM methyltransferase (MT2), is oxygen stable, and ATP is not required in its activity (20). It transfers the methyl group of the bound corrinoid of MT1 to HS-CoM. The activity of MT2 is not limited to the bound methylated corrinoid of MT,; free methylcorrinoids with either 5-hydroxybenzimidazole (HBI) (16) or 5,6-dimethylbenzimidazole (DMBI) as the a-ligand could be demethylated (19, 20). Here we report on the purification and pro...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.