The specificity of a 7 alpha-hydroxy steroid dehydrogenase from Escherichia coli.
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ABSTRACT:
SUBMITTER: Haslewood ES
PROVIDER: S-EPMC1163832 | biostudies-other | 1976 Jul
REPOSITORIES: biostudies-other
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Project description:Commensal microbes are exposed to enterohepatically circulated steroids such as bile acids and hormones in the gastrointestinal, vaginal, and urinary tracts. Since commensal microbes are exposed to these molecules exclusively in association with their host, we hypothesized that they may serve as effectors to identify and characterize genetic pathways involved in the commensal-host relationship. Host specific and some ingested steroids (phytoestrogens) are enterohepatically circulated through the lumen of the small intestine. Bile acid steroids are present at high concentrations, approximating 4 to 20 mM in the duodenum, and are released in bile from the common bile duct. Steroid hormones are secreted in bile at levels approximating 6 to 13 mg per day once conjugated to glucuronide or sulfate by the liver. Re-absorption of steroids by the terminal ileum is an incomplete process: for example, 200 to 600 mg of bile acid steroids per day in humans escape to the colon where complex microbial populations exist. We have shown that steroid hormones estradiol, progesterone, and hydrocortisone serve as strong substrates for the major RND- and MFS-type (except hydrocortisone) tripartite multidrug efflux systems, AcrAB-TolC and EmrAB-TolC respectively, even though such molecules fail to demonstrate antimicrobial properties in this bacterial background (Elkins and Mullis [2006] J. Bacteriol. 188:1191-1195). Although bile acids are subject to efflux, they are also known to directly interact with intracellular global regulators such as MarR (Prouty et al. [2004] Microbiol. 150:775-783) and Rob (Rosenberg et al., [2003] Mol. Microbiol. 48:1609-1619) consequently altering antimicrobial and bile resistance profiles. In our present study, whole-genome DNA microarrays of E. coli were used to determine what general effect steroids, both bile acid and hormones, may have on the transcriptome in relation to the human commensal environment. Keywords: Comparative Chemical Class Treatment
2008-03-14 | GSE10811 | GEO
Project description:Aldehyde dehydrogenases catalyze the oxidation of aldehyde substrates to the corresponding carboxylic acids. Lactaldehyde dehydrogenase from Escherichia coli (aldA gene product, P25553) is an NAD(+)-dependent enzyme implicated in the metabolism of l-fucose and l-rhamnose. During the heterologous expression and purification of taxadiene synthase from the Pacific yew, lactaldehyde dehydrogenase from E. coli was identified as a minor (</=5%) side-product subsequent to its unexpected crystallization. Accordingly, we now report the serendipitous crystal structure determination of unliganded lactaldehyde dehydrogenase from E. coli determined by the technique of multiple isomorphous replacement using anomalous scattering at 2.2 A resolution. Additionally, we report the crystal structure of the ternary enzyme complex with products lactate and NADH at 2.1 A resolution, and the crystal structure of the enzyme complex with NADPH at 2.7 A resolution. The structure of the ternary complex reveals that the nicotinamide ring of the cofactor is disordered between two conformations: one with the ring positioned in the active site in the so-called hydrolysis conformation, and another with the ring extended out of the active site into the solvent region, designated the out conformation. This represents the first crystal structure of an aldehyde dehydrogenase-product complex. The active site pocket in which lactate binds is more constricted than that of medium-chain dehydrogenases such as the YdcW gene product of E. coli. The structure of the binary complex with NADPH reveals the first view of the structural basis of specificity for NADH: the negatively charged carboxylate group of E179 destabilizes the binding of the 2'-phosphate group of NADPH sterically and electrostatically, thereby accounting for the lack of enzyme activity with this cofactor.
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Project description:Treatment of pregnant rats with human chorionic gonadotrophin, luteotrophin (luteinizing hormone), luteotrophin-releasing hormone, prostaglandin F2alpha, aminoglutethimide, or by foetoplacental removal or hysterectomy achieved a common multiple-response pattern, namely increased activity of luteal 20alpha-hydroxy steroid dehydrogenase with decreased activity of delta5-3beta-hydroxy steriod dehydrogenase and release of delta4-3-oxo steroids in vitro. 2. Similar effects of foetoplacental removal are noted in pregnant mice. 3. Gonadotrophin induced lower activities of 20alpha-hydroxy steroid dehydrogenase, except at the very end of pregnancy, and partly inhibited the induction caused by foetoplacental removal. 4. The results suggest that existence of a placental factor that restrains these changes until the end of normal pregnancy, which is produced in amounts proportional to the number of placentae and is conveyed to the ovary via the blood. 5. This factor was not replaced by prolactin. 6. It is argued that neither placental lactogen nor pituitary luteotrophin participate in the induction of 20alpha-hydroxy steroid dehydrogenase at late pregnancy in the rat. 7. Aminoglutethimide induced 20alpha-hydroxy steroid dehydrogenase only in late pregnancy. This was partly reversed by progesterone, wholly reversed by progesterone plus oestrogen, and did not involve the pituitary.
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Project description:The behaviour of various C(19) and C(18) steroids as substrates for crystalline preparations of cortisone reductase (EC 1.1.1.53) is described. 3alpha(Axial,3R)-, 3alpha(equatorial,3R)- and 3beta(axial,3S)-hydroxy steroid-NAD oxidoreductase activities are demonstrated. Four pairs of the substrates differed only in the shape of the a/b ring junction, three pairs differed only in substitution at C-10, and four pairs differed only in substitution in ring d. The shape of the substrate molecule and certain substituents (e.g. 10beta-methyl, 17beta-hydroxy, 16-oxo or 17-oxo) altered substrate behaviour, but steroids differing considerably in shape nevertheless acted as substrates, suggesting the possibility of a large or flexible binding site. K(m) values varied about 10-fold, many being approx. 140mum. V(max.) values covered a greater range (about 200-fold) and the good substrates had high V(max.) values rather than low K(m) values.
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Project description:1. The inhibition of NAD-linked 3alpha-hydroxy steroid dehydrogenase in vitro by some of the more therapeutically effective antimetabolic drugs has been investigated. 2. Antipurine and antifolic acid drugs inhibit this system, as does folic acid. 3. Combinations of antifolic acid drugs and folic acid itself produce an additive but not synergic inhibition. 4. Antimetabolic drugs give inhibitions that are additive to those produced by antagonistic steroid hormones.
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Project description:1. The specificity of 3 oestradiol-binding proteins was studied. Two of these proteins are naturally occurring (rat alpha-foetoprotein and rat liver microsomal 17beta-hydroxy steroid dehydrogenase) and the third is an artificially induced model, anti-(oestradiol-6-carboxymethyloxime-bovine serum albumin) gamma-globulins. 2. A specific binding procedure for each protein model permitted a determination of its affinity for oestradiol and for 30 other steroids. 3. The results obtained have brought to light the different areas of the steroid molecule that are important for its recognition by each of the three proteins. The two naturally occurring proteins (alpha-foetoprotein and 17beta-hydroxy steroid dehydrogenase) recognize the edge of the steroid defined by C-4, C-6, C-8 and C-15. On the other hand, the gamma-globulins recognize the opposite edge, i.e. that defined by C-2, C-10, C-11 and C-17. 4. Diethylstilboestrol, whose structure is analogous to that of a steroid, is only recognized by the two naturally occurring proteins.
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Project description:Glucose 6-Phosphate Dehydrogenases (G6PDHs) from different sources show varying specificities towards NAD+ and NADP+ as cofactors. However, it is not known to what extent structural determinants of cofactor preference are conserved in the G6PDH family. In this work, molecular simulations, kinetic characterization of site-directed mutants and phylogenetic analyses were used to study the structural basis for the strong preference towards NADP+ shown by the G6PDH from Escherichia coli. Molecular Dynamics trajectories of homology models showed a highly favorable binding energy for residues K18 and R50 when interacting with the 2'-phosphate of NADP+, but the same residues formed no observable interactions in the case of NAD+. Alanine mutants of both residues were kinetically characterized and analyzed with respect to the binding energy of the transition state, according to the kcat/KM value determined for each cofactor. Whereas both residues contribute to the binding energy of NADP+, only R50 makes a contribution (about -1 kcal/mol) to NAD+ binding. In the absence of both positive charges the enzyme was unable to discriminate NADP+ from NAD+. Although kinetic data is sparse, the observed distribution of cofactor preferences within the phylogenetic tree is sufficient to rule out the possibility that the known NADP+-specific G6PDHs form a monophyletic group. While the β1-α1 loop shows no strict conservation of K18, (rather, S and T seem to be more frequent), in the case of the β2-α2 loop, different degrees of conservation are observed for R50. Noteworthy is the fact that a K18T mutant is indistinguishable from K18A in terms of cofactor preference. We conclude that the structural determinants for the strict discrimination against NAD+ in the case of the NADP+-specific enzymes have evolved independently through different means during the evolution of the G6PDH family. We further suggest that other regions in the cofactor binding pocket, besides the β1-α1 and β2-α2 loops, play a role in determining cofactor preference.
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Project description:Escherichia coli (strain K-12, substrain MG1655) glycerol dehydrogenase (GldA) is required to catalyze the first step in fermentative glycerol metabolism. The protein was expressed and purified to homogeneity using a simple combination of heat-shock and chromatographic methods. The high yield of the protein (?250?mg per litre of culture) allows large-scale production for potential industrial applications. Purified GldA exhibited a homogeneous tetrameric state (?161?kDa) in solution and relatively high thermostability (Tm = 65.6°C). Sitting-drop sparse-matrix screens were used for protein crystallization. An optimized condition with ammonium sulfate (2?M) provided crystals suitable for diffraction, and a binary structure containing glycerol in the active site was solved at 2.8?Å resolution. Each GldA monomer consists of nine ?-strands, thirteen ?-helices, two 310-helices and several loops organized into two domains, the N- and C-terminal domains; the active site is located in a deep cleft between the two domains. The N-terminal domain contains a classic Rossmann fold for NAD+ binding. The O1 and O2 atoms of glycerol serve as ligands for the tetrahedrally coordinated Zn2+ ion. The orientation of the glycerol within the active site is mainly stabilized by van der Waals and electrostatic interactions with the benzyl ring of Phe245. Computer modeling suggests that the glycerol molecule is sandwiched by the Zn2+ and NAD+ ions. Based on this, the mechanism for the relaxed substrate specificity of this enzyme is also discussed.
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Project description:1. The 17beta-hydroxy steroid dehydrogenase was solubilized during haemolysis of erythrocytes and was isolated from the membrane-free haemolysate. Membrane preparations isolated in different ways did not contain 17beta-hydroxy steroid dehydrogenase activity. The 17beta-hydroxy steroid dehydrogenase activity in the haemolysate was concentrated by repeated ammonium sulphate precipitation and gel filtration on Sephadex G-150. The 17beta-hydroxy steroid dehydrogenase activity of the purified preparation per unit weight of protein was 350-3000 times higher than the activity of the crude erythrocyte haemolysate. The 20alpha-hydroxy steroid dehydrogenase activity was lost during this purification procedure. 2. The 17beta-hydroxy steroid dehydrogenase was NADP-dependent and had a pH optimum for conversion of testosterone between 8.5 and 10. For the molecular weight of the enzyme a value of 64000 was calculated from Sephadex chromatography results. 3. p-Chloromercuribenzoate inhibited the enzymic activity. The oxidative activity of the enzyme for the 17beta-hydroxyl group was only partly inhibited when a large excess of 17-oxo steroids was added. The catalysing activity of the enzyme was influenced by the NADP(+)/NADPH ratio. The oxidation of the 17beta-hydroxyl group in the presence of NADP(+) proceeded faster than the reduction of the 17-oxo group with NADPH. When both reduced and oxidized cofactors were present the oxidation of the 17beta-hydroxyl group was inhibited to a considerable extent. 4. The enzyme had a broad substrate specificity and not only catalysed the conversion of androstanes with a 17beta-hydroxyl group, or 17-oxo group, but also the conversion oestradiolleft arrow over right arrowoestrone. In addition the steroid conjugates dehydroepiandrosterone sulphate and oestrone sulphate were also converted. There were no indications that more than one 17beta-hydroxy steroid dehydrogenase was present in the partially purified preparation.
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Project description:The Escherichia coli isocitrate dehydrogenase (ICDH) is one of the tricarboxylic acid cycle enzymes, playing key roles in energy production and carbon flux regulation. E. coli ICDH was the first bacterial enzyme shown to be regulated by reversible phosphorylation. However, the effect of lysine acetylation on E. coli ICDH, which has no sequence similarity with its counterparts in eukaryotes, is still unclear. Based on previous studies of E. coli acetylome and ICDH crystal structures, eight lysine residues were selected for mutational and kinetic analyses. They were replaced with acetyllysine by the genetic code expansion strategy or substituted with glutamine as a classic approach. Although acetylation decreased the overall ICDH activity, its effects were different site by site. Deacetylation tests demonstrated that the CobB deacetylase could deacetylate ICDH both in vivo and in vitro, but CobB was only specific for lysine residues at the protein surface. On the other hand, ICDH could be acetylated by acetyl-phosphate chemically in vitro. And in vivo acetylation tests indicated that the acetylation level of ICDH was correlated with the amounts of intracellular acetyl-phosphate. This study nicely complements previous proteomic studies to provide direct biochemical evidence for ICDH acetylation.
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