Alteration of biliary ursodeoxycholic acid in guinea pig during early stages of cholestyramine feeding

The effects of cholestyramine feeding on biliary ursodeoxycholic acid, fecal excretion of bile acids and neutral sterols on cholesterol 7α-hydroxylase and hepatic HMG-CoA reductase were examined in the guinea pig. In the bile there was a 57% decrease in the concentration of ursodeoxycholic acid while an increase was observed in the concentration of chenodeoxycholic acid. Cholestyramine feeding for ten days resulted in a decrease in plasma cholesterol levels and an increase in both hepatic HMG-CoA reductase and cholesterol 7α-hydroxylase activities. The fecal excretion of both bile acids and neutral sterols was significantly increased.     

Characterization of the lys2 gene of Penicillium chrysogenum encoding α -aminoadipic acid reductase

A DNA fragment containing a gene homologous to LYS2 gene of Saccharomyces cerevisiae was cloned from a genomic DNA library of Penicillium chrysogenum AS-P-78. It encodes a protein of 1409 amino acids (Mr^ 154 859) with strong similarity to the S. cerevisiae (49.9% identity) Schizosaccharomycespombe (51.3% identity) and Candida albicans (48.12% identity) α-aminoadipate reductases and a lesser degree of identity to the amino acid-activating domains of the non-ribosomal peptide synthetases, including the α-aminoadipate-activating domain of the α-aminoadipyl-cysteinyl-valine synthetase of P. chrysogenum (12.4% identical amino acids). The lys2 gene contained one intron in the 5′-region and other in the 3′-region, as shown by comparing the nucleotide sequences of the cDNA and genomic DNA, and was transcribed as a 4.7-kb monocistronic mRNA. The lys2 gene was localized on chromosome III (7.5 Mb) in P. chrysogenum AS-P-78 and on chromosome IV (5.6 Mb) in strain P2, whereas the penicillin gene cluster is known to be located in chromosome I in both strains. The lys2-encoded protein is a member of the aminoacyladenylate-forming enzyme family with a reductase domain in its C-terminal region. Received: 26 January 1998 / Accepted: 4 May 1998     

Biosorption of reactive and basic dyes using fermentation waste <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>: the effects of pH and salt concentration and characterization of the binding sites

A low cost biosorbent, Corynebacterium glutamicum, was studied for the sorption of Reactive Red 4 (RR 4) and Methylene Blue (MB). The equilibrium isotherm data were well described by the Langmuir model. pH edge experiments showed that pH of the solution was an important controlling parameter in the sorption process. In the case of RR 4, with increases in the pH from 2 to 10, the uptake decreased from 52 to 1 mg/g; conversely, the uptake of MB increased and the maximum MB uptake was obtained at pH ≥ 9. An increase in the salt concentration strongly influenced the uptake of MB, but had no effect on that of RR 4. In order to identify the binding sites for the dye molecules, the biosorbent was potentiometrically titrated, the results of which showed the presents of four major functional group types on the biomass surface, which were confirmed by FTIR analysis. It was found that positively charged amine groups (Biomass-NH₃ ⁺) were the likely binding sites for anionic RR 4, and negatively charged carboxyl (Biomass-COO⁻) and phosphate groups (Biomass-HPO₄ ⁻) played a role in the electrostatic attraction of cationic MB.     

Distal end of 105-125 loop - A putative reductase binding domain of phthalate dioxygenase

The phthalate dioxygenase system consists of the dioxygenase, PDO, which contains a Rieske [2Fe-2S] center and a Fe(II)-mononuclear center, and the reductase, PDR. Involvement of the distal end of the 105-125 loop of PDO in its interaction with PDR was tested by substituting charged residues in the loop with alanines and by replacing the conserved tryptophan-94. Compared to wild-type PDO, all variants had lower catalytic activity and the Rieske centers were reduced more slowly by reduced PDR. The rates of oxidation of the Rieske centers by oxygen, which represent electron transfer between the Rieske and mononuclear centers, were essentially unaffected. These results suggest that positively charged residues of the distal end of the 105-125 loop are collectively involved in PDR binding with the PDO. Contrary to expectations, Trp94 variants were not directly involved in electron transfer between PDR and PDO. The tryptophan appears to have mainly a structural role, apparently preserving the hydrophilic environment of the Rieske center.     

Receptor independent and receptor dependent CoMSA modeling with IVE-PLS: application to CBG benchmark steroids and reductase activators

Comparative molecular surface analysis (CoMSA) with robust IVE-PLS variable elimination if tested for the benchmark CBG steroid series provides highly predictive RI 3D QSAR models, but failed however to model the activity of sulforaphane (SP) activators of quinone reductase. The application of the SP poses obtained from multipose molecular docking to model the RD IVE-PLS CoMSA resulted in a predictive form. This model indicated lipophilic potential as the activity determinant. The individual molecular surface areas of the highest contribution to the SP activity was identified and visualized by CoMSA contour plots.     

Crystal structure of bifunctional 5,10-methylenetetrahydrofolate dehydrogenase/cyclohydrolase from Thermoplasma acidophilum

Folate co-enzymes play a pivotal role in one-carbon transfer cellular processes. Many eukaryotes encode the tri-functional tetrahydrofolate dehydrogenase/cyclohydrolase/synthetase (deh/cyc/syn) enzyme, which consists of a N-terminal bifunctional domain (deh/cyc) and a C-terminal monofunctional domain (syn). Here, we report the first analogous archeal enzyme structures, for the bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase from Thermoplasma acidophilum (TaMTHFDC) as the native protein and also as its NADP complex. The TaMTHFDC structure is a dimer with a polar interface, as well as a NADP binding site that shows minor conformational change. The orientations of the residues in the NADP binding site do not change on ligand binding, incorporating three water molecules which are hydrogen bonded with phosphate groups of NADP in the structure of the complex. Our structural information will contribute to an improved understanding of the basis of THF and one-carbon metabolism.     

Deciphering the role of histidine 252 in mycobacterial adenosine 5'-phosphosulfate (APS) reductase catalysis

Mycobacterium tuberculosis adenosine 5'-phosphosulfate reductase (APR) catalyzes the first committed step in sulfate reduction for the biosynthesis of cysteine and is essential for survival in the latent phase of tuberculosis infection. The reaction catalyzed by APR involves the nucleophilic attack by conserved Cys-249 on adenosine 5'-phosphosulfate, resulting in a covalent S-sulfocysteine intermediate that is reduced in subsequent steps by thioredoxin to yield the sulfite product. Cys-249 resides on a mobile active site lid at the C terminus, within a K(R/T)ECG(L/I)H motif. Owing to its strict conservation among sulfonucleotide reductases and its proximity to the active site cysteine, it has been suggested that His-252 plays a key role in APR catalysis, specifically as a general base to deprotonate Cys-249. Using site-directed mutagenesis, we have changed His-252 to an alanine residue and analyzed the effect of this mutation on the kinetic parameters, pH rate profile, and ionization of Cys-249 of APR. Interestingly, our data demonstrate that His-252 does not perturb the pK(a) of Cys-249 or play a direct role in rate-limiting chemical steps of the reaction. Rather, we show that His-252 enhances substrate affinity via interaction with the α-phosphate and the endocyclic ribose oxygen. These findings were further supported by isothermal titration calorimetry to provide a thermodynamic profile of ligand-protein interactions. From an applied standpoint, our study suggests that small-molecules targeting residues in the dynamic C-terminal segment, particularly His-252, may lead to inhibitors with improved binding affinity.     

Atomic structure of salutaridine reductase from the opium poppy (Papaver somniferum)

The opium poppy (Papaver somniferum L.) is one of the oldest known medicinal plants. In the biosynthetic pathway for morphine and codeine, salutaridine is reduced to salutaridinol by salutaridine reductase (SalR; EC 1.1.1.248) using NADPH as coenzyme. Here, we report the atomic structure of SalR to a resolution of ∼1.9 Å in the presence of NADPH. The core structure is highly homologous to other members of the short chain dehydrogenase/reductase family. The major difference is that the nicotinamide moiety and the substrate-binding pocket are covered by a loop (residues 265–279), on top of which lies a large “flap”-like domain (residues 105–140). This configuration appears to be a combination of the two common structural themes found in other members of the short chain dehydrogenase/reductase family. Previous modeling studies suggested that substrate inhibition is due to mutually exclusive productive and nonproductive modes of substrate binding in the active site. This model was tested via site-directed mutagenesis, and a number of these mutations abrogated substrate inhibition. However, the atomic structure of SalR shows that these mutated residues are instead distributed over a wide area of the enzyme, and many are not in the active site. To explain how residues distal to the active site might affect catalysis, a model is presented whereby SalR may undergo significant conformational changes during catalytic turnover.     

Insig Proteins Mediate Feedback Inhibition of Cholesterol Synthesis in the Intestine

Enterocytes are the only cell type that must balance the de novo synthesis and absorption of cholesterol, although the coordinate regulation of these processes is not well understood. Our previous studies demonstrated that enterocytes respond to the pharmacological blockade of cholesterol absorption by ramping up de novo sterol synthesis through activation of sterol regulatory element-binding protein-2 (SREBP-2). Here, we genetically disrupt both Insig1 and Insig2 in the intestine, two closely related proteins that are required for the feedback inhibition of SREBP and HMG-CoA reductase (HMGR). This double knock-out was achieved by generating mice with an intestine-specific deletion of Insig1 using Villin-Cre in combination with a germ line deletion of Insig2. Deficiency of both Insigs in enterocytes resulted in constitutive activation of SREBP and HMGR, leading to an 11-fold increase in sterol synthesis in the small intestine and producing lipidosis of the intestinal crypts. The intestine-derived cholesterol accumulated in plasma and liver, leading to secondary feedback inhibition of hepatic SREBP2 activity. Pharmacological blockade of cholesterol absorption was unable to further induce the already elevated activities of SREBP-2 or HMGR in Insig-deficient enterocytes. These studies confirm the essential role of Insig proteins in the sterol homeostasis of enterocytes.