Assessing consumers' perception and demand on the community pharmacists' dispensing.

BACKGROUND: This study aimed to assess the general public's perception of services provided by community pharmacies, their willingness to utilize these services, their satisfaction with and understanding of community pharmacists, and their views on dispensing separation and pharmacy medicines (P medicines). METHODS: An online cross-sectional study was conducted, in which questionnaires were distributed among the general public. A novel questionnaire was designed and validated specifically for this study. It was composed of six sections: demographics, pharmacy usage and service preferences, understanding and satisfaction with pharmacists, views on dispensing separation, private community pharmacies, and knowledge of P medicines. Statistical analyses such as one-way ANOVA, independent t test, and binary logistic regression were employed, with a p value of < 0.05 considered statistically significant. RESULTS: The study received 222 responses. The majority of the respondents were females within the 20-29-year-old age group (62.2%). Most respondents preferred to consult doctors for medical treatment, with their primary reason for visiting community pharmacies being to collect prescribed medicines. About 52.7% of respondents expressed their willingness to avail of screening services and treatment for minor illnesses at community pharmacies. A statistically significant difference was found among different age groups regarding their views on the dispensing separation system, with those aged 41-50 years demonstrating higher scores. However, the binary logistic regression analysis did not reveal any statistical significance when comparing the understanding of P medicines among respondents. CONCLUSIONS: In general, the public prefers to consult doctors for medical treatment and visit community pharmacies predominantly to collect prescriptions or purchase over-the-counter medications. Nonetheless, they are also open to utilizing services provided by community pharmacists, particularly screening services and treatment for minor illnesses.

Drug-related problems in hospitalized patients with type 2 diabetes mellitus: A systematic review.

Introduction: Type 2 diabetes mellitus (T2DM) is one of the non-communicable diseases which continues to rise in prevalence and mortality rate throughout the years. Drug-related problems (DRPs) are more prevalent among T2DM patients especially those with co-morbidities. Objective: The objective of this study was to review and assess the prevalence and characteristics of DRPs among hospitalized type 2 diabetes mellitus patients. Methods: The systematic review of the literature was carried out using five online databases: PubMed, Scopus, Google Scholar, Web of Science, and Cochrane Library from the inception of the database until June 2022. Studies included in the review were published in English or Malay language. The data were extracted and assessed using the Joanna Briggs Institute (JBI) critical appraisal tools. Results: A total of 939 studies were identified with 20 studies that met inclusion criteria and were included in this systematic review. The overall prevalence of DRPs in all 20 studies ranged from 7% to 94%. The most common DRPs included drug-drug interaction (DDI), adverse drug reaction (ADR), therapeutic effectiveness problems, and inappropriate medication use. Conclusion: The most common drug classes involved were antidiabetics (metformin), antihypertensives, antiplatelets and antibiotics. The risk factors contributing to DRPs included the presence of comorbidities, the number of medications, and polypharmacy. To conclude, the rate of DRPs incidence in hospitalized T2DM patients was observed to be high. Further future studies with appropriate study designs and methods of detecting DRPs will be necessary to reduce and prevent DRPs occurrences.

Cassava (Manihot esculenta Crantz): A Systematic Review for the Pharmacological Activities, Traditional Uses, Nutritional Values, and Phytochemistry.

Cassava (Manihot esculenta Crantz) is considered one of the essential tuber crops, serving as a dietary staple food for various populations. This systematic review provides a comprehensive summary of the nutritional and therapeutic properties of cassava, which is an important dietary staple and traditional medicine. The review aims to evaluate and summarize the phytochemical components of cassava and their association with pharmacological activities, traditional uses, and nutritional importance in global food crises. To collect all relevant information, electronic databases; Cochrane Library, PubMed, Scopus, Web of Science, Google Scholar, and Preprint Platforms were searched for studies on cassava from inception until October 2022. A total of 1582 studies were screened, while only 34 were included in this review. The results of the review indicate that cassava has diverse pharmacological activities, including anti-bacterial, anti-cancer, anti-diabetic, anti-diarrheal, anti-inflammatory, hypocholesterolemic effects, and wound healing properties. However, more studies that aim to isolate the phytochemicals in cassava extracts and evaluate their pharmacological property are necessary to further validate their medical and nutritional values.

Role of Immunotherapy in the Treatment of Cancer: A Systematic Review.

Tremendous progress has been made in cancer research over the years, and, as a result, immunotherapy has emerged as an important therapy for the treatment of cancer, either as a stand-alone treatment or in conjunction with other cancer therapies. Immunotherapy has demonstrated encouraging outcomes and offers a viable strategy for not only enhancing the quality of life but also dramatically boosting the overall survival rate of cancer patients. The objective of this systematic review was to assess the efficacy of immunotherapy in the treatment of cancer. Databases such as PubMed and Science Direct were searched from their inception until September 2021, using the following keywords: cancer immunotherapy, cancer recurrence, cancer treatment options, and cancer therapies. The systematic review was conducted in accordance with the PRISMA protocol. There were a total of 599 articles; however, after applying the inclusion and exclusion criteria, the final review ended up with 34 publications. In conclusion, the studies have demonstrated that immunotherapy is a viable alternative treatment option for patients with recurrent or metastatic cancer, since the overall survival rate and progression-free survival rate were shown to be successful.

In Silico Analysis of PORD Mutations on the 3D Structure of P450 Oxidoreductase.

Cytochrome P450 oxidoreductase (POR) is a membrane-bound flavoprotein that helps in transferring electrons from its NADPH domain to all cytochrome P450 (CYP450) enzymes. Mutations in the POR gene could severely affect the metabolism of steroid hormones and the development of skeletal muscles, a condition known as Cytochrome P450 oxidoreductase deficiency (PORD). PORD is associated with clinical presentations of disorders of sex development, Antley and Bixler's syndrome (ABS), as well as an abnormal steroid hormone profile. We have performed an in silico analysis of POR 3D X-ray protein crystal structure to study the effects of reported mutations on the POR enzyme structure. A total of 32 missense mutations were identified, from 170 PORD patients, and mapped on the 3D crystal structure of the POR enzyme. In addition, five of the missense mutations (R457H, A287P, D210G, Y181D and Y607C) were further selected for an in-depth in silico analysis to correlate the observed changes in POR protein structure with the clinical phenotypes observed in PORD patients. Overall, missense mutations found in the binding sites of POR cofactors could lead to a severe form of PORD, emphasizing the importance of POR cofactor binding domains in transferring electrons to the CYP450 enzyme family.

Phytochemicals With Anti 5-alpha-reductase Activity: A Prospective For Prostate Cancer Treatment.

Prostate cancer (CaP) is one of the leading causes of death in men worldwide. Much attention has been given on its prevention and treatment strategies, including targeting the regulation of 5-alpha-Reductase (5αR) enzyme activity, aimed to limit the progression of CaP by inhibiting the conversion of potent androgen dihydrotestosterone from testosterone that is thought to play a role in pathogenesis of CaP, by using the 5-alpha-Reductase inhibitors (5αRis) such as finasteride and dutasteride. However, 5αRis are reported to exhibit numerous adverse side effects, for instance erectile dysfunction, ejaculatory dysfunction and loss of libido. This has led to a surge of interests on plant-derived alternatives that might offer favourable side effects and less toxic profiles. Phytochemicals from plants are shown to exhibit numerous medicinal properties in various studies targeting many major illnesses including CaP. Therefore, in this review, we aim to discuss on the use of phytochemicals namely phytosterols, polyphenols and fatty acids, found in various plants with proven anti-CaP properties, as an alternative herbal CaP medicines as well as to outline their inhibitory activities on 5αRs isozymes based on their structural similarities with current 5αRis as part of CaP treatment approaches.

Towards a systematic analysis of human short-chain dehydrogenases/reductases (SDR): Ligand identification and structure-activity relationships.

Short-chain dehydrogenases/reductases (SDRs) constitute a large, functionally diverse branch of enzymes within the class of NAD(P)(H) dependent oxidoreductases. In humans, over 80 genes have been identified with distinct metabolic roles in carbohydrate, amino acid, lipid, retinoid and steroid hormone metabolism, frequently associated with inherited genetic defects. Besides metabolic functions, a subset of atypical SDR proteins appears to play critical roles in adapting to redox status or RNA processing, and thereby controlling metabolic pathways. Here we present an update on the human SDR superfamily and a ligand identification strategy using differential scanning fluorimetry (DSF) with a focused library of oxidoreductase and metabolic ligands to identify substrate classes and inhibitor chemotypes. This method is applicable to investigate structure-activity relationships of oxidoreductases and ultimately to better understand their physiological roles.

Adenosine-5'-phosphosulfate--a multifaceted modulator of bifunctional 3'-phospho-adenosine-5'-phosphosulfate synthases and related enzymes.

All sulfation reactions rely on active sulfate in the form of 3'-phospho-adenosine-5'-phosphosulfate (PAPS). In fungi, bacteria, and plants, the enzymes responsible for PAPS synthesis, ATP sulfurylase and adenosine-5'-phosphosulfate (APS) kinase, reside on separate polypeptide chains. In metazoans, however, bifunctional PAPS synthases catalyze the consecutive steps of sulfate activation by converting sulfate to PAPS via the intermediate APS. This intricate molecule and the related nucleotides PAPS and 3'-phospho-adenosine-5'-phosphate modulate the function of various enzymes from sulfation pathways, and these effects are summarized in this review. On the ATP sulfurylase domain that initially produces APS from sulfate and ATP, APS acts as a potent product inhibitor, being competitive with both ATP and sulfate. For the APS kinase domain that phosphorylates APS to PAPS, APS is an uncompetitive substrate inhibitor that can bind both at the ATP/ADP-binding site and the PAPS/APS-binding site. For human PAPS synthase 1, the steady-state concentration of APS has been modelled to be 1.6 µM, but this may increase up to 60 µM under conditions of sulfate excess. It is noteworthy that the APS concentration for maximal APS kinase activity is 15 µM. Finally, we recognized APS as a highly specific stabilizer of bifunctional PAPS synthases. APS most likely stabilizes the APS kinase part of these proteins by forming a dead-end enzyme-ADP-APS complex at APS concentrations between 0.5 and 5 µM; at higher concentrations, APS may bind to the catalytic centers of ATP sulfurylase. Based on the assumption that cellular concentrations of APS fluctuate within this range, APS can therefore be regarded as a key modulator of PAPS synthase functions.

Insight into S-adenosylmethionine biosynthesis from the crystal structures of the human methionine adenosyltransferase catalytic and regulatory subunits.

MAT (methionine adenosyltransferase) utilizes L-methionine and ATP to form SAM (S-adenosylmethionine), the principal methyl donor in biological methylation. Mammals encode a liver-specific isoenzyme, MAT1A, that is genetically linked with an inborn metabolic disorder of hypermethioninaemia, as well as a ubiquitously expressed isoenzyme, MAT2A, whose enzymatic activity is regulated by an associated subunit MAT2B. To understand the molecular mechanism of MAT functions and interactions, we have crystallized the ligand-bound complexes of human MAT1A, MAT2A and MAT2B. The structures of MAT1A and MAT2A in binary complexes with their product SAM allow for a comparison with the Escherichia coli and rat structures. This facilitates the understanding of the different substrate or product conformations, mediated by the neighbouring gating loop, which can be accommodated by the compact active site during catalysis. The structure of MAT2B reveals an SDR (short-chain dehydrogenase/reductase) core with specificity for the NADP/H cofactor, and harbours the SDR catalytic triad (YxxxKS). Extended from the MAT2B core is a second domain with homology with an SDR sub-family that binds nucleotide-sugar substrates, although the equivalent region in MAT2B presents a more open and extended surface which may endow a different ligand/protein-binding capability. Together, the results of the present study provide a framework to assign structural features to the functional and catalytic properties of the human MAT proteins, and facilitate future studies to probe new catalytic and binding functions.

A structural mapping of mutations causing succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency.

Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency is a rare inherited metabolic disorder of ketone metabolism, characterized by ketoacidotic episodes and often permanent ketosis. To date there are ~20 disease-associated alleles on the OXCT1 gene that encodes the mitochondrial enzyme SCOT. SCOT catalyzes the first, rate-limiting step of ketone body utilization in peripheral tissues, by transferring a CoA moiety from succinyl-CoA to form acetoacetyl-CoA, for entry into the tricarboxylic acid cycle for energy production. We have determined the crystal structure of human SCOT, providing a molecular understanding of the reported mutations based on their potential structural effects. An interactive version of this manuscript (which may contain additional mutations appended after acceptance of this manuscript) may be found on the web address: http://www.thesgc.org/jimd/SCOT .

Structure and kinetic characterization of human sperm-specific glyceraldehyde-3-phosphate dehydrogenase, GAPDS.

hGAPDS (human sperm-specific glyceraldehyde-3-phosphate dehydrogenase) is a glycolytic enzyme essential for the survival of spermatozoa, and constitutes a potential target for non-hormonal contraception. However, enzyme characterization of GAPDS has been hampered by the difficulty in producing soluble recombinant protein. In the present study, we have overexpressed in Escherichia coli a highly soluble form of hGAPDS truncated at the N-terminus (hGAPDSΔN), and crystallized the homotetrameric enzyme in two ligand complexes. The hGAPDSΔN-NAD+-phosphate structure maps the two anion-recognition sites within the catalytic pocket that correspond to the conserved Ps site and the newly recognized Pi site identified in other organisms. The hGAPDSΔN-NAD+-glycerol structure shows serendipitous binding of glycerol at the Ps and new Pi sites, demonstrating the propensity of these anion-recognition sites to bind non-physiologically relevant ligands. A comparison of kinetic profiles between hGAPDSΔN and its somatic equivalent reveals a 3-fold increase in catalytic efficiency for hGAPDSΔN. This may be attributable to subtle amino acid substitutions peripheral to the active centre that influence the charge properties and protonation states of catalytic residues. Our data therefore elucidate structural and kinetic features of hGAPDS that might provide insightful information towards inhibitor development.

Kinetic and structural evidence of the alkenal/one reductase specificity of human ζ-crystallin.

Human ζ-crystallin is a Zn(2+)-lacking medium-chain dehydrogenase/reductase (MDR) included in the quinone oxidoreductase (QOR) family because of its activity with quinones. In the present work a novel enzymatic activity was characterized: the double bond α,ß-hydrogenation of medium-chain 2-alkenals and 3-alkenones. The enzyme is especially active with lipid peroxidation products such as 4-hydroxyhexenal, and a role in their detoxification is discussed. This specificity is novel in the QOR family, and it is similar to that described in the distantly related alkenal/one reductase family. Moreover, we report the X-ray structure of ζ-crystallin, which represents the first structure solved for a tetrameric Zn(2+)-lacking MDR, and which allowed the identification of the active-site lining residues. Docking simulations suggest a role for Tyr53 and Tyr59 in catalysis. The kinetics of Tyr53Phe and Tyr59Phe mutants support the implication of Tyr53 in binding/catalysis of alkenal/one substrates, while Tyr59 is involved in the recognition of 4-OH-alkenals.

Crystal structures of human HMG-CoA synthase isoforms provide insights into inherited ketogenesis disorders and inhibitor design.

3-Hydroxy-3-methylglutaryl coenzyme A (CoA) synthase (HMGCS) catalyzes the condensation of acetyl-CoA and acetoacetyl-CoA into 3-hydroxy-3-methylglutaryl CoA. It is ubiquitous across the phylogenetic tree and is broadly classified into three classes. The prokaryotic isoform is essential in Gram-positive bacteria for isoprenoid synthesis via the mevalonate pathway. The eukaryotic cytosolic isoform also participates in the mevalonate pathway but its end product is cholesterol. Mammals also contain a mitochondrial isoform; its deficiency results in an inherited disorder of ketone body formation. Here, we report high-resolution crystal structures of the human cytosolic (hHMGCS1) and mitochondrial (hHMGCS2) isoforms in binary product complexes. Our data represent the first structures solved for human HMGCS and the mitochondrial isoform, allowing for the first time structural comparison among the three isoforms. This serves as a starting point for the development of isoform-specific inhibitors that have potential cholesterol-reducing and antibiotic applications. In addition, missense mutations that cause mitochondrial HMGCS deficiency have been mapped onto the hHMGCS2 structure to rationalize the structural basis for the disease pathology.

A non-enzymatic function of 17beta-hydroxysteroid dehydrogenase type 10 is required for mitochondrial integrity and cell survival.

Deficiency of the mitochondrial enzyme 2-methyl-3-hydroxybutyryl-CoA dehydrogenase involved in isoleucine metabolism causes an organic aciduria with atypical neurodegenerative course. The disease-causing gene is HSD17B10 and encodes 17beta-hydroxysteroid dehydrogenase type 10 (HSD10), a protein also implicated in the pathogenesis of Alzheimer's disease. Here we show that clinical symptoms in patients are not correlated with residual enzymatic activity of mutated HSD10. Loss-of-function and rescue experiments in Xenopus embryos and cells derived from conditional Hsd17b10(-/-) mice demonstrate that a property of HSD10 independent of its enzymatic activity is essential for structural and functional integrity of mitochondria. Impairment of this function in neural cells causes apoptotic cell death whilst the enzymatic activity of HSD10 is not required for cell survival. This finding indicates that the symptoms in patients with mutations in the HSD17B10 gene are unrelated to accumulation of toxic metabolites in the isoleucine pathway and, rather, related to defects in general mitochondrial function. Therefore alternative therapeutic approaches to an isoleucine-restricted diet are required.

Three-dimensional structure and enzymatic function of proapoptotic human p53-inducible quinone oxidoreductase PIG3.

Tumor suppressor p53 regulates the expression of p53-induced genes (PIG) that trigger apoptosis. PIG3 or TP53I3 is the only known member of the medium chain dehydrogenase/reductase superfamily induced by p53 and is used as a proapoptotic marker. Although the participation of PIG3 in the apoptotic pathway is proven, the protein and its mechanism of action were never characterized. We analyzed human PIG3 enzymatic function and found NADPH-dependent reductase activity with ortho-quinones, which is consistent with the classification of PIG3 in the quinone oxidoreductase family. However, the activity is much lower than that of zeta-crystallin, a better known quinone oxidoreductase. In addition, we report the crystallographic structure of PIG3, which allowed the identification of substrate- and cofactor-binding sites, with residues fully conserved from bacteria to human. Tyr-59 in zeta-crystallin (Tyr-51 in PIG3) was suggested to participate in the catalysis of quinone reduction. However, kinetics of Tyr/Phe and Tyr/Ala mutants of both enzymes demonstrated that the active site Tyr is not catalytic but may participate in substrate binding, consistent with a mechanism based on propinquity effects. It has been proposed that PIG3 contribution to apoptosis would be through oxidative stress generation. We found that in vitro activity and in vivo overexpression of PIG3 accumulate reactive oxygen species. Accordingly, an inactive PIG3 mutant (S151V) did not produce reactive oxygen species in cells, indicating that enzymatically active protein is necessary for this function. This supports that PIG3 action is through oxidative stress produced by its enzymatic activity and provides essential knowledge for eventual control of apoptosis.

Evaluation of micro-parallel liquid chromatography as a method for HTS-coupled actives verification.

The identification of biologically active compounds from high-throughput screening (HTS) can involve considerable postscreening analysis to verify the nature of the sample activity. In this study we evaluated the performance of micro-parallel liquid chromatography (microPLC) as a separation-based enzyme assay platform for follow-up of compound activities found in quantitative HTS of two different targets, a hydrolase and an oxidoreductase. In an effort to couple secondary analysis to primary screening we explored the application of microPLC immediately after a primary screen. In microPLC, up to 24 samples can be loaded and analyzed simultaneously via high-performance liquid chromatography within a specially designed cartridge. In a proof-of-concept experiment for screen-coupled actives verification, we identified, selected, and consolidated the contents of "active" wells from a 1,536-well format HTS experiment into a 384-well plate and subsequently analyzed these samples by a 24-channel microPLC system. The method utilized 0.6% of the original 6-microl 1,536-well assay for the analysis. The analysis revealed several non-biological-based "positive" samples. The main examples included "false" enzyme activators resulting from an increase in well fluorescence due to fluorescent compound or impurity. The microPLC analysis also provided a verification of the activity of two activators of glucocerebrosidase. We discuss the benefits of microPLC and its limitations from the standpoint of ease of use and integration into a seamless postscreen workflow.

Active site variability of type 1 11beta-hydroxysteroid dehydrogenase revealed by selective inhibitors and cross-species comparisons.

The NADPH-dependent enzyme type 1 11beta-hydroxysteroid dehydrogenase (11beta-HSD1) activates in a tissue-specific manner circulating pro-glucocorticoid hormones (cortisone in humans) to the 11beta-OH ligand (cortisol in humans), which is able to bind to its cognate receptor and regulate gene transcription. Modulation of this pre-receptor activation mechanism by selective enzyme inhibitors is a desirable goal in the treatment of insulin resistance and related metabolic disorders. Like most other hydroxysteroid dehydrogenases 11beta-HSD1 belongs to the evolutionarily conserved enzyme superfamily of short-chain dehydrogenases/reductases (SDR). The enzyme is anchored within the endoplasmic reticulum through an N-terminal transmembrane domain. In this study we aimed to characterize the active site of mammalian 11beta-HSD1 by determining primary structures from several mammalian lines (cat, hamster, cynomolgus, chimpanzee, dog) thus increasing substantially available sequence information, and allowing us to determine highly variable and constant parts within the primary structure. These regions were mapped to the recently determined three-dimensional structure and are mostly found around the substrate binding site. Furthermore we performed inhibition studies by using different series of inhibitors, comprising 11beta-HSD1 selective arylsulfonamidothiazoles and the unselective steroid-based compound carbenoxolone. The different arylsulfonamidothiazoles display distinct inhibition profiles versus the mammalian species tested, with several tight binding inhibitors for the human enzyme (Ki approximately 50 nM), intermediate for mouse, and weak or not binding inhibitors for rat and guinea pig (Ki>3 microM). Analysis of the inhibition mode reveals that the tight binding inhibitor BVT.528 is a competitive inhibitor for the human form, whereas the related compound BVT.2733 displays a mixed-type inhibition pattern versus the mouse enzyme. Taken together, this structure-activity study provides increased insight into active site complexity and catalytic mechanism of 11beta-HSD1, useful for further inhibitor design.

Expanded substrate screenings of human and Drosophila type 10 17beta-hydroxysteroid dehydrogenases (HSDs) reveal multiple specificities in bile acid and steroid hormone metabolism: characterization of multifunctional 3alpha/7alpha/7beta/17beta/20beta/21-HSD.

17beta-hydroxysteroid dehydrogenases (17beta-HSDs) catalyse the conversion of 17beta-OH (-hydroxy)/17-oxo groups of steroids, and are essential in mammalian hormone physiology. At present, eleven 17beta-HSD isoforms have been defined in mammals, with different tissue-expression and substrate-conversion patterns. We analysed 17beta-HSD type 10 (17beta-HSD10) from humans and Drosophila, the latter known to be essential in development. In addition to the known hydroxyacyl-CoA dehydrogenase, and 3alpha-OH and 17beta-OH activities with sex steroids, we here demonstrate novel activities of 17beta-HSD10. Both species variants oxidize the 20beta-OH and 21-OH groups in C21 steroids, and act as 7beta-OH dehydrogenases of ursodeoxycholic or isoursodeoxycholic acid (also known as 7beta-hydroxylithocholic acid or 7beta-hydroxyisolithocholic acid respectively). Additionally, the human orthologue oxidizes the 7alpha-OH of chenodeoxycholic acid (5beta-cholanic acid, 3alpha,7alpha-diol) and cholic acid (5beta-cholanic acid). These novel substrate specificities are explained by homology models based on the orthologous rat crystal structure, showing a wide hydrophobic cleft, capable of accommodating steroids in different orientations. These properties suggest that the human enzyme is involved in glucocorticoid and gestagen catabolism, and participates in bile acid isomerization. Confocal microscopy and electron microscopy studies reveal that the human form is localized to mitochondria, whereas Drosophila 17beta-HSD10 shows a cytosolic localization pattern, possibly due to an N-terminal sequence difference that in human 17beta-HSD10 constitutes a mitochondrial targeting signal, extending into the Rossmann-fold motif.

Short-chain dehydrogenases/reductases (SDR): the 2002 update.

Short-chain dehydrogenases/reductases (SDR) form a large, functionally heterogeneous protein family presently with about 3000 primary and about 30 3D structures deposited in databases. Despite low sequence identities between different forms (about 15-30%), the 3D structures display highly similar alpha/beta folding patterns with a central beta-sheet, typical of the Rossmann-fold. Based on distinct sequence motifs functional assignments and classifications are possible, making it possible to build a general nomenclature system. Recent mutagenetic and structural studies considerably extend the knowledge on the general reaction mechanism, thereby establishing a catalytic tetrad of Asn-Ser-Tyr-Lys residues, which presumably form the framework for a proton relay system including the 2'-OH of the nicotinamide ribose, similar to the mechanism found in horse liver ADH. Based on their cellular functions, several SDR enzymes appear as possible and promising pharmacological targets with application areas spanning hormone-dependent cancer forms or metabolic diseases such as obesity and diabetes, and infectious diseases.

Comparative enzymology of 11 beta -hydroxysteroid dehydrogenase type 1 from glucocorticoid resistant (Guinea pig) versus sensitive (human) species.

Type 1 11 beta-hydroxysteroid dehydrogenase constitutes a prereceptor control mechanism through its ability to reduce dehydroglucocorticoids to the receptor ligands cortisol and corticosterone in vivo. We compared kinetic characteristics of the human and guinea pig 11 beta-hydroxysteroid dehydrogenase isozymes derived from species differing in glucocorticoid sensitivity. Both orthologs were successfully expressed as full-length enzymes in yeast and COS7 cells and as soluble transmembrane-deleted constructs in Escherichia coli. Both isozymes display Michaelis-Menten kinetics in intact cells and homogenates and show low apparent micromolar K(m) values in homogenates, which are lowered by approximately one order of magnitude in intact cells, allowing corticosteroid activation at physiological glucocorticoid levels. Recombinant soluble proteins were expressed and purified with high specific dehydrogenase and reductase activities, revealing several hundred-fold higher specificity constants than those reported earlier for the purified native enzyme. Importantly, these purified soluble enzymes also display a hyperbolic dependence of reaction velocity versus substrate concentration in 11-oxoreduction with K(m) values of 0.8 microm (human) and 0.6 microm (guinea pig), close to the values obtained from intact cells. Active site titration was carried out with the human enzyme using a novel inhibitor compound and reveals a fraction of 40-50% active sites/mol total enzyme. The kinetic data obtained argue against the involvement of 11 beta-hydroxysteroid dehydrogenase as a modulating factor for the glucocorticoid resistance observed in guinea pigs. Instead, the expression of 11 beta-hydroxysteroid dehydrogenase type 1 in the Zona glomerulosa of the guinea pig adrenal gland suggests a role of this enzyme in mineralocorticoid synthesis in this hypercortisolic species.