Structural role of conserved Asn179 in the short-chain dehydrogenase/reductase scaffold.

Short-chain dehydrogenases/reductases (SDR) constitute a large family of enzymes found in all forms of life. Despite a low level of sequence identity, the three-dimensional structures determined display a nearly superimposable alpha/beta folding pattern. We identified a conserved asparagine residue located within strand betaF and analyzed its role in the short-chain dehydrogenase/reductase architecture. Mutagenetic replacement of Asn179 by Ala in bacterial 3beta/17beta-hydroxysteroid dehydrogenase yields a folded, but enzymatically inactive enzyme, which is significantly more resistant to denaturation by guanidinium hydrochloride. Crystallographic analysis of the wild-type enzyme at 1.2-A resolution reveals a hydrogen bonding network, including a buried and well-ordered water molecule connecting strands betaE to betaF, a common feature found in 16 of 21 known three-dimensional structures of the family. Based on these results, we hypothesize that in mammalian 11beta-hydroxysteroid dehydrogenase the essential Asn-linked glycosylation site, which corresponds to the conserved segment, displays similar structural features and has a central role to maintain the SDR scaffold.

Forms and functions of human SDR enzymes.

Short-chain dehydrogenases/reductases (SDR) are defined by distinct, common sequence motifs but constitute a functionally heterogenous superfamily of enzymes. At present, well over 1600 members from all forms of life are annotated in databases. Using the defined sequence motifs as queries, 37 distinct human members of the SDR family can be retrieved. The functional assignments of these forms fall minimally into three main groups, enzymes involved in intermediary metabolism, enzymes participating in lipid hormone and mediator metabolism, and open reading frames (ORFs) of yet undeciphered function. This overview, prepared just before completion of the human genome project, gives the different human SDR forms and relates them to human diseases.

Subcellular targeting analysis of SDR-type hydroxysteroid dehydrogenases.

Most mammalian hydroxysteroid dehydrogenases known thus far belong to the protein superfamilies of short-chain dehydrogenases/reductases (SDR) and aldo-keto reductases (AKR). Whereas members of the AKR family are soluble, cytoplasmic enzymes, SDR-type hydroxysteroid dehydrogenases are also located to other subcellular compartments, i.e. endoplasmic reticulum, mitochondria or peroxisomes. Differential localization might play an important role in influencing the reaction direction of hydroxy dehydrogenase/oxo reductase pathways by determining the available nucleotide cofactor pool. Targeting signals for different subcellular organelles in human hydroxysteroid dehydrogenases have been identified, however, in several enzymes localization signals remain to be determined.

Lack of quinone reductase activity suggests that amyloid-beta peptide/ERAB induced lipid peroxidation is not directly related to production of reactive oxygen species by redoxcycling.

Mitochondrial type II hydroxyacyl-CoA dehydrogenase (ERAB) has recently been shown to mediate amyloid-beta peptide (Abeta) induced apoptosis and neurodegeneration. The precise mechanism of cell death induction is unknown, however, Abeta inhibits ERAB activities and as a result of ERAB-Abeta interactions, enhanced formation of lipid peroxidation products occur. The possibility that ERAB mediates quinone reduction is therefore investigated, thus giving the potential of redoxcycling and production of reactive oxygen species, leading to lipid peroxidation. Recombinant human ERAB was produced in a bacterial expression system and enzymological properties were evaluated. Using several orthoquinones as substrates, no ERAB mediated quinone reductase activity was found either in the presence or absence of Abeta, suggesting that the observed in vivo lipid peroxidation is a result of other mechanisms than redoxcycling by quinones.

Active site directed mutagenesis of 3 beta/17 beta-hydroxysteroid dehydrogenase establishes differential effects on short-chain dehydrogenase/reductase reactions.

Mutagenetic replacements of conserved residues within the active site of the short-chain dehydrogenase/reductase (SDR) superfamily were studied using prokaryotic 3 beta/17 beta-hydroxysteroid dehydrogenase (3 beta/17 beta-HSD) from Comamonas testosteroni as a model system. The results provide novel data to establish Ser 138 as a member of a catalytically important "triad" of residues also involving Tyr151 and Lys155. A Ser-->Ala exchange at position 138 results in an almost complete (> 99.9%) loss of enzymatic activity, which is not observed with a Ser-->Thr replacement. This indicates that an essential factor for catalysis is the ability of side chain 138 to form hydrogen bond interactions. Mutations in the NAD(H) binding region, in strands beta A, beta D, and adjacent turns, reveal two additional residues, Thr12 and Asn87, which are important for correct binding of the coenzyme and with a differential effect on the reactions catalyzed. Thus, mutation of Thr12 to Ala results in a complete loss of the 3 beta-dehydrogenase activity, whereas the 3-oxoreductase activity remains unchanged. On the other hand, a T12S substitution yields a protein with unaltered catalytic constants for both reactions, revealing that a specific hydrogen bond is critical for the dehydrogenase activity. Our interpretation of the available crystal structure of 3 alpha/20 beta-HSD from Streptomyces hydrogenans suggests a hydrogen bond in that enzyme between the Thr12 side chain and the backbone NH of Asn87 rather than the coenzyme, indicating that this hydrogen bond to the beta D strand might determine a crucial difference between the reductive and the oxidative reaction types. Similarly, mutation of Asn87 to Ala results in an 80% reduction of kcat/Km in the dehydrogenase direction but also unchanged 3-oxoreductase properties. It appears that the binding of NAD+ to the protein is influenced by local structural changes involving strand beta D and turn beta A to alpha B.

Data discrepancies and their origins. An evaluation of a family practice residency program using a naturalistic inquiry paradigm.

The results of an evaluation of a family practice residency program are presented. The program evaluation employed a naturalistic inquiry paradigm and a variety of qualitative methods to ensure credibility of results. An important outcome of the data collection and analysis was the identification of significant discrepancies between data collected from interviews with residents and from direct observations of a variety of the educational components of the program. Two major explanations are proposed for interpreting these discrepancies: variations in standards and variations in definition of goals and clients. The discussion supports the position that naturalistic inquiry promotes expansionist rather than reductionist outcomes, which are particularly appropriate for formative program evaluation goals.

Multiple strategies for studying medical clerkship experiences: a case study.

As part of a study reported here, the authors explored the utility of combining information generated by using various methodological approaches to study medical clerkship education. The clerkship consisted of two six-week rotations on two separate medical services. Three trained observers followed three third-year medical students for six consecutive days during the first and last week of each rotation. Observers went everywhere with students during each observational period, including 24-hour on-call days. The medical students also kept diaries of their daily experience during the study period. Diary data were combined with data from field observations and interviews to illustrate the enhanced understanding gained from a multiple-strategy approach.

Research approaches in health professions education: problems and prospects.

Research in health professions education has been dominated by a research paradigm emphasizing controlled experimentation. Widespread criticism has been voiced by leaders in the research community concerning the adequacy of the paradigm to deal with complex human behavior occurring in complex social environments. This article is concerned with exploring the implications of controlled studies for health professions education and proposing an alternative research strategy.