DSD – An integrated, web-accessible database of Dehydrogenase Enzyme Stereospecificities
© Toseland et al. 2005
Received: 27 June 2005
Accepted: 30 November 2005
Published: 30 November 2005
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© Toseland et al. 2005
Received: 27 June 2005
Accepted: 30 November 2005
Published: 30 November 2005
Dehydrogenase enzymes belong to the oxidoreductase class and utilise the coenzymes NAD and NADP. Stereo-selectivity is focused on the C4 hydrogen atoms of the nicotinamide ring of NAD(P). Depending upon which hydrogen is transferred at the C4 location, the enzyme is designated as A or B stereospecific.
The Dehydrogenase Stereospecificity Database v1.0 (DSD) provides a compilation of enzyme stereochemical data, as sourced from the primary literature, in the form of a web-accessible database. There are two search engines, a menu driven search and a BLAST search. The entries are also linked to several external databases, including the NCBI and the Protein Data Bank, providing wide background information. The database is freely available online at: http://www.jenner.ac.uk/DSD/
DSD is a unique compilation available on-line for the first time which provides a key resource for the comparative analysis of reductase hydrogen transfer stereospecificity. As databases increasingly form the backbone of science, largely complete databases such as DSD, are a vital addition.
The structure of NAD(P) allows presentation of either hydrogen depending on the orientation of the nicotinamide ring, which can be bound in either syn- or anti- conformations, which are related by a 180 degree rotation about the glycosidic bond linking the nicotinamide base to the C1 of ribose  (Figure 1b). There have been several designations for the C4 hydrogens since the 1950's, however the approved systems are A/B  and pro R/pro S . The pro R/pro S system is superior to A/B as it denotes the absolute configuration of the atom. The A/B system works by comparing enzyme stereo-selectivity with that of yeast alcohol dehydrogenase, which is an A stereospecific enzyme. Enzymes with the same stereospecificity are designated as A, while other enzymes are denoted as B stereospecific . For comparison between the two systems, A is essentially the same as pro R and B is the same as pro S .
Fisher  was the first to observe these stereospecific reactions. It was shown that yeast alcohol dehydrogenase catalysed the hydride transfer between the substrate and NAD. Further studies were able to show that such stereospecificity was not unique to alcohol dehydrogenases; the same hydride transfer was seen in malate  and lactate  dehydrogenases. However, it was not until 1955 that B stereospecific enzymes were discovered: β-hydroxysteroid dehydrogenase  and transhydrogenase . These initial investigations were conducted using mass spectroscopy, with deuterium-labelled coenzymes . Tritium-labelled coenzymes were later used with scintillation counting [11, 12]. The C4 location of the nicotinamide ring is labelled and the isotope level determined . Both of these techniques were not adequate for large-scale studies, as they are laborious, time-consuming and require multiple purification steps . In 1976, Arnold  introduced a technique based on 1H Nuclear Magnetic Resonance (NMR) spectroscopy. This method was accurate and allowed direct measurement of deuterium content at the reaction site, as well as being safer and more facile than previous techniques.
The determination of stereospecificity is essential for gaining an understanding of the reaction mechanism of an enzyme. Most important of all, is the fact that knowledge of the stereochemistry based on hydride transfer can provide an insight into the relative substrate and coenzyme binding orientations; this can be instrumental in designing strategies to control and regulate reactions . We have therefore accumulated a comprehensive compendium of hydride transfer stereospecificity data, and placed the information in a searchable database. The Dehydrogenase Stereospecificity Database (DSD) v1.0 database designates enzymes as either A or B stereospecific, for a given coenzyme. Two decades ago, You  compiled available stereospecificity data in a landmark review; we have used this and added experimental data determined during the past 20 years, to bring the compilation up to date. This provides a unique and accurate collection of dehydrogenase enzyme stereospecificities.
The data was sourced from a previous compilation  and exhaustive searching of the primary literature. The archive consists of entries containing the enzyme name, the species from which it was derived, experimental technique, coenzymes and the stereospecificity. For simplicity, DSD utilises the A/B naming system , which was adopted by You .
Contents of the database entries.
The relevant protein and provides a link to the NCBI Entrez-Protein sequence
PDB identification code, plus a link to the equivalent structure
The Enzymes Commissions identification number, plus a link to the external database
Species in which the protein is found
The coenzyme used in the experimental determination e.g. NAD(P)
Given according to the A/B nomenclature
Experiment techniques used to obtain data e.g. NMR
Temperature at which the experiment was carried out
Range or fixed pH at which the experiment were carried out
Concentrations, etc used in the experiment
Full literature reference with link to the PubMed database
The usefulness of a database is governed by the accuracy of the data it contains. The data contained in DSD v1.0 was compiled manually from previously published, peer-reviewed articles, and verified, where possible, from the original literature. This suggests that, compared to some other databases, DSD will be accurate and reliable. Moreover, to maintain this accuracy, data was only added from the primary literature for widely accepted experimental techniques, such as NMR spectroscopy. Experimental conditions have also been included, with 113 temperature measurements, and 117 pH values, and 135 concentration measurements. As logistical considerations preclude us from undertaking independent verification of the data, we are obliged to trust the data reported in the literature.
The alternative search interface is based on BLAST . A local database of protein sequences found in DSD was compiled from SWISS-PROT  and an additional postgreSQL table was created to hold this data. The local database is searched using the NCBI BLASTP and BLASTX programs , allowing for input of protein or nucleotide sequences. The HTML Front-End connects to a web server-based PL/CGI scripts which interacts with the BLASTP or BLASTX programs. In the output, DSD entries are linked to SWISS-PROT  using accession codes.
Overview of the DSD data.
A-stereospecific enzymes may have evolved before B-stereospecific enzymes, since A-stereospecific enzyme substrates are smaller and less complex compounds than those of B-stereospecific enzymes . Prebiotic chemical processes would favour simpler molecules, while primitive organisms would have had rudimentary catabolic systems able to derive energy from small molecules. Due to structural conservation, it is also possible to deduce evolutionary correlations between diverse catalysts by comparing the binding orientations of the coenzymes, substrate and enzyme active site . On this basis, Benner [19, 20] postulated that A stereospecific enzymes will bind NAD(P) with the ring in the anti-conformation, while B stereospecific enzymes will bind NAD(P) with the ring in the syn-conformation (Figure 1b). With respect to dehydrogenase enzymes, which have divergently evolved from a common precursor, the stereospecificity will be maintained for as long as the protein fold of the catalytic domain is conserved, for a given substrate . An A to B transformation in stereospecificity, or vice versa, would require a total rearrangement of the essential functional residues, to allow the correct binding orientation. This is based on the structural constraints mentioned above and therefore such an exchange is inconceivable during divergent evolution . Binding constraints may not be an overall determinant, as several groups of enzymes from different sources having contrasting stereospecificities .
A relationship between the stereospecificity and equilibrium constant of a hydride transfer has also been proposed: reactions with pKeq > 11.3 correlate with A stereospecificity and reactions with pKeq <11.3 correlate with B stereospecificity. However, as with many postulated generalisations, there are exceptions, such as 3α-hydroxysteroid dehydrogenase. The enzyme reaction has a pKeq ~ 8, within the B range yet it is A stereospecific . This highlights the problems associated with forming generalisations about these reactions.
With respect to future work, the database needs to be maintained and developed further, ensuring our links to external databases remain current and newly published data is added. Initially, as with all databases, random errors will have occurred due to human error during the data accumulation or will be extant within the original experimental data. The database will be assessed for errors and inconsistencies, thus maintaining, as far as possible, the overall veracity of our data. Moreover, feedback on the search interfaces and the general infrastructure will allow us to address issues raised and revise the database accordingly.
You  included data on several other pyridine nucleotide coenzymes, such as 3-acetylpyridine adenine Dinucleotide ((3AcPy)AD), 3-cyanopyridine adenine Dinucleotide ((CNPy)AD), thionicotinamide adenine dinucleotide ((TN)AD) and flavin adenine dinucleotide (FAD). We will seek to incorporate data on these and similar coenzymes in future versions of the database. Likewise, we will seek to enhance search capabilities within the database by including the ability to search for types of interaction sets of amino acids within user-defined distances of NAD(P) coenzymes. We will complement this with a simple visual summary.
The DSD v1.0 database is a unique, up to date compilation of dehydrogenase stereospecificities, which has advanced on reviews put together over 20 years ago. We see the database as being relatively small, due to the constraints of information, but largely complete. As new data becomes available, the database will increase in size. The ease of access to the data is of great importance and the bespoke search system and the inclusion of a BLAST search greatly facilitates this. The addition of the cross-references to several external databases provides expansive background information. We hope the database will provide an important resource which will help enhance our understanding of enzyme mechanisms. In an age when databases are increasingly forming the backbone of science, largely complete databases, such as DSD, are an important addition.
The database is available at http://www.jenner.ac.uk/DSD/ suitable for most graphical web browser.
We should like to thank Andrew Worth for his technical assistance and Martin Blythe for help with programming. The Edward Jenner Institute for Vaccine Research wishes to thank its sponsors: GlaxoSmithKline, the Medical Research Council, the Biotechnology and Biological Sciences Research Council, and the UK Department of Health.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.