Mammalian Sorbitol Dehydrogenase Research Articles

A hydrogen-bonding network in mammalian sorbitol dehydrogenase stabilizes the tetrameric state and is essential for the catalytic power.

Subunit interaction in sorbitol dehydrogenase (SDH) has been studied with in vitro and in silico methods identifying a vital hydrogen-bonding network, which is strictly conserved among mammalian SDH proteins. Mutation of one of the residues in the hydrogen-bonding network, Tyr110Phe, abolished the enzymatic activity and destabilized the protein into tetramers, dimers and monomers as judged from gel filtration experiments at different temperatures compared to only tetramers for the wild-type protein below 307 K. The determined equilibrium constants revealed a large difference in Gibbs energy (8 kJ/mol) for the tetramer stability between wild-type SDH and the mutated form Tyr110Phe SDH. The results focus on a network of coupled hydrogen bonds in wild-type SDH that uphold the protein interface, which is specific and favorable to electrostatic, van der Waals and hydrogen-bond interactions between subunits, interactions that are crucial for the catalytic power of SDH.

The metabolic role and evolution of L-arabinitol 4-dehydrogenase of Hypocrea jecorina.

L-Arabinitol 4-dehydrogenase (Lad1) of the cellulolytic and hemicellulolytic fungus Hypocrea jecorina (anamorph: Trichoderma reesei) has been implicated in the catabolism of L-arabinose, and genetic evidence also shows that it is involved in the catabolism of D-xylose in xylitol dehydrogenase (xdh1) mutants and of D-galactose in galactokinase (gal1) mutants of H. jecorina. In order to identify the substrate specificity of Lad1, we have recombinantly produced the enzyme in Escherichia coli and purified it to physical homogeneity. The resulting enzyme preparation catalyzed the oxidation of pentitols (L-arabinitol) and hexitols (D-allitol, D-sorbitol, L-iditol, L-mannitol) to the same corresponding ketoses as mammalian sorbitol dehydrogenase (SDH), albeit with different catalytic efficacies, showing highest k(cat)/K(m) for L-arabinitol. However, it oxidized galactitol and D-talitol at C4 exclusively, yielding L-xylo-3-hexulose and D-arabino-3-hexulose, respectively. Phylogenetic analysis of Lad1 showed that it is a member of a terminal clade of putative fungal arabinitol dehydrogenase orthologues which separated during evolution of SDHs. Juxtapositioning of the Lad1 3D structure over that of SDH revealed major amino acid exchanges at topologies flanking the binding pocket for d-sorbitol. A lad1 gene disruptant was almost unable to grow on L-arabinose, grew extremely weakly on L-arabinitol, D-talitol and galactitol, showed reduced growth on D-sorbitol and D-galactose and a slightly reduced growth on D-glucose. The weak growth on L-arabinitol was completely eliminated in a mutant in which the xdh1 gene had also been disrupted. These data show not only that Lad1 is indeed essential for the catabolism of L-arabinose, but also that it constitutes an essential step in the catabolism of several hexoses; this emphasizes the importance of such reductive pathways of catabolism in fungi.

Sorbitol Dehydrogenase of Drosophila: GENE, PROTEIN, AND EXPRESSION DATA SHOW A TWO-GENE SYSTEM

The Drosophila melanogaster sorbitol dehydrogenase (SDH) is characterized as a two-enzyme system of the medium chain dehydrogenase/reductase family (MDR). The SDH-1 enzyme has an enzymology with Km and kcat values an order of magnitude higher than those for the human enzyme but with a similar kcat/Km ratio. It is a tetramer with identical subunits of approximately 38 kDa. At the genomic level, two genes, Sdh-1 and Sdh-2, have a single transcriptional start site and no functional TATA box. Expression is greater in larvae and adults than in pupae, where it is very low. At all three stages, Sdh-1 constitutes the major transcript. Sdh-1 and Sdh-2 genes were located at positions 84E-F and 86D in polytene chromosomes. The deduced amino acid sequences of the two genes show 90% residue identity. Evaluation of the sequence and modeling of the structure toward that of class I alcohol dehydrogenase (ADH) show altered loop and gap arrangements as in mammalian SDH and establishes that SDH, despite gene multiplicity and larger variability than the "constant" ADH of class III, is an enzyme conserved over wide ranges.

Sorbitol Metabolism in Growing Tissues of Peach

Sorbitol is the major photosynthetic product in peach. In sink tissues, sorbitol is converted to fructose via the NAD-dependent enzyme sorbitol dehydrogenase (SDH). A new assay is described that allows rapid, simple quantitation of SDH activity in growing shoot tips, root tips, and fruits. The activity was measured on the crude extract desalted with a Saphadex G-25 column to eliminate small molecules such as sugars and nucleotides. Optimum buffer type and pH for the enzyme as well as degradation by proteolytic enzymes and stability over time were determined in the present study. Inhibition by dithiothreitol (DTT) was detected at an inhibitor concentration as low as 2 mM, proving the similarity with mammalian SDH. Storage of samples at 4°C overnight resulted in significant loss of enzyme activity. Using this assay, we also correlated SDH activity with sink strength in peach.

Structure of the Bombyx sorbitol dehydrogenase gene: a possible alternative use of the promoter.

In an initial effort to understand the molecular mechanism of how low temperature induces sorbitol dehydrogenase gene expression in diapause eggs of the silkworm, the sorbitol dehydrogenase gene was isolated from a Bombyx genomic library using a cDNA encoding the Bombyx homologue of mammalian sorbitol dehydrogenase as a probe. The gene extended for about 10 kb, consisting of eight exons and seven introns. Four TATA motifs were found in the 5' upstream region of the gene, without CCAAT. AATTAA, instead of AATAAA, was localized in the upstream region of the polyadenylation site. Although a single copy of this gene was present per haploid genome, 1.2 kb and 1.1 kb transcripts were found from yolk cells in diapause eggs and from larval fat-body cells, respectively. The two major transcription initiation sites corresponding to both transcripts were localized at 355 and 226 base pairs upstream from the transition start site, indicating an alternative use of promoter. The 5'-upstream region of the gene contained a consensus sequence, TGA(A/T)AA(A/G/T), that has been found in insect genes expressed mainly in larval and pupal fat bodies. It also contained three kinds of sequences similar to cis-elements recognized by members of the steroid receptor superfamily, such as chicken ovalbumin upstream promoter transcription factor (COUP-TF)/Drosophila Seven up (SVP), Drosophila hormone receptor 39 (DHR39) and Bombyx fushi tarazu transcriptional factor 1 (BmFTZ-F1).

Developmental profile of the gene expression of a Bombyx homolog of mammalian sorbitol dehydrogenase during embryogenesis in non-diapause eggs

1. 1.|By using semi-quantitative PCR analysis, changes in the amount of the transcript for a Bombyx homolog of mammalian sorbitol dehydrogenase (BmSDH) were examined during embryogenesis in non-diapause eggs. 2. 2.|Occurrence of the transcript for BmSDH correlated with the two developmental phases, the growth of embryo and the formation of larval tissues. 3. 3.|In the first phase, an increase in the amount of the transcript for BmSDH resulted from embryonic cells rather than yolk cells. 4. 4.|In the second phase, the transcript was suggested to be abundant in fat-body cells of pharate larva, because it was abundant in fat-bodies of 5th instar larvae. 5. 5.|The difference in the expression of BmSDH gene between diapause and non-diapause eggs is discussed.

Variability within mammalian sorbitol dehydrogenases

European Journal of BiochemistryVolume 186, Issue 3 p. 550-550 Free Access Variability within mammalian sorbitol dehydrogenases The primary structure of the human liver enzyme Christina KARLSSON, Search for more papers by this authorWolfgang MARET, Search for more papers by this authorDouglas S. AULD, Search for more papers by this authorJan-Olov HÖÖG, Search for more papers by this authorHans JÖRNVALL, Search for more papers by this author Christina KARLSSON, Search for more papers by this authorWolfgang MARET, Search for more papers by this authorDouglas S. AULD, Search for more papers by this authorJan-Olov HÖÖG, Search for more papers by this authorHans JÖRNVALL, Search for more papers by this author First published: December 1989 https://doi.org/10.1111/j.1432-1033.1989.tb15241.xAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume186, Issue3December 1989Pages 550-550 RelatedInformation

Variability within mammalian sorbitol dehydrogenases. The primary structure of the human liver enzyme.

The primary structure of sorbitol dehydrogenase from human liver has been determined by peptide analysis in order to relate the variability of this enzyme to that of the others within the alcohol dehydrogenase family. The structure obtained reveals 355 residues with an acyl-blocked N-terminus and an unexpected microheterogeneity at position 237 (Gln/Leu). The residue identity between sheep and human liver sorbitol dehydrogenase is 89%. This variability is similar to that of class I alcohol dehydrogenases, but distinctly different from that of class III alcohol dehydrogenases, the structures of which are much more conserved. Consequently, class III alcohol dehydrogenase is thus far unique within this family of dehydrogenases, suggesting a particularly strict requirement for that structure. The variability within sorbitol dehydrogenase involves all segments of the molecule but is largely at surface positions and clusters in one such region, covering positions 214-240, corresponding to a segment of the coenzyme-binding domain. Ligands to the active-site zinc and most residues lining the coenzyme-binding and substrate-binding pockets are conserved. However, provided conformational models are reliable, a charge difference may affect the interactions at the inner part of the substrate pocket, another charge difference may affect the interdomain region, and a size difference the adenine pocket. The primary structure of human liver sorbitol dehydrogenase further shows that the absence of three of the four ligands to a second zinc atom present in alcohol dehydrogenases is a general property of sorbitol dehydrogenase.