title: Functional assignment of gene AAC16202.1 from Rhodobacter capsulatus SB1003: new insights into the bacterial SDR sorbitol dehydrogenases family.
abstract: Short-chain dehydrogenases/reductases (SDR) constitute one of the largest enzyme superfamilies with over 60,000 non-redundant sequences in the database, many of which need a correct functional assignment. Among them, the gene AAC16202.1 (NCBI) from Rhodobacter capsulatus SB1003 has been assigned in Uniprot both as a sorbitol dehydrogenase (#D5AUY1) and, as an N-acetyl-d-mannosamine dehydrogenase (#O66112), both enzymes being of biotechnological interest. When the gene was overexpressed in Escherichia coli Rosetta (DE3)pLys, the purified enzyme was not active toward N-acetyl-d-mannosamine, whereas it was active toward d-sorbitol and d-fructose. However, the relative activities toward xylitol and l-iditol (0.45 and 6.9%, respectively) were low compared with that toward d-sorbitol. Thus, the enzyme could be considered sorbitol dehydrogenase (SDH) with very low activity toward xylitol, which could increase its biotechnological interest for determining sorbitol without the unspecific cross-determination of added xylitol in food and pharma compositions. The tetrameric enzyme (120 kDa) showed similar catalytic efficiency (2.2 × 10(3) M(-1) s(-1)) to other sorbitol dehydrogenases for d-sorbitol, with an optimum pH of 9.0 and an optimum temperature of 37 °C. The enzyme was also more thermostable than other reported SDH, ammonium sulfate being the best stabilizer in this respect, increasing the melting temperature (T(m)) up to 52.9 °C. The enzyme can also be considered as a new member of the Zn(2+) independent SDH family since no effect on activity was detected in the presence of divalent cations or chelating agents. Finally, its in silico analysis enabled the specific conserved sequence blocks that are the fingerprints of bacterial sorbitol dehydrogenases and mainly located at C-terminal of the protein, to be determined for the first time. This knowledge will facilitate future data curation of present databases and a better functional assignment of newly described sequences.
authors: Department of Biochemistry and Molecular Biology-A, Faculty of Biology, Regional Campus of International Excellence Campus Mare Nostrum, University of Murcia, Campus Espinardo, E-30100 MURCIA, Spain.
authors: Agustín
authors: A
authors: Sola-Carvajal
authors: María Inmaculada
authors: MI
authors: García-García
authors: Guiomar
authors: G
authors: Sánchez-Carrón
authors: Francisco
authors: F
authors: García-Carmona
authors: Alvaro
authors: A
authors: Sánchez-Ferrer
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D019295:Computational Biology; D006595:Hexosamines / Q000378:metabolism; D007700:Kinetics; D007064:L-Iditol 2-Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D016354:Rhodobacter capsulatus / Q000201:enzymology* / Q000235:genetics*; D016415:Sequence Alignment; D013012:Sorbitol / Q000378:metabolism
chemical_list: D006595:Hexosamines; D013012:Sorbitol; D007064:L-Iditol 2-Dehydrogenase; C002022:N-acetylmannosamine