1.Bioproduction of D-Tagatose from D-Galactose Using Phosphoglucose Isomerase from Pseudomonas aeruginosa PAO1.
Patel MJ1, Patel AT1, Akhani R1, Dedania S1, Patel DH2. Appl Biochem Biotechnol. 2016 Feb 27. [Epub ahead of print]
Pseudomonas aeruginosa PAO1 phosphoglucose isomerase was purified as an active soluble form by a single-step purification using Ni-NTA chromatography that showed homogeneity on SDS-PAGE with molecular mass ∼62 kDa. The optimum temperature and pH for the maximum isomerization activity with D-galactose were 60 °C and 7.0, respectively. Generally, sugar phosphate isomerases show metal-independent activity but PA-PGI exhibited metal-dependent isomerization activity with aldosugars and optimally catalyzed the D-galactose isomerization in the presence of 1.0 mM MnCl2. The apparent Km and Vmax for D-galactose under standardized conditions were calculated to be 1029 mM (±31.30 with S.E.) and 5.95 U/mg (±0.9 with S.E.), respectively. Equilibrium reached after 180 min with production of 567.51 μM D-tagatose from 1000 mM of D-galactose. Though, the bioconversion ratio is low but it can be increased by immobilization and enzyme engineering. Although various L-arabinose isomerases have been characterized for bioproduction of D-tagatose, P.
2.ATP-binding Cassette (ABC) Transport System Solute-binding Protein-guided Identification of Novel d-Altritol and Galactitol Catabolic Pathways in Agrobacterium tumefaciens C58.
Wichelecki DJ1, Vetting MW2, Chou L1, Al-Obaidi N2, Bouvier JT1, Almo SC2, Gerlt JA3. J Biol Chem. 2015 Nov 27;290(48):28963-76. doi: 10.1074/jbc.M115.686857. Epub 2015 Oct 15.
Innovations in the discovery of the functions of uncharacterized proteins/enzymes have become increasingly important as advances in sequencing technology flood protein databases with an exponentially growing number of open reading frames. This study documents one such innovation developed by the Enzyme Function Initiative (EFI; U54GM093342), the use of solute-binding proteins for transport systems to identify novel metabolic pathways. In a previous study, this strategy was applied to the tripartite ATP-independent periplasmic transporters. Here, we apply this strategy to the ATP-binding cassette transporters and report the discovery of novel catabolic pathways for d-altritol and galactitol in Agrobacterium tumefaciens C58. These efforts resulted in the description of three novel enzymatic reactions as follows: 1) oxidation of d-altritol to d-tagatose via a dehydrogenase in Pfam family PF00107, a previously unknown reaction; 2) phosphorylation of d-tagatose to d-tagatose 6-phosphate via a kinase in Pfam family PF00294, a previously orphan EC number; and 3) epimerization of d-tagatose 6-phosphate C-4 to d-fructose 6-phosphate via a member of Pfam family PF08013, another previously unknown reaction.
3.Novel mode of inhibition by D-tagatose 6-phosphate through a Heyns rearrangement in the active site of transaldolase B variants.
Stellmacher L1, Sandalova T2, Schneider S1, Schneider G3, Sprenger GA1, Samland AK1. Acta Crystallogr D Struct Biol. 2016 Apr 1;72(Pt 4):467-76. doi: 10.1107/S2059798316001170. Epub 2016 Mar 24.
Transaldolase B (TalB) and D-fructose-6-phosphate aldolase A (FSAA) from Escherichia coli are C-C bond-forming enzymes. Using kinetic inhibition studies and mass spectrometry, it is shown that enzyme variants of FSAA and TalB that exhibit D-fructose-6-phosphate aldolase activity are inhibited covalently and irreversibly by D-tagatose 6-phosphate (D-T6P), whereas no inhibition was observed for wild-type transaldolase B from E. coli. The crystal structure of the variant TalB(F178Y) with bound sugar phosphate was solved to a resolution of 1.46 Å and revealed a novel mode of covalent inhibition. The sugar is bound covalently via its C2 atom to the ℇ-NH2 group of the active-site residue Lys132. It is neither bound in the open-chain form nor as the closed-ring form of D-T6P, but has been converted to β-D-galactofuranose 6-phosphate (D-G6P), a five-membered ring structure. The furanose ring of the covalent adduct is formed via a Heyns rearrangement and subsequent hemiacetal formation.
4.Biocatalytic Production of D-Tagatose: A Potential Rare Sugar with Versatile Applications.
J J1, P G1, G S1, M C1. Crit Rev Food Sci Nutr. 2016 Jan 8:0. [Epub ahead of print]
D-Tagatose is a naturally existing rare monosaccharide having prebiotic properties. Minimal absorption, low metabolizing energy and unique clinical properties are the characteristics of D-tagatose. D-Tagatose gained international attention by matching the purpose of alternate sweeteners that is much needed for the control of diabetes among world population. Recent efforts in understanding tagatose bioconversion has generated essential information regarding its production and application. This article reviews the evolution of D-Tagatose as an important rare sugar by appreciable improvements in production results and its significant applications resulted of its unique physical, chemical, biological and clinical properties thus considering it an appropriate product for requisite improvements in technical viability. Based on current knowledge and technology projections, the commercialization of D-Tagatose rare sugar as food additive is close to reality.