Biosynthesis and Enzymology

Carbohydrate Enzymology and Biosynthesis. Our work in this area has covered a broad range of topics and techniques. We have studied mechanisms for glycosyltransfer enzymes called sialyltransferases and trans-sialidases, using deuterium and heavy-atom kinetic isotope effects as a probative mechanistic technique to elucidate features of the transition states for these reactions. Another aspect of the work was design and synthesis of glycosidase inhibitors, based on the observation that nitrogen in the ring analogs of common sugars are often potent inhibitors. Indeed, such inhibitory compounds as exemplified by the well known deoxynojirimycin, inspired us to pursue investigation of their biosynthetic path. We have identified a three gene signature cluster consisting of a transaminase, phosphatase, and NADH dependent dehydrogenase that are capable of converting fructose-6-phosphate into mannojirimycin-6-phosphate. Present work is aimed at the enzymology of these enzymes and identification of remaining enzymes in the pathway. This three gene signature has also allowed us to identify similar pathways in other bacterial species.

  1. Wu, Y.; Arciola, J.; Horenstein, N.A. (2014) “Medium-Chain Dehydrogenases with New Specificity: Amino Mannitol Dehydrogenases on the Azasugar Biosynthetic Pathway” Prot. Pept. Lett. 21, 10-14.

  2. Clark, L.F.; Johnson, J.V.;  Horenstein, N.A.(2011) “Identification of a gene cluster that initiates azasugar biosynthesis in Bacillus amyloliquefaciens.” ChemBiochem. 12, 2147-2150.

  3. Pierdominici-Sottile, G., Horenstein N.A.; Roitberg, A.E.(2011) “Free energy study of the catalytic mechanism of Trypanosoma cruzi trans-sialidase. From the Michaelis complex to the covalent intermediate. Biochemistry. 50,10150-10158.

  4. Leonik, F.M.; Ghiviriga,I.; and Horenstein,N.A. (2010) ”Synthesis of 3,5-diazabicyclo[5.1.0]octenes. A new platform to mimic glycosidase transition states.”Tetrahedron, 66 (30) 5566-5572.

  5. Sun,H.; Abboud,K.A., Horenstein, N.A. (2005) “A new route into hexahydro-cyclopenta[b]pyrrole-cis-3a,6-diols. Synthesis of constrained bicyclic analogues of pyrrolidine azasugars.”Tetrahedron, 61 10462-10469.

  6. Burke, E. and Horenstein, N.A. (2004) Enzymatic Synthesis of [1-14C-N-acetyl, P18O2] Cytidine Monophosphate Neuraminic Acid” J. Label. Compd. Radiopharm. 47, 1007-1017.

  7. Horenstein,B.A.; Bruner,M. (2002)”Characterization of α(26)-sialyltransferase reaction intermediates: Use of alternative substrates to unmask kinetic isotope effects. Meth. Enzymol., 354, 159-168.

  8. Yang,J.; Schenkman,S.; Horenstein, B.A.(2000)”Primary13C and β-secondary 2H KIEs for Trans- sialidase. A snapshot of nucleophilic participation during catalysis.” Biochemistry, 39, 5902-5910.