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Altered Substrate Selection of the Melibiose Transporter (MelY) of Enterobacter cloacae Involving Point Mutations in Leu-88, Leu-91, and Ala-182 That Confer Enhanced Maltose Transport. Steven G. Shinnick, 2003.We isolated mutants of Escherichia coli HS4006 containing the melibiose-H+ symporter (MelY) from Enterobacter cloacae that had enhanced fermentation on 1% maltose MacConkey plates . DNA sequencing revealed three site classes of mutations: L-88-P, L-91-P, and A-182-P . The mutants L-88-P and L-91-P had 3.6- and 5.1-fold greater maltose uptake than the wild type and enhanced apparent affinities for maltose . Energy-coupled transport was defective for melibiose accumulation, but detectable maltose accumulation for the mutants indicated that active transport is dependent upon the substrate transported through the carrier . We conclude that the residues Leu-88, Leu-91 (transmembrane segment 3 [TMS-3]), and Ala-182 (TMS-6) of MelY mediate sugar selection . These data represent the first MelY mutations that confer changes in sugar selection . Probing the Catalytic Activity of a Cell Division-Specific Transpeptidase In Vivo with ß-Lactams. Christian Eberhardt, 2003.Penicillin-binding protein 3 (PBP3; also called FtsI) is a transpeptidase that catalyzes cross-linking of the peptidoglycan cell wall in the division septum of Escherichia coli . To determine whether the catalytic activity of PBP3 is activated during division, we assayed acylation of PBP3 with three ß-lactams (cephalexin, aztreonam, and piperacillin) in growing cells . Acylation of PBP3 with cephalexin, but not aztreonam or piperacillin, appeared to be stimulated by cell division . Specifically, cephalexin acylated PBP3 about 50% faster in a population of dividing cells than in a population of filamentous cells in which division was inhibited by inactivation or depletion of FtsZ, FtsA, FtsQ, FtsW, or FtsN . However, in a simpler in vitro system using isolated membranes, acylation with cephalexin was not impaired by depletion of FtsW or FtsN . A conflicting previous report that the ftsA3(Ts) allele interferes with acylation of PBP3 was found to be due to the presence of a thermolabile PBP3 in the strain used in that study . The new findings presented here are discussed in light of the hypothesis that the catalytic activity of PBP3 is stimulated by interaction(s) with other division proteins . We suggest that there might be allosteric activation of substrate binding . Purification and Properties of a Feruloyl Esterase Involved in Lignocellulose Degradation by Aureobasidium pullulans. Karl Rumbold, 2003.The lignocellulolytic fungus Aureobasidium pullulans NRRL Y 2311-1 produces feruloyl esterase activity when grown on birchwood xylan . Feruloyl esterase was purified from culture supernatant by ultrafiltration and anion-exchange, hydrophobic interaction, and gel filtration chromatography . The pure enzyme is a monomer with an estimated molecular mass of 210 kDa in both native and denatured forms and has an apparent degree of glycosylation of 48% . The enzyme has a pI of 6.5, and maximum activity is observed at pH 6.7 and 60°C . Specific activities for methyl ferulate, methyl p-coumarate, methyl sinapate, and methyl caffeate are 21.6, 35.3, 12.9, and 30.4 µmol/min/mg, respectively . The pure feruloyl esterase transforms both 2-O and 5-O arabinofuranosidase-linked ferulate equally well and also shows high activity on the substrates 4-O-trans-feruloyl-xylopyranoside, O-{5-O-[(E)-feruloyl]-  -L-arabinofuranosyl}-(1,3)-O-ß-D-xylopyranosyl-(1,4)-D-xylopyranose, and p-nitrophenyl-acetate but reveals only low activity on p-nitrophenyl-butyrate . The catalytic efficiency (kcat/Km) of the enzyme was highest on methyl p-coumarate of all the substrates tested . Sequencing revealed the following eight N-terminal amino acids: AVYTLDGD .
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