PubMed · 8688632
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L Katz, S Donadio. 1995. Macrolides.. https://doi.org/10.1016/b978-0-7506-9095-9.50023-x
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The role of various residues in the conserved structural elements of the Actinomadura R39 penicillin-sensitive dd-peptidase has been studied by site-directed mutagenesis. Replacement of Ser-298 of the 'SDN loop' by Ala or Gly significantly decreased the kcat/Km value for the peptide substrate, but only by a factor of 15 and had little effect on the other catalytic properties. Mutations of Asn-300 of the same loop and of Lys-410 of the KTG triad yielded very unstable proteins. However, the N300S mutant could be purified as a fusion protein with thioredoxin that exhibited decreased rates of acylation by the peptide substrate and various cephalosporins. Similar fusion proteins obtained with the N300A, K410H and K410N mutants were unstable and their catalytic and penicillin-binding properties were very strongly affected. In transpeptidation reactions, the presence of the acceptor influenced the kcat/Km values, which suggested a catalytic pathway more complex than a simple partition of the acyl-enzyme between hydrolysis and aminolysis. These results are compared with those obtained with two other penicillin-sensitive enzymes, the Streptomyces R61 dd-peptidase and Escherichia coli penicillin-binding protein (PBP) 5.
Alpha-mannosidase was isolated from the culture liquid of Oerskovia sp. The purified enzyme had a molecular mass of 480 kDa and comprises four identical subunits. The enzyme cleaves bonds in side chains of yeast mannan (Km = 0.08 mM, k(cat) = 1.02 micromol x min(-1) x mg(-1)) and reveals a low activity towards p-nitrophenyl alpha-D-mannopyranoside. The alpha-mannosidase is a Ca2+-dependent enzyme and is inhibited by EDTA. The enzyme possess no endo-mannosidase activity releasing only mannose in the reaction with the inversion of anomeric configuration and could be classified as exo-alpha-mannanase. The enzyme revealed a high deglycosylating activity towards the short mannose-rich O-linked carbohydrate chains of glycoproteins.