A chemoenzymatic route to mannosamine derivatives bearing different N-acyl groups.
The chemoenzymatic route to 2-deoxy-2-propionamido-D-mannose (1b), 2-butyramido-2-deoxy-D-mannose (2b) and 2-deoxy-2-phenylacetamido-D-mannose (3b) involved N-acylation of 2-amino-2-deoxy-D-glucose followed by alkaline C-2 epimerization and selective microbial removal of the epimers with gluco-configuration. The latter step employed whole cells of Rhodococcus equi A4 able to degrade 2-deoxy-2-propionamido-D-glucose (1a), 2-butyramido-2-deoxy-D-glucose (2a) and 2-deoxy-2-phenylacetamido-D-glucose (3a) but inactive towards the corresponding manno-isomers. The metabolism of the gluco-isomers probably involved phosphorylation and subsequent deacylation. 2-Acetamido-2-deoxy-6-O-phospho-D-glucose amidohydrolase [EC 3.5.1.25] but not 2-acetamido-2-deoxy-D-glucose amidohydrolase was detected in the cell extract, the former enzyme being partially purified (15.8-fold with an overall yield of 18.1% and a specific activity of 0.95 units mg-1 protein). According to SDS-PAGE electrophoresis, gel filtration and mass spectrometry, the enzyme was a monomer with an apparent molecular mass of approximately 42 kDa. The optimum temperature and pH of the enzyme were 60 degrees C and 8.0-9.0, respectively. 2-Acetamido-2-deoxy-6-O-phospho-D-glucose and 2-acetamido-2-deoxy-6-O-sulfo-D-glucose but not 2-acetamido-2-deoxy-1-O-phospho-D-glucose or 2-acetamido-2-deoxy-D-glucose were substrates of the enzyme. Its activity was slightly inhibited by the addition of 1 mM Al3+, Ca2+, Co2+, Cu2+, Mn2+ or Zn2+ and activated by 1 mM Mg2+. The concentrated enzyme is highly stable at 4 degrees C in the presence of 0.1 M ammonium sulfate.
Kristová
Veronika
V
Institute of Microbiology, Academy of Sciences of the Czech Republic, Laboratory of Biotransformation, Vídenská 1083, CZ-142 20 Prague 4, Czech Republic.
Martínková
Ludmila
L
Husáková
Lucie
L
Kuzma
Marek
M
Rauvolfová
Jana
J
Kavan
Daniel
D
Pompach
Petr
P
Bezouska
Karel
K
Kren
Vladimír
V
D000215:Acylation; D000581:Amidohydrolases / Q000378:metabolism*; D004789:Enzyme Activation; D004795:Enzyme Stability; D005947:Glucose / Q000378:metabolism*; D006595:Hexosamines / Q000096:biosynthesis*; D006863:Hydrogen-Ion Concentration; D016929:Rhodococcus equi / Q000201:enzymology*; D013696:Temperature
D006595:Hexosamines; C008322:mannosamine; D000581:Amidohydrolases; D005947:Glucose