Construction and analyses of hybrid Azotobacter vinelandii mannuronan C-5 epimerases with new epimerization pattern characteristics.
The secreted mannuronan C-5 epimerases from Azotobacter vinelandii form a family of seven homologous modular type enzymes, which appear to have evolved through duplications and point mutations in the individual modules. The catalytic A modules of these enzymes are responsible for generating the characteristic sequence distribution patterns of G residues in the industrially important polymer alginate by epimerizing M (beta-D-mannuronic acid) moieties to G (alpha-L-guluronic acid). Forty-six different hybrid enzymes were constructed by exchanging parts of the sequences encoding the A modules of AlgE2 (generates consecutive stretches of G residues) and AlgE4 (generates alternating structures). These hybrid enzymes introduce a variety of new monomer-sequence patterns into their substrates, and some regions important for the subsite specificity or processivity of the enzymes were identified. By using time-resolved NMR spectroscopy, it became clear that the rates for introducing alternating structures and consecutive stretches of G residues are different for each enzyme, and that it is the ratio between these rates that determines the overall epimerization pattern. These findings open up new possibilities in biotechnology and in studies of the many biological functions of alginates.
Bjerkan
Tonje M
TM
Department of Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway.
Lillehov
Bjørn E
BE
Strand
Wenche I
WI
Skjåk-Braek
Gudmund
G
Valla
Svein
S
Ertesvåg
Helga
H
D000595:Amino Acid Sequence / Q000235:genetics; D016948:Azotobacter vinelandii / Q000201:enzymology* / Q000235:genetics; D002238:Carbohydrate Epimerases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D005998:Glycine / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000235:genetics
D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D002238:Carbohydrate Epimerases; C086519:mannuronan c-5-epimerase; D005998:Glycine