Molecular characterization of Alg8, a putative glycosyltransferase, involved in alginate polymerisation.
The topology of Alg8, the proposed catalytic subunit of the alginate polymerase, was assessed using PhoA and LacZ fusion protein analysis. This analysis suggested that the periplasmic loop comprises only three amino acid residues with the adjacent transmembrane helices at positions 361-387 and 393-416. Accordingly, the extended cytosolic loop could be located at positions 71-361 and was proposed to contain important catalytic residues. Further experimental evidence for this cytosolic domain was obtained by independently demonstrating this protein region as purified soluble protein domain. The soluble protein domain was identified by MALDI-TOF/MS and presumably represents the cytosolic catalytic domain of Alg8. Site-directed mutagenesis of 11 conserved residues in the cytosolic loop showed that D-188/D-190 (DXD motif), D-295/D-296 (acid-base catalysts) and K-297 were each essential for in vivo polymerase activity, whereas D-179/D-181 (DXD motif), C-244, R-263, D-279, and E-282 were not directly involved in the polymerisation reaction. The role of these amino acid residues with respect to the catalysed alginate polymerisation reaction was discussed with the aid of the recently developed structural model of Alg8.
Institute of Molecular BioSciences, Massey University, Private Bag 11222, Palmerston North, New Zealand.
Uwe
U
Remminghorst
Iain D
ID
Hay
Bernd H A
BH
Rehm
D000464:Alginates / Q000378:metabolism*; D000469:Alkaline Phosphatase / Q000378:metabolism*; D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D020723:Glucuronic Acid / Q000378:metabolism; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism; D006603:Hexuronic Acids / Q000378:metabolism; D008957:Models, Genetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D000464:Alginates; D006603:Hexuronic Acids; D011993:Recombinant Fusion Proteins; D020723:Glucuronic Acid; D016695:Glycosyltransferases; D000469:Alkaline Phosphatase; D001616:beta-Galactosidase