Amino acid sequence of the cold-active alkaline phosphatase from Atlantic cod (Gadus morhua).
Atlantic cod is a marine fish that lives at low temperatures of 0-10 degrees C and contains a cold-adapted alkaline phosphatase (AP). Preparations of AP from either the lower part of the intestines or the pyloric caeca area were subjected to proteolytic digestion, mass spectrometry and amino acid sequencing by Edman degradation. The primary structure exhibits greatest similarity to human tissue non-specific AP (80%), and approximately 30% similarity to AP from Escherichia coli. The key residues required for catalysis are conserved in the cod AP, except for the third metal binding site, where cod AP has the same variable residues as mammalian APs (His153 and His328 by E. coli AP numbering). General comparison of the amino acid composition with mammalian APs showed that cod AP contains fewer Cys, Leu, Met and Ser, but proportionally more Asn, Asp, Ile, Lys, Trp and Tyr residues. Three N-linked glycosylation sites were found. The glycan structure was determined as complex biantennary in type with fucose and sialic acid attached, although a trace of complex tri-antennary structure was also observed. A three-dimensional model was obtained by homology modelling using the human placental AP scaffold. Cod AP has fewer charged and hydrophobic residues, but more polar residues at the intersubunit surface. The N-terminal helix arm that embraces the second subunit in dimeric APs may be more flexible due to a replaced Pro at its base. One disulfide bridge was found instead of the two present in most other APs. This may invoke greater movement in the structure that together with weaker subunit contacts leads to improved catalytic efficiency.
Asgeirsson
Bjarni
B
Department of Chemistry, Science Institute, University of Iceland, Dunhaga 3, Reykjavik IS-107, Iceland. bjarni@raunvis.hi.is
Nielsen
Berit Noesgaard
BN
Højrup
Peter
P
D000222:Adaptation, Physiological; D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D003080:Cold Temperature; D019143:Evolution, Molecular; D005399:Fishes / Q000235:genetics*; D041981:Gastrointestinal Tract / Q000201:enzymology; D006031:Glycosylation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry; D020539:Sequence Analysis, Protein; D017385:Sequence Homology; D040681:Structural Homology, Protein
D011134:Polysaccharides; D000469:Alkaline Phosphatase