Structural analysis of periplasmic carbonic anhydrase 1 of Chlamydomonas reinhardtii.
Periplasmic carbonic anhydrase 1 of the unicellular green alga Chlamydomonas reinhardtii is a heterotetrameric glycoprotein consisting of two large subunits and two small subunits [Kamo, K., Shimogawara, K., Fukuzawa, H., Muto, S. & Miyachi, S. (1990) Eur. J. Biochem. 192, 557-562]. The cDNA sequence showed that the two subunits are cotranslated as a 377-amino-acid precursor polypeptide (41.6 kDa) consisting of an N-terminal 20-amino-acid signal peptide, a large subunit (35.6 kDa) and a small subunit (4.1 kDa) [Fukuzawa, H., Fujiwara, S., Yamamoto, Y., DionisioSese, M. L. & Miyachi, S. (1990) Proc. Natl Acad. Sci. USA 87, 4383-4387]. In the present study, amino-acid-sequence analysis of the carbonic anhydrase 1 was carried out to determine the sites of disulfide bonds and N-glycosylation and the C-terminal amino acid of the large subunit. Disulfide bonds were detected between Cys21 and Cys21, Cys61 and Cys264, Cys194 and Cys198, and Cys296 and Cys351 in the amino acid sequence deduced from the cDNA. Cys21 and Cys21 link two large subunits and Cys296 and Cys351 link a large subunit to a small subunit. Thus, the holoenzyme is constructed of two disulfide-bound large subunits, each of which joins to a small subunit by a disulfide bond. The C-terminal amino acid residue of the large subunit was determined as Ala305. This indicates that the peptide consisting of 35 amino acid residues between the large and the small subunit is deleted from the precursor during maturation, since the N-terminus of the small subunit is Ala341. Three potential N-glycosylation sites in the large subunit were all glycosylated.
Ishida
S
S
Institute of Applied Microbiology, University of Tokyo, Japan.
Muto
S
S
Miyachi
S
S
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000378:metabolism; D016825:Chlamydomonas reinhardtii / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D004229:Dithiothreitol / Q000494:pharmacology; D006031:Glycosylation; D008969:Molecular Sequence Data; D017421:Sequence Analysis; D012697:Serine Endopeptidases / Q000378:metabolism
D000596:Amino Acids; D012697:Serine Endopeptidases; C036813:lysyl endopeptidase; D002256:Carbonic Anhydrases; D004229:Dithiothreitol