Identification, cDNA cloning, expression, crystallization and preliminary X-ray analysis of an exceptionally halotolerant carbonic anhydrase from Dunaliella salina.
An extracellular alpha-type carbonic anhydrase (dCAII) from the salt-tolerant alga Dunaliella salina differs from its mesophilic counterparts in remaining active from zero to multimolar salt concentrations. To gain insight into the outstanding salt tolerance of dCAII, the enzyme was functionally overexpressed in Escherichia coli, purified by affinity chromatography and crystallized by the hanging-drop method. The crystals belonged to space group P2(1), with unit-cell parameters a = 47.0, b = 119.9, c = 58.5 A, beta = 94.2 degrees. Data from a single crystal were collected to 2.4 A resolution under cryogenic conditions (120 K) using an R-AXIS IV(++) detector mounted on a Rigaku RU-H3R rotating-anode generator. The asymmetric unit contains two molecules of the protein, which corresponds to V(M) = 2.65 A(3) Da(-1) and a solvent content of 52.7%.
Premkumar
Lakshmanane
L
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
Greenblatt
Harry M
HM
Bageshwar
Umesh K
UK
Savchenko
Tatyana
T
Gokhman
Irena
I
Zamir
Ada
A
Sussman
Joel L
JL
D000595:Amino Acid Sequence; D002256:Carbonic Anhydrases / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D000460:Chlorophyta / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D014961:X-Ray Diffraction
D018076:DNA, Complementary; D002256:Carbonic Anhydrases