title: Expression, purification, crystallization and preliminary X-ray crystallographic studies of a cold-adapted aspartate carbamoyltransferase from Moritella profunda.
abstract: Aspartate carbamoyltransferase (ATCase) catalyzes the carbamoylation of the alpha-amino group of L-aspartate by carbamoyl phosphate (CP) to yield N-carbamoyl-L-aspartate and orthophosphate in the first step of de novo pyrimidine biosynthesis. Apart from its key role in nucleotide metabolism, the enzyme is generally regarded as a model system in the study of proteins exhibiting allosteric behaviour. Here, the successful preparation, crystallization and diffraction data collection of the ATCase from the psychrophilic bacterium Moritella profunda are reported. To date, there is no structural representative of a cold-adapted ATCase. The structure of M. profunda ATCase is thus expected to provide important insights into the molecular basis of allosteric activity at low temperatures. Furthermore, through comparisons with the recently reported structure of an extremely thermostable ATCase from Sulfolobus acidocaldarius, it is hoped to contribute to general principles governing protein adaptation to extreme environments. A complete native data to 2.85 A resolution showed that the crystal belongs to space group P3(2)21, with unit-cell parameters a = 129.25, b = 129.25, c = 207.23 A, alpha = beta = 90, gamma = 120 degrees, and that it contains three catalytic and three regulatory subunits per asymmetric unit. The three-dimensional structure of the Escherichia coli ATCase was sufficient to solve the structure of the M. profunda ATCase via the molecular-replacement method and to obtain electron density of good quality.
authors.affiliation: Laboratorium voor Eiwitbiochemie en Eiwitengineering, Universiteit Gent, K. L. Ledeganckstraat 35, Ghent, Belgium.
authors.forename: Dirk
authors.initials: D
authors.lastname: De Vos
authors.forename: Paco
authors.initials: P
authors.lastname: Hulpiau
authors.forename: Bjorn
authors.initials: B
authors.lastname: Vergauwen
authors.forename: Savvas N
authors.initials: SN
authors.lastname: Savvides
authors.forename: Jozef
authors.initials: J
authors.lastname: Van Beeumen
mesh_terms: D000064:Acclimatization; D000595:Amino Acid Sequence; D001221:Aspartate Carbamoyltransferase / Q000737:chemistry* / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology; D008969:Molecular Sequence Data; D034221:Moritella / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D010446:Peptide Fragments; D021122:Protein Subunits; D011994:Recombinant Proteins; D001221:Aspartate Carbamoyltransferase