Expression, purification, crystallization and preliminary X-ray crystallographic studies of a cold-adapted aspartate carbamoyltransferase from Moritella profunda

Crystals of the aspartate carbamoyltransferase of the psychrophile M. profunda diffract X-rays to 2.85 Å. Three catalytic and three regulatory subunits are predicted per asymmetric unit. Aspartate carbamoyltransferase (ATCase) catalyzes the carbamoylation of the α-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 Å resolution showed that the crystal belongs to space group P3₂21, with unit-cell parameters a = 129.25, b = 129.25, c = 207.23 Å, α = β = 90, γ = 120°, 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