Characterization of the Aspartate Transcarbamoylase fromMethanococcus jannaschii

Emily S Hack,Tatyana Vorobyova,Jessica B Sakash,Jay M West,Christine P Macol,Guy Hervé,Mark K Williams,Evan R Kantrowitz

doi:10.1074/jbc.m909220199

Abstract

The genes from the thermophilic archaeabacterium Methanococcus jannaschii that code for the putative catalytic and regulatory chains of aspartate transcarbamoylase were expressed at high levels in Escherichia coli. Only the M. jannaschii PyrB (Mj-PyrB) gene product exhibited catalytic activity. A purification protocol was devised for the Mj-PyrB and M. jannaschii PyrI (Mj-PyrI) gene products. Molecular weight measurements of the Mj-PyrB and Mj-PyrI gene products revealed that the Mj-PyrB gene product is a trimer and the Mj-PyrI gene product is a dimer. Preliminary characterization of the aspartate transcarbamoylase from M. jannaschii cell-free extract revealed that the enzyme has a similar molecular weight to that of the E. coli holoenzyme. Kinetic analysis of the M. jannaschii aspartate transcarbamoylase from the cell-free extract indicates that the enzyme exhibited limited homotropic cooperativity and little if any regulatory properties. The purified Mj-catalytic trimer exhibited hyperbolic kinetics, with an activation energy similar to that observed for the E. coli catalytic trimer. Homology models of the Mj-PyrB and Mj-PyrI gene products were constructed based on the three-dimensional structures of the homologous E. coli proteins. The residues known to be critical for catalysis, regulation, and formation of the quaternary structure from the well characterized E. coli aspartate transcarbamoylase were compared.

Highlights

The genes from the thermophilic archaeabacterium Methanococcus jannaschii that code for the putative catalytic and regulatory chains of aspartate transcarbamoylase were expressed at high levels in Escherichia coli
In order to study the M. jannaschii aspartate transcarbamoylase, the M. jannaschii pyrB (Mj-pyrB)2 and M. jannaschii pyrI (Mj-pyrI) genes were inserted into an expression system that yielded substantial amounts of both gene products
A protein purification scheme was developed for the M. jannaschii PyrB (Mj-PyrB) and M. jannaschii PyrI (Mj-PyrI) gene products, and the quaternary structure of the M. jannaschii aspartate transcarbamoylase found in vivo was analyzed and kinetically characterized

Summary

EXPERIMENTAL PROCEDURES

Materials—Agarose, ATP, CTP, L-aspartate, N-carbamoyl-L-aspartate, 2-mercaptoethanol, isopropyl-␤-thiogalactosidase, potassium di-. Plasmids pEK400, pEK401, and pET23a were digested with NdeI and SacI endonucleases to remove the Mj-pyrB and Mj-pyrI genes These DNA fragments were separated by agarose gel electrophoresis, isolated using glass beads, and were individually mixed with NdeI- and SacI-digested pET23a. The protein was eluted from the column using 300 ml of column buffer followed by a 600-ml linear gradient of column buffer to column buffer containing 0.5 M NaCl. The column fractions containing the Mj-PyrB gene product, as determined by A280 measurements and by SDS-PAGE, were pooled and dialyzed into a buffer solution of 40 mM KH2PO4, 2 mM 2-mercaptoethanol, and 1.3 M ammonium sulfate, pH 7.0. Purification of the M. jannaschii PyrI Gene Product —A 2-liter cell culture was centrifuged at 4,500 ϫ g for 20 min, and the pellet was resuspended in 16 ml of ice-cold 0.1 M Tris-Cl, 0.1 mM zinc acetate buffer, pH 9.2. Final analysis of the models was performed in QUANTA (Molecular Simulations, Inc.)

RESULTS AND DISCUSSION

Total Protein

Carbamoyl phosphate saturatione

Characterization of the Aspartate Transcarbamoylase fromMethanococcus jannaschii