Abstract
Aquifex aeolicus, an organism that flourishes at 95 degrees C, is one of the most thermophilic eubacteria thus far described. The A. aeolicus pyrB gene encoding aspartate transcarbamoylase (ATCase) was cloned, overexpressed in Escherichia coli, and purified by affinity chromatography to a homogeneous form that could be crystallized. Chemical cross-linking and size exclusion chromatography showed that the protein was a homotrimer of 34-kDa catalytic chains. The activity of A. aeolicus ATCase increased dramatically with increasing temperature due to an increase in kcat with little change in the Km for the substrates, carbamoyl phosphate and aspartate. The Km for both substrates was 30-40-fold lower than the corresponding values for the homologous E. coli ATCase catalytic subunit. Although rapidly degraded at high temperature, the carbamoyl phosphate generated in situ by A. aeolicus carbamoyl phosphate synthetase (CPSase) was channeled to ATCase. The transient time for carbamoyl aspartate formation was 26 s, compared with the much longer transient times observed when A. aeolicus CPSase was coupled to E. coli ATCase. Several other approaches provided strong evidence for channeling and transient complex formation between A. aeolicus ATCase and CPSase. The high affinity for substrates combined with channeling ensures the efficient transfer of carbamoyl phosphate from the active site of CPSase to that of ATCase, thus preserving it from degradation and preventing the formation of toxic cyanate.
Highlights
Aquifex aeolicus, one of the most hyperthermophilic eubacteria far discovered, is classified as a hydrogen-oxidizing, microaerophilic, obligate chemolithoautotroph [1]
The effect of PALA on the aspartate transcarbamoylase (ATCase) reaction, using exogenous carbamoyl phosphate and aspartate, was measured in the presence and absence of A. aeolicus occupies the deepest branch of the eubacterial phylogenetic tree, the polypeptide encoded by the pyrB gene closely resembles the catalytic chain of ATCases from other organisms in size and sequence
No homotropic or heterotropic interactions were found in A. aeolicus ATCase, an observation that is consistent with the absence of regulatory subunits
Summary
Materials—Pfu Turbo DNA polymerase was obtained from Stratagene; DNA ligase was from Invitrogen; aspartate, carbamoyl phosphate, carbamoyl aspartate, antipyrine, diacetyl monoxime, triethoanolamine (TEA), spectinomycin, pyridoxal phosphate, and sodium borohydride were from Sigma; dimethyl suberimidate was from Pierce; [14C]carbamoyl phosphate (7 Ci/mol) was from American Radiolabeled Chemicals; and N-(phosphonacetyl)-L-aspartate (PALA) was provided by Drs V. Fractions containing pure A. aeolicus ATCase were applied to a Nick Spin Column (Amersham Biosciences) equilibrated with 50 mM Tris-HCl, pH 8, to eliminate NaCl and imidazole from the buffer. Size Exclusion Chromatography—The molecular mass of the recombinant enzyme was determined by size exclusion chromatography [27] on a 1 ϫ 50-cm column of Sephacryl S-300 High Resolution equilibrated with 50 mM Tris-HCl, pH 8, in the presence or absence of 100 mM NaCl. 1 ml of A. aeolicus ATCase at a concentration of 3 mg/ml was applied to the column. Chemical Cross-linking—A reaction mixture containing 35 g of A. aeolicus ATCase, 10 mM dimethyl suberimidate [28], and 100 mM TEA, pH 8.5, in a final volume of 40 l, was incubated at room temperature and quenched at the designated times by the addition of 4 l of 1 M Tris-HCl, pH 8. Internal consistency checks in the MODELLER4 software package and the program PROCHECK were used to assess the quality of the model
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