The Carbamoyl-phosphate Synthetase of Pyrococcus furiosus Is Enzymologically and Structurally a Carbamate Kinase

Matxalen Uriarte,Alberto Marina,Santiago Ramón-Maiques,Ignacio Fita,Vicente Rubio

doi:10.1074/jbc.274.23.16295

Abstract

The hyperthermophiles Pyrococcus furiosus and Pyrococcus abyssi make pyrimidines and arginine from carbamoyl phosphate (CP) synthesized by an enzyme that differs from other carbamoyl-phosphate synthetases and that resembles carbamate kinase (CK) in polypeptide mass, amino acid sequence, and oligomeric organization. This enzyme was reported to use ammonia, bicarbonate, and two ATP molecules as carbamoyl-phosphate synthetases to make CP and to exhibit bicarbonatedependent ATPase activity. We have reexamined these findings using the enzyme of P. furiosus expressed in Escherichia coli from the corresponding gene cloned in a plasmid. We show that the enzyme uses chemically made carbamate rather than ammonia and bicarbonate and catalyzes a reaction with the stoichiometry and equilibrium that are typical for CK. Furthermore, the enzyme catalyzes actively full reversion of the CK reaction and exhibits little bicarbonate-dependent ATPase. In addition, it cross-reacts with antibodies raised against CK from Enterococcus faecium, and its three-dimensional structure, judged by x-ray crystallography of enzyme crystals, is very similar to that of CK. Thus, the enzyme is, in all respects other than its function in vivo, a CK. Because in other organisms the function of CK is to make ATP from ADP and CP derived from arginine catabolism, this is the first example of using CK for making rather than using CP. The reasons for this use and the adaptation of the enzyme to this new function are discussed.

Highlights

Two types of enzymes, carbamate kinase (CK)1 and carbamoyl-phosphate synthetase (CPS), are known to synthesize carbamoyl phosphate (CP) from mixtures of ATP, bicarbonate, and ammonia
This interpretation was confirmed by the drastic increase in the production of the recombinant enzyme that was observed when the pCPS184-carrying E. coli cells were transformed with plasmid pSJS1240 (20, 21), which encodes the tRNAs for these rare codons (Fig. 1)
The present results clearly show that the CP-synthesizing activity previously reported in P. furiosus (13, 14) is due to an enzyme that uses chemically made carbamate and a single ATP molecule to synthesize CP reversibly

Summary

EXPERIMENTAL PROCEDURES

Materials—Recombinant enterococcal CK was isolated from E. coli BL21 (DE3) cells (obtained from Novagen) transformed with the plasmid pCK41 exactly as described (6). Of Biochemistry, University of Georgia, Athens, GA) was used as a template for polymerase chain reaction amplification of the CPS gene using a high fidelity proofreading thermostable DNA polymerase (Deep Vent, New England Biolabs) and the primers 5Ј-GTGGTTTCCATGGGTAAGAGGGTAGTGATTGC-3Ј and 5Ј-GCATTCGCTAAGCTGGGTCTTCTAAAGTTCCTCAGG-3Ј. Molecular replacement was performed with the AMoRe program (25) using polyalanine models of the structures of enterococcal CK (11) and the N-terminal 315 residues of E. coli biotin carboxylase (Protein Data Bank, entry code: 1bcn) as search models. Other Assays—Indirect enzyme-linked immunosorbent assays in 96microwell plastic plates (from Costar) were carried out as described in Ref. 17 using phosphate-buffered saline solutions containing 2 ␮g/ml protein antigens and 2% bovine serum albumin with 0.1% defatted dry milk, respectively, for coating and blocking the wells. Cross-linking with dimethyl suberimidate and SDS-PAGE of the covalent adducts was done according to Davies and Stark (34)

RESULTS

Enzyme CPS

Enterococcal CK

DISCUSSION