Carbamoyl phosphate synthetases (CPSs) utilize either glutamine or ammonia for the ATP-dependent generation of carbamoyl phosphate. In glutamine-utilizing CPSs (e.g. the single Escherichia coli CPS and mammalian CPS II), the hydrolysis of glutamine to yield ammonia is catalyzed at a triad-type glutamine amidotransferase domain. Non-glutamine-utilizing CPSs (e.g. rat and human CPS I), lacking the catalytic cysteine residue, can generate carbamoyl phosphate only in the presence of free ammonia. Frog CPS I (fCPS I), unlike mammalian CPS Is, retains most of the glutamine amidotransferase residues conserved in glutamine-utilizing CPSs, including an intact catalytic triad, and could therefore be expected to use glutamine. Our work with native fCPS I provides the first demonstration of the inability of this enzyme to bind/utilize glutamine. To determine why fCPS I is unable to utilize glutamine, we compared sequences of glutamine-using and non-glutamine-using CPSs to identify residues that are present or conservatively substituted in all glutamine-utilizing CPSs but absent in fCPS I. We constructed the sitedirected mutants Q273E, L270K, Q273E/N240S, and Q273E/L270K in E. coli CPS and have determined that simultaneous occurrence of the two substitutions, Gln 3 Glu and Leu 3 Lys, found in the frog CPS I glutamine amidotransferase domain are sufficient to eliminate glutamine utilization by the E. coli enzyme. Formation of carbamoyl phosphate (CP)1 is the first step in the synthesis of arginine and pyrimidines and, in higher organisms, is also critical in nitric oxide formation and in ammonia detoxification via the urea cycle. Carbamoyl phosphate synthetases (CPSs) are made up of a two-domain glutamine amidotransferase (GAT) component and a four-domain synthetase (SYN) component (1-5). The N-terminal domain of the GAT component is required for interaction with the SYN component (6, 7), and the C-terminal domain has a triad-type GAT structure (5, 8). In glutamine-utilizing CPSs (e.g. the single Escherichia coli CPS and mammalian CPS II), the hydrolysis of glutamine to yield ammonia is catalyzed at the GAT domain, where the cysteine of the Cys-His-Glu triad carries out a nucleophilic attack on the amide carbonyl of glutamine (9 -12). The ammonia thus liberated is subsequently channeled to the SYN component where it, together with bicarbonate, is used to generate CP with an energy expenditure of two ATP molecules per CP synthesized. The SYN component consists of two ATPutilizing domains (13), one allosteric interaction domain (14 -17), and one domain for interaction with the GAT component (6, 7). In the absence of other CPS substrates, the GAT domain is able to catalyze a glutaminase reaction, and in the absence of glutamine, the SYN component is able to synthesize CP from NH 3 (18 -20). However, each component can sense occupancy of the other, and when all substrates are present, the glutaminase activity is increased 600-fold, and the ATPase activity is increased 15-fold. The final rates of the partial reactions involved in ...

Depending on their physiological role, carbamoyl phosphate synthetases (CPSs) use either glutamine or free ammonia as the nitrogen donor for carbamoyl phosphate synthesis. Sequence analysis of known CPSs indicates that, regardless of whether they are ammonia-or glutamine-specific, all CPSs contain the structural equivalent of a triad-type glutamine amidotransferase (GAT) domain. In ammonia-specific CPSs, such as those of rat or human, the catalytic inactivity of the GAT domain can be rationalized by the substitution of the Triad cysteine residue by serine (1). The ammonia-specific CPS of Rana catesbeiana (fCPS) presents an interesting anomaly in that, despite its retention of the entire catalytic triad (2) and almost all other residues conserved in Triad GATs, it is unable to utilize glutamine as a nitrogen-donating substrate (3). Based on our earlier work with the glutamine-utilizing E. coli CPS (eCPS), we have targeted residues Lys 258 and Glu 261 in the fCPS GAT domain as critical for preventing GAT function. Previously we have shown that substitution of the corresponding residues in eCPS by their fCPS counterparts (Leu 3 Lys and Gln 3 Glu) resulted in complete loss of GAT function in eCPS (3). To examine the role of these residues in the fCPS GAT component, we have cloned the full-length fCPS gene from R. catesbeiana liver. Here we report the first heterologous expression of an ammonia-specific CPS and show that a single mutation of the frog enzyme, K258L, yields a gain of glutaminase function.

We have developed a system for rapid and reliable assessment of gene essentiality in Haemophilus influenzae Rd strain KW20. We constructed two "suicide" complementation vectors (pASK5 and pASK6) containing 5 and 3 regions of the nonessential ompP1 gene flanking a multiple cloning site and a selectable marker (a chloramphenicol resistance gene or a tetracycline resistance cassette). Transformation of H. influenzae with the complementation constructs directs chromosomal integration of a gene of interest into the ompP1 locus, where the strong, constitutive ompP1 promoter drives its expression. This single-copy, chromosome-based complementation system is useful for confirming the essentiality of disrupted genes of interest. It allows genetic analysis in a background free of interference from any upstream or downstream genetic elements and enables conclusive assignment of essentiality. We validated this system by using the riboflavin synthase gene (ribC), a component of the riboflavin biosynthetic pathway. Our results confirmed the essentiality of ribC for survival of H. influenzae Rd strain KW20 and demonstrated that a complementing copy of ribC placed under control of the ompP1 promoter reverses the lethal phenotype of a strain with ribC deleted.

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