Converting Catabolic Ornithine Carbamoyltransferase to an Anabolic Enzyme*

Pseudomonas aeruginosa has an anabolic and a catabolic ornithine carbamoyltransferase (OTCase). In vitro, these homologous enzymes catalyze the same reaction (ornithine + carbamoyl phosphate (CP) in equilibrium citrulline + Pi), yet in vivo they function unidirectionally owing to specific kinetic properties. The catabolic OTC-ase cannot promote the anabolic reaction (citrulline formation) in vivo because of a sigmoidal CP saturation curve and a high CP concentration for half-maximal velocity. The structural basis for this kinetic specialization was examined. The catabolic OTCase lost most of its homotropic cooperativity and gained anabolic activity when an amino acid residue near the CP binding site, Glu-106, was replaced by alanine or glycine. In the anabolic OTCase of Escherichia coli the glutamine residue corresponding to Glu-106 was exchanged for glutamate; however, in this case no CP cooperativity was acquired. Thus, in catabolic OTCase, sequence features in addition to Glu-106 are important for sigmoidal CP saturation, and such a sequence was identified in the C-terminal part. By an in vivo gene fusion technique the 9 C-terminal amino acids of catabolic OTCase were replaced by the homologous 8 amino acids from anabolic OTCase of E. coli; the hybrid enzyme had a markedly reduced homotropic cooperativity. This gene fusion method should be generally useful for directed enzyme evolution.

In uitro, these homologous enzymes catalyze the same reaction (ornithine + carbamoyl phosphate (CP) F= citrulline + Pi), yet in uiuo they function unidirectionally owing to specific kinetic properties.
The catabolic OTCase cannot promote the anabolic reaction (citrulline formation) in uiuo because of a sigmoidal CP saturation curve and a high CP concentration for half-maximal velocity.
The structural basis for this kinetic specialization was examined.
The catabolic OTCase lost most of its homotropic cooperativity and gained anabolic activity when an amino acid residue near the CP binding site, Glu-106, was replaced by alanine or glycine. In the anabolic OTCase of Escherichia coli the glutamine residue corresponding to Glu-106 was exchanged for glutamate; however, in this case no CP cooperativity was acquired.
Thus, in catabolic OTCase, sequence features in addition to Glu-106 are important for sigmoidal CP saturation, and such a sequence was identified in the C-terminal part. By an in uiuo gene fusion technique the 9 C-terminal amino acids of catabolic OTCase were replaced by the homologous 8 amino acids from anabolic OTCase of E. coli; the hybrid enzyme had a markedly reduced homotropic cooperativity. This gene fusion method should be generally useful for directed enzyme evolution.
Anabolic OTCases promote citrulline formation from ornithine as part of arginine biosynthesis (1) catabolic OTCase which participates in the anaerobic degradation of arginine via citrulline to ornithine (l-3). OTCases are structurally related to aspartate carbamoyltransferase (ATCase; EC 2.1.3.2) of Escherichia coli, an allosteric enzyme par excellence (4-7). Because of these sequence identities, the residues involved in carbamoyl phosphate (CP) binding and the secondary structures of OTCases can be predicted on the basis of the known ATCase structure (4-11). The catalytic subunit of ATCase (the pyrB gene product) forms an active trimer, which displays Michaelis-Menten kinetics. In vivo, two catalytic ATCase trimers are complexed with three regulatory dimers, and this holoenzyme has allosteric properties and a sigmoidal aspartate saturation curve (4,5). The anabolic OTCases of E. coli (the argF and argZ products) and P.
aeruginosa (the argF product) are trimers resembling the ATCase catalytic trimer (4, 6,8,12,13). The substrate saturation curves of the E. coli anabolic OTCase and of the catalytic ATCase trimer are changed from hyperbolic to sigmoidal when a conserved residue, Arg-108, involved in CP binding (4), is converted to a nonpolar amino acid residue by site-directed mutagenesis (14,15). * We are interested in the reciprocal situation. How can we strip the catabolic OTCase of its cooperativity? The catabolic OTCase of P. aeruginosa (the arcB product) is a trimeric enzyme aggregated to a complex form, i.e. a nonamer or dodecamer (16). The CP saturation curve is strongly sigmoidal and a high CP concentration is needed for half-maximal velocity, [S]$.f (17,18). Therefore, the wild-type enzyme cannot perform the anabolic reaction in vivo, but mutant enzymes have been obtained which can (17,18). This study characterizes the sequences and kinetics of such mutant enzymes. Then, an in viuo recombination method is described which allows the isolation of arcB-argF gene fusions. This technique, which should be widely applicable, has led to the identification of further residues involved in homotropic cooperativity of catabolic OTCase.

AND DISCUSSION
The initial aim was to isolate new mutants of P. aeruginosa which had a modified catabolic OTCase capable of catalyzing the anabolic reaction. The argF mutant PA0532 having an inactive anabolic OTCase was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine and arginine prototrophic derivatives were obtained. By transductional analysis (18) we identified those arginine prototrophs which had retained the original argF mutation but carried a mutation in arcB (designated arcB(Su), for suppression of argF), assuming that these strains contained a mutant catabolic OTCase. The arcB(Su) alleles of four independent isolates (PA06238, PA06240, PA06241, and PA06254) were transferred from the chromosome to an arc recombinant plasmid by an in vivo cloning method (see "Experimental Procedures"). Moreover, the arcBl(Su) allele of a mutant previously obtained (PA0524; Ref. 18) was cloned in vitro. The five arcB(Su) mutations were characterized by sequencing; in two cases (PA0524, PA06254), an A + G transition led to a replacement Glu-106 + Gly-106 in catabolic OTCase, whereas in the other three cases the same A had undergone a transversion to C, giving Glu-106 -+ Ala-106. It was verified that these were the only mutational changes in the arcB(Su) genes. P. aeruginosa strains carrying these genes were able to utilize arginine anaerobically, indicating that the modified catabolic OTCases still functioned in the catabolic direction. The Gly-106 and Ala-106 enzymes were purified and their kinetic properties compared to those of the wild-type catabolic enzyme. Both modified OTCases had a strongly reduced cooperativity for CP (as expressed by the Hill coefficient rzn), a markedly lower [S]:.: value, and an improved specific activity (Table I). Thus, they were much more efficient in citrulline synthesis than was the wild-type enzyme (Table I), and this is consistent with the arginine prototrophy of the arcB(Su) strains. Other effects of the arcB(Su) mutations were a twofold increase in the apparent K,,, for ornithine (at 10 mM CP) and a shift of the pH optimum by -1 pH unit toward alkaline values (Table I). With respect to the pH optimum, the modified catabolic OTCases resembled the anabolic E. coli OTCase ( Table I). The main conclusion is that Glu-106 in the catabolic enzyme is essential for CP cooperativity.
Inorganic phosphate is an activator of wild-type catabolic OTCase (17,18) (Table I) than had the Pi-activated wild-type catabolic OTCase. The catabolic OTCase of I? aeruginosa has more amino acid sequence identity with the anabolic OTCase of E. coli than with the anabolic enzyme of P. aeruginosa (13). We have therefore focused on the first two enzymes. Glu-106 of the catabolic OTCase corresponds to a glutamine residue in the urgF enzyme of E. coli. Does a Gln-106 + Glu replacement" confer CP cooperativity on the anabolic OTCase? This was tested by site-directed mutagenesis of the E. coli argF gene cloned into a pBR322 derivative (pME3604; Fig. 1). However, the engineered urgF (Gln-106 += Glu) enzyme, in extracts from E. coli, retained Michaelis-Menten kinetics and, in fact, had a lower apparent K,,, value for CP (0.06 mM, at pH 8.5) than had the wild-type argF enzyme (Kc,' = 0.36 mM, at pH 8.5). Thus, in the context of the anabolic OTCase of E. coli, Glu-106 did not produce a sigmoidal CP saturation curve, and the cooperativity of the catabolic OTCase must be due to particular sequence features in addition to Glu-106. To identify such sequences we constructed urcB-urgF fusions in uiuo (Fig. 2a). A truncated arcB' gene (with a 0.05kb deletion in the distal part) and a truncated 'urgF gene (with a 0.12-kb deletion in the proximal part) were inserted into the multi-copy, broad host range plasmid vector pKT240 in the same orientation.
These shortened genes were separated by 1.8 kb (Fig. 2~) and neither specified an active OTCase. The E. coli host used (SClSOO; argI90 A(proAB luc argF)) was recombination-proficient and OTCase-negative. Selection for arginine prototrophy, i.e. for OTCase function, resulted in the recovery of arcB-urcF fusions (Fig. 2a) at frequencies of low6 to 10m7. Recombination occurred between arcB' and 'argF in three different 30-base pair regions having -80% nucleotide sequence identities (data not shown), the overall arcB/argF identity being 63% (16). One recombinant, pME3606, was characterized in detail; it specified a modified catabolic OTCase with 8 C-terminal amino acids originating from the anabolic OTCase (Fig. 2b). This hybrid enzyme functioned in the anabolic direction in both E. coli and P. ueruginosa, owing to lowered [S]Eg and nn values (Table I).
Thus, the C terminus of catabolic OTCase is important for cooperativity.
All arcB(Su) enzymes including the one specified by pME3606 had the same molecular weight as wild-type catabolic OTCase (-420,000), as judged by Sepharose 4B gel filtration and Phast gel electrophoresis under nondenaturing conditions.
We have no evidence for dissociation of these enzymes into catalytically active, low molecular weight forms (e.g. trimers) without cooperative properties. However, we cannot exclude this type of dissociation during enzyme assays when protein concentrations were -100 times lower than during gel filtration.