Aerobic Gram negative bacteria such as Pseudomonas putida were reported to possess class A ATCases and to have a M.W. of 360 kD. The nucleotide sequence of the P. putida pyrBC was determined to answer this question once and for all. The expected regulatory gene was not found. It is shown that the P. putida pyrB gene is overlapped by pyrC by 4 bp. The P.putida pyrB is 1005 bp (335 aa) in length and the pyrC is 1275 bp (425 aa) long. Both of these genes complement E. coli mutants with their respective genotypes. Another finding borne out from the sequence is an effector binding site at the N-terminus of pyrB of P. putIda. The binding site shows that effectors compete with carbamoylphosphate for the active site. In this dissertation, it is shown that the ATCase of P.putida is a trimer of M.W. of 109 kD (3 x 36.4 kD) and that the gene encoding pyrB is overlapped by the pyrC gene which encodes DHOase. It is also shown that the pyrBC encoded enzymes copurify as a dodecameric complex with a M.W. of 484 kD.

Aspartate transcarbamoylase (ATCase) was purified from 16 selected bacterial species including existing Pseudomonas species and former species reassigned to new genera. An enormous diversity was seen among the 16 enzymes with each class of ATCase being represented. The smallest class, class C, with a catalytically active homotrimer, at 100 kDa, was found in Bacillus and other Gram positive bacteria. In this report, the ATCases from the Gram negatives, Shewanella putrefaciens and Stenotrophomonas maltophilia were added to class C membership. The enteric bacteria typify class B ATCases at 310 kDa, with a dodecameric structure composed of two catalytic trimers coupled to three regulatory dimers. A key feature of class B ATCases is the dissociability of the holoenzyme into regulatory and catalytic subunits which were enzymatically active. In this report, the ATCase from Pseudomonas indigofera was added to class B ATCases. The largest class, at 480 kDa, class A, contains the fluorescent Pseudomonas including most members of the 16S rRNA homology group I. Two polypeptides are produced from overlapping pyrBC' genes. The former, pyrB, encodes a 34 kDa catalytic polypeptide while pyrC' encodes a 45 kDa dihydroorotase-like polypeptide. Two non active trimers are made from six 34 kDa chains which are cemented by six 45 kDa chains to form the active dodecameric structure. Dissociation of the holoenyzme into its separate active subunits has not been possible. In this report, the ATCases from Comamonas acidovorans and C. testosteroni, were added to the class A enzymes. An even larger class of ATCase than class A at 600 kDa was discovered in Burkholderia cepacia. Stoichiometric measurements predict a dodecamer of six 39 kDa polypeptides and six 60 kDa polypeptides. Unlike other large pseudomonads ATCases, the enzyme from B. cepacia was dissociable into smaller active forms. Both the holoenzyme and its dissociated forms were regulated by nucleotide effectors. A new class of ATCase was proposed for B. cepacia type enzymes.

Bacterial aspartate transcarbamoylases (ATCase's) are divided into three classes that correspond to taxonomic relationships within the bacteria. The opportunistic pathogen Moraxeila catarrhalis has undergone several reclassifications based on traditional microbiological criteria. The previously uncharacterized ATCase from M. catarrhalis was purified to homogeneity and its chemical properties characterized. The ATCase from M. catarrhalis is a class C ATCase with an apparent molecular mass of 480-520 kDa. The M. catarrhalis ATCase is a dodecomer composed of six 35 kDa polypeptides and six 45 kDa polypeptides. The enzyme has an unusually high pH optimum of greater than pH 10. The enzyme exhibited hyperbolic kinetic with a Km for aspartate of 2 mM. A single, separate 78 kDa dihydroorotase from M. catarrhalis was identified and it was not associated with ATCase. These data support the reclassification of M. catarrhalis out of the Neisseriaceae family.

Aspartate transcarbamoylase (ATCase) of Pseudomonas putida consists of two different polypeptides, PyrB and PyrC' (Schurr et al, 1995). The role of the PyrC' and the assembly of PyrB and PyrC' have been studied. The ATCase made in vitro of P.putida PyrB with P.putida PyrC', and of E.coli PyrB with P.putida PyrC ' were generated under two different conditions, denaturation and renaturation, and untreated. It was found that PyrC' plays a role in the enzymatic regulation by ATP, CTP and UTP. In addition to playing a role in substrate binding, the PyrB polypeptide is also involved in effector binding (Kumar et al., manuscript in preparation). The most energetically preferred form of the P.putida WT is a dodecamer with a molecular mass of 480 kDa. The ratio between the PyrB and the PyrC' is 1:1. In studies of nucleotide binding, it was discovered that the P.putida PyrB was phosphorylated by a protein kinase in the cell extract. In the presence of 20 mM EDTA, this phosphorylation was inhibited and the inhibition could be overcome by the addition of divalent cations such as Zn2+ and Mg2+. This result suggested that the phosphorylation reaction required divalent cations. In the CAD complex of eukaryotes, phosphorylations of the CPSase and the linker region between ATCase and DHOase did not occur in the presence of UTP and it was hypothesized (Carrey, 1993) that UTP and phosphorylation(s) regulated the conformational change in the enzyme complex. Therefore, the same idea was approached with P.putida ATCase, where it was found that 1.0 mM UTP inhibited the phosphorylation of PyrB by more than 50%. These results suggested that the regulation of the conformational change of the P.putida ATCase might be similar to that of CAD. Furthermore, peptide mapping for phosphorylation sites was performed on P.putida ATCase WT, WT --11 amino acids and WT --34 amino acids from the N-terminus of the PyrB polypeptide. The results showed that the phosphorylation sites were located on the fragment that contained amino acid number-35 to amino acid number-112 from the N-terminus of the PyrB polypeptide.

The Pseudomonas aeruginosa ATCase was cloned and sequenced to determine the correct size, subunit composition and architecture of this pivotal enzyme in pyrimidine biosynthesis. During the course of this work, it was determined that the ATCase of Pseudomonas was not 360,000 Da but rather present in a complex of 484,000 Da consisting of two different polypeptides (36,000 Da and 44,000 Da) with an architecture similar to that of E. coli ATCase, 2(C3):3(r2). However, there was no regulatory polypeptide found in the Pseudomonas ATCase.

The enzyme that catalyzes the committed step in pyrimidine biosynthesis, aspartate transcarbamoylase, has been compared in selected endospore-forming organisms and in morphologically similar control organisms. The ATCases and pyrimidine salvage from Sporosarcina ureae, Sporolactobacillus inulinus, Lactobacillus fermentum, and Micrococcus luteus were compared to those of Bacillus subtilis. While the ATCases from Sporosarcina ureae, Sporolactobacillus inulinus, and L. fermentum were found to exhibit characteristics to that of Bacillus with respect to molecular weight and kinetics, M. luteus ATCase was larger at approximately 480 kDa. Furthermore, pyrimidine salvage in Sporosarcina ureae and M. luteus was identical to those of B. subtilis, while pyrimidine salvage of Sporolactobacillus inulinus and L. fermentum resembled that of the pseudomonads.

The enzyme, aspartate transcarbamoylase (ATCase) from Moraxella (Branhamella) catarrhalis, has been purified. The holoenzyme has a molecular mass of approximately 510kDa, harbors predominantly positive charges and is hydrophobic in nature. The holoenzyme possesses two subunits, a smaller one of 40 kDa and a larger one of 45 kDa. A third polypeptide has been found to contribute to the overall enzymatic activity, having an approximate mass of 55 kDa. The ATCase purification included the generation of cell-free extract, streptomycin sulfate cut, 60 °C heat step, ammonium sulfate cut, dialysis and ion, gel-filtration and hydrophobic interaction chromatography. The enzyme's performance throughout purification steps was analyzed on activity and SDS-PAGE gradient gels. Its enzymatic, specific activities, yield and fold purification, were also determined.

Aspartate transcarbamoylase (ATCase) catalyzes the first committed step in the pyrimidine biosynthetic pathway. Bacterial ATCases have been divided into three classes, class A, B, and C, based on their molecular weight, holoenzyme architecture, and enzyme kinetics. Moraxella bovis is a fastidious organism, the etiologic agent of infectious bovine keratoconjunctivitis (IBK). The M. bovis ATCase was purified and characterized for the first time. It is a class A enzyme with a molecular mass of 480 to 520 kDa. It has a pH optimum of 9.5 and is stable at high temperatures. The ATCase holoenzyme is inhibited by CTP > ATP > UTP. The Km for aspartate is 1.8 mM and the Vmax 1.04 µmol per min, where the Km for carbamoylphosphate is 1.05 mM and the Vmax 1.74 µmol per min.

The pyrimidine salvage enzyme, nucleoside hydrolase, is catalyzes the irreversible hydrolysis of nucleosides into the free nucleic acid base and D-ribose. Nucleoside hydrolases have varying degrees of specificity towards purine and pyrimidine nucleosides. In E. coli, three genes were found that encode homologues of several known nucleoside hydrolases in protozoa. All three genes (designated yaaF, yeiK, and ybeK) were amplified by PCR and cloned. Two of the gene products (yeiK and ybeK) encode pyrimidine-specific nucleoside hydrolases, while the third (yaaF) encodes a nonspecific nucleoside hydrolase. All three were expressed at low levels and had different modes of regulation. As a comparative analysis, the homologous genes of Pseudomonas aeruginosa and P. fluorescens (designated nuh) were cloned. Both were determined to encode nonspecific nucleoside hydrolases. The nucleoside hydrolases of the pseudomonads exhibited markedly different modes of regulation. Both have unique promoter structures and genetic organization. Furthermore, both pseudomonad nucleoside hydrolase were found to contain an N-terminal extension of 30-35 amino acids that is shown to act as a periplasmic-signaling sequence. These are the first two nucleoside hydrolases, to date,that have been conclusively demonstrated to be exported to the periplasmic space. The physiological relevance of this is explained.

The regulation of pyrimidine biosynthesis was studied in Pseudomonas putida. The biosynthetic and salvage pathways provide pyrimidine nucleotides for RNA, DNA, cell membrane and cell wall biosynthesis. Pyrimidine metabolism is intensely studied because many of its enzymes are targets for chemotheraphy. Four aspects of pyrimidine regulation are described in this dissertation. Chapter I compares the salvage pathways of Escherichia coli and P. putida. Surprisingly, P. putida lacks several salvage enzymes including nucleoside kinases, uridine phosphorylase and cytidine deaminase. Without a functional nucleoside kinase, it was impossible to feed exogenous uridine to P. putida. To obviate this problem, uridine kinase was transferred to P. putida from E. coli and shown to function in this heterologous host. Chapter II details the enzymology of Pseudomonas aspartate transcarbamoylase (ATCase), its allosteric regulation and how it is assembled. The E. coli ATCase is a dodecamer of two different polypeptides, encoded by pyrBI. Six regulatory (PyrI) and six catalytic (PyrB) polypeptides assemble from two preformed trimers (B3) and three preformed regulatory dimers (I2) in the conserved 2B3:3I2 molecular structure. The Pseudomonas ATCase also assembles from two different polypeptides encoded by pyrBC'. However, a PyrB polypeptide combines with a PyrC. polypeptide to form a PyrB:PyrC. protomer; six of these assemble into a dodecamer of structure 2B3:3C'2. pyrC' encodes an inactive dihydroorotase with pyrB and pyrC' overlapping by 4 bp. Chapter III explores how catabolite repression affects pyrimidine metabolism. The global catabolite repression control protein, Crc, has been shown to affect pyrimidine metabolism in a number of ways. This includes orotate transport for use as pyrimidine, carbon and nitrogen sources. Orotate is important because it interacts with PyrR in repressing the pyr genes. Chapter IV describes PyrR, the positive activator of the pyrimidine pathway. As with other positive activator proteins, when pyrimidine nucleotides are depleted, PyrR binds to DNA thereby enhancing expression of pyrD, pyrE and pyrF genes. When pyrimidine nucleotides are in excess, the PyrR apoprotein binds to orotate, its co-repressor, to shut down all the pyrimidine genes. Like many positive activators, PyrR is subject to autoregulation and has catalytic activity for uracil phosphoribosyltransferase inducible by orotate.