Thesis advisor: Evan Kantrowitz / Aspartate transcarbamoylase (ATCase) catalyzes the reaction between carbamoyl phosphate (CP) and aspartate to form N-carbamoyl-L-aspartate (CA) and inorganic phosphate (Pi). In Escherichia coli, it catalyzes the committed step of pyrimidine nucleotide biosynthesis. It is heterotropically activated by ATP, and is inhibited by CTP and UTP. X-ray studies have revealed valuable information regarding the catalytic mechanism, as well as insights into the homotropic and heterotropic interactions. However, localized changes and changes under biological conditions are difficult to study this way. The expression of a viable and active fluorescent mutant of ATCase containing a 7-hydroxycoumarin amino acid will allow for studies of the signal transduction process involved in the negative cooperativity observed in the binding of allosteric effectors. The fluorescent intensity should correlate to the binding of the NTPs at close proximity to the unnatural fluorescent amino acid. Furthermore, TR-SAXS will be used to observe transiently stable intermediates of ATCase formed during the T to R transition. This method’s typically observed poor time resolution caused by the dead time of the stopped-flow mixer will be addressed by the used of a caged aspartate, 4-methoxy-7-nitroindolinyl-L-aspartate, synthesized in this work. / Thesis (MS) — Boston College, 2008. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Aspartate transcarbamoylase (ATCase) catalyzes the first committed step in pyrimidine biosynthesis. Bacterial ATCases are divided into three classes, A, B and C. Class A ATCases are largest at 450-500, are. dodecamers and represented by Pseudomonas ATCase. The overlapping pyrBC' genes encode the Pseudomonases ATCase, which is active only as a 480 kDa dodecamer and requires an inactive pyrC'-encoded DHOase for ATCase activity. ATCase has been studied in two non-pathogenic members of Mycobacterium, M. smegmatis and M. phlei. Their ATCases are dodecamers of molecular weight 480 kDa, composed of six PyrB and six PyrC polypeptides. Unlike the Pseudomonas ATCase, the PyrC polypeptide in these mycobacteria encodes an active DHOase. Moreover, the ATCase: DHOase complex in M. smegmatis is active both as the native 480 kDa and as a 390 kDa complex. The latter lacks two PyrC polypeptides yet retains ATCase activity. The ATCase from M. phlei is similar, except that it is active as the native 480 kDa form but also as 450,410 and 380 kDa forms. These complexes lack one, two, and three PyrC polypeptides, respectively. By contrast,.ATCases from pathogenic mycobacteria are active only at 480 kDa. Mycobacterial ATCases contain active DHOases and accordingly. are placed in class A1 . The class A1 ATCases contain active DHOases while class A2 ATCases contain inactive DHOases. ATCase has also been purified from Burkholderia cepacia and from an E. coli strain in which the cloned pyrB of B. cepacia was expressed. The B. cepacia ATCase has a molecular mass of 550 kDa, with two different polypeptides, PyrB (52 kDa) and PyrC of (39 kDa). The enzyme is active both as the native enzyme at 550 kDa and as smaller molecular forms including 240 kDa and 165 kDa. The ATCase synthesized by the cloned pyrB gene has a molecular weight of 165 kDa composed of three identical PyrB and no PyrC polypeptides. Nucleotide effectors ATP, CTP, and UTP inhibited all forms of enzymes. Because of its size and its activity as a trimer and smaller than native forms, the B. cepacia enzyme is placed in a new class.

Like its enteric counterpart, aspartate transcarbamoylase (ATCase) from Pseudomonas putida is a dodecamer of two different polypeptides. Unlike the enterics, the Pseudomonas ATCase lacks regulatory polypeptides but employs instead inactive dihydroorotases for an active dodecamer. Previous work showed that PyrB contains not only the active site but also the effector binding sites for ATP, UTP and CTP at its N-terminus. In this work, 11 amino acids were deleted from the N-terminus of PyrB and the ATCase with the truncated protein was expressed in E. coli pyrB- and purified. The wild type enzyme was similarly treated. Velocity-substrate plots without effectors gave Michaelis-Menten kinetics in all cases. Deleting 11 amino acids did not affect dodecameric assembly but altered effector responses. When carbamoylphosphate was varied, the mutant enzyme was inhibited by UTP while the wild type enzyme was activated 2-fold. When the aspartate was varied, CTP had no effect on the mutant enzyme but strongly inhibited the wild type enzyme.

Burkholderia cepacia is a common soil bacterium of significance in agriculture and bioremediation. B. cepacia is also an opportunistic pathogen of humans causing highly communicable pulmonary infections in cystic fibrosis and immunocompromized patients. The pyrB gene encoding ATCase was cloned and ATCase was purified by the glutathione S-transferase gene fusion system. The ATCase in B. cepacia has been previously classified as a class A enzyme by Bethell and Jones. ATCase activity gels showed that B. cepacia contained a holoenzyme pyrBC complex of 550 kDa comprised of 47 kDa pyrB and 45 kDa pyrC subunits. In the course of purifying the enzyme, trimeric subunits of 140 kDa and 120 kDa were observed as well as a unique proteolysis of the enzyme. The 47 kDa ATCase subunits were cleaved to 40 kDa proteins, which still demonstrated high activity as trimers. The proteolysis site is between Ser74 and Val75 residues. To confirm this, we converted the Ser74 residue to an Ala and to an Arg by site-directed mutagenesis. After this primary sequence changed, the proteolysis of ATCase was not observed. To further investigate the characteristics of B. cepacia pyrB gene, a pyrB knock-out (pyrB-) was constructed by in vitro mutagenesis. In the assay, the 550 kDa holoenzyme and 140 kDa and 120 kDa trimers disappeared and were replaced with a previously unseen 480 kDa holoenzyme pyrB- strain. The results suggest that B. cepacia has two genes that encode ATCase. ATC1 is constitutive and ATC2 is expressed only in the absence of ATC1 activity. To check for the virulence of these two strains, a eukaryotic model virulence test was performed using Caenorhabditis elegans (C. elegans). The pyrB1+pyrB2+ (wild type) B cepacia killed the nematode but pyrB1-pyrB2+ B. cepacia had lost its virulence against C. elegans. This suggests that ATC1 (pyrB1) is involved in virulence in B.cepacia and ATC2 (pyrB2) is not.

The pyrimidine biosynthetic pathway is an essential pathway for most organisms. Previous research on the pyrimidine pathway in Pseudomonas aeruginosa (PAO1) has shown that a block in the third step of the pathway resulted in both a requirement for exogenous pyrimidines and decreased ability to produce virulence factors. In this work an organism with a mutation in the second step of the pathway, aspartate transcarbamoylase (ATCase), was created. Assays for pyrimidine intermediates, and virulence factors were performed. Results showed that the production of pigments, haemolysin, and rhamnolipids were significantly decreased from PAO1. Elastase and casein protease production were also moderately decreased. In the Caenorhabditis elegans infection model the nematodes fed the ATCase mutant had increased mortality, as compared to nematodes fed wild type bacteria. These findings lend support to the hypothesis that changes in the pyrimidine biosynthetic pathway contribute to the organism's ability to effect pathogenicity.

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.

Thesis advisor: Evan R. Kantrowitz / A full understanding of an enzyme's catalytic mechanism and a crystal structure representative of its in vivo form are powerful tools in computational drug screening and design. In the case of aspartate transcarbamoylase (ATCase), an allosteric enzyme, the mechanism and allosteric transition are still being explored. The crystallization of the ATCase mutant Asp236 to alanine, a T-state destabilized mutant, in the presence of phosphonoacetamide (PAM) by microdialysis was successful at pH 5.7. The enzyme crystallized in the R-state in the presence of only one substrate analogue. Globally the enzyme had converted to R, but the active site domains are more open than previously observed. Due to the ordered nature of the reaction, the R-state active site exists with a variety of small molecules bound at different times through out the course of the reaction. This structure shows an R-state active site with only one substrate analogue bound, and may therefore represent the R active site after catalysis has occurred and the active site is binding new substrates to perform its reaction again. Docking studies of small molecules can be conducted using this more open, emptier active site as it may be more representative of an in vivo conformation of the enzyme just before catalysis. Additionally, Arg296, previously unobserved as part of the active site, makes a hydrogen bonding interaction with the PAM molecule. The role of this residue will require further investigation. / Thesis (BS) — Boston College, 2005. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Chemistry. / Discipline: College Honors Program.

Thesis advisor: Evan R. Kantrowitz / The enzyme aspartate transcarbamoylase (ATCase) is an enzyme in the pyrimidine nucleotide biosynthetic pathway. It was once an attractive target for anti-proliferation drugs but has since become a teaching model due to kinetic properties such as cooperativity and allostery exhibited by the Escherichia coli form of the enzyme. ATCase from E. coli has been extensively studied over that last 60 years and is the textbook example of allosteric enzymes. Through this past research it is understood that ATCase is allosterically inhibited by CTP, the end product of pyrimidine biosynthesis, and allosterically activated by ATP, the end product of the parallel purine biosynthetic pathway. Part of the work discussed in this dissertation involves further understanding the catalytic properties of ATCase by examining an unregulated trimeric form from Bacillus subtilis, a bacterial ATCase that more closely resembles the mammalian form than E. coli ATCase. Through X-ray crystallography and molecular modeling, the complete catalytic cycle of B. subtilis ATCase was visualized, which provided new insights into the manifestation of properties such as cooperativity and allostery in forms of ATCase that are regulated. Most of the work described in the following chapters involves understanding allostery in E. coli ATCase. The work here progressively builds a new model of allostery through new X-ray structures of ATCase*NTP complexes. Throughout these studies it has been determined that the allosteric site is bigger than previously thought and that metal ions play a significant role in the kinetic response of the enzyme to nucleotide effectors. This work proves that what is known about ATCase regulation is inaccurate and that currently accepted, and taught, models of allostery are wrong. This new model of allostery for E. coli ATCase unifies all old and current data for ATCase regulation, and has clarified many previously unexplainable results. / Thesis (PhD) — Boston College, 2013. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Aspartate transcarbamoylase (ATCase; encoded by pyrBI genes) is one of the most studied regulatory enzymes in bacteria. It is feedback inhibited by cytidine triphosphate (CTP) and activated by adenosine triphosphate (ATP). Much is known about the catalytic site of the enzyme, not nearly as much about the regulatory site, to which CTP binds. Until now a positive selection for feedback-modified mutants was not available. The selection we have developed involves the use of a pyrA deletion in S. typhimurium. This strain lacks carbamoylphosphate and requires both a pyrimidine and arginine for growth. In this strain citrulline is used to satisfy the pyrimidine and arginine requirements. The minimal flow through the pyrimidine pathway from the citrulline-produced carbamoylphosphate is exquisitely sensitive to feedback control of ATCase by CTP. By elevating the CTP pool, via exogenous cytidine, in a strain that also contains a cytidine deaminase mutant (cdd) growth can be stopped completely, indicating 100% inhibition. It was therefore possible to measure in vivo feedback inhibition of ATCase among the citrulline users and to isolate a family of ATCase regulatory mutants with either modified or no response to effectors.

This dissertation comprises three parts and is presented in two chapters. Chapter 1 concerns Arthrobacter, a bacterium with an intriguing growth cycle. Whereas most bacteria exist as either a rod or coccus, this bacterium shares the rod/coccus lifestyle. It therefore seemed important to examine the growth regulatory pathways from the rod and coccus. The committed step, that catalyzed by aspartate transcarbamoylase (ATCase), in the pyrimidine biosynthetic pathway was chosen. The ATCase in Arthrobacter is like the well known Pseudomonas enzyme except that it has an active dihydroorotase (DHOase) associated. Included in Chapter 1 is the description of a microorganism, Burkholderia cepacia, whose ATCase has characteristics that are at once reminiscent of bacteria, mammals, and fungi. It differs in size or aggregation based on environmental conditions. In addition, it has an active DHOase associated with the ATCase, like Arthrobacter. B. cepacia is important both medically and for bioremediation. Since B. cepacia is resistant to most antibiotics, its unique ATCase is a prime target for inhibition. Whereas the first chapter deals with the de novo pathway to making pyrimidines, which is found mainly in the lag and log phase, Chapter 2 addresses the salvage pathway, which comes more into play during the stationary phase. This section focuses on the isolation, identification, and grouping of a number of natural soil bacteria from various soil locations. These organisms are important agriculturally, medically, and industrially. Addition of these soil isolates to poor soils has been found to improve the soil. In a previous study by D.A. Beck, the salvage schemes for a number of laboratory strains of microorganisms were determined. Nine separate classes of salvage were designated by determining the salvage enzymes present. In this study emphasis has been placed on soil bacteria, which had not previously been analyzed. A number of species of soil bacteria were identified using the MIDI. The salvage enzymes were then determined for these organisms and a comparison of these isolates to the previous study was performed in order to group the new organisms into 19 salvage schemes, that is 10 more than in the previous study.