Expression, purification, crystallization and preliminary X-ray diffraction analysis of the dihydroorotase domain of human CAD.

CAD is a 243 kDa eukaryotic multifunctional polypeptide that catalyzes the first three reactions of de novo pyrimidine biosynthesis: glutamine-dependent carbamyl phosphate synthetase, aspartate transcarbamylase and dihydroorotase (DHO). In prokaryotes, these activities are associated with monofunctional proteins, for which crystal structures are available. However, there is no detailed structural information on the full-length CAD protein or any of its functional domains apart from that it associates to form a homohexamer of ∼1.5 MDa. Here, the expression, purification and crystallization of the DHO domain of human CAD are reported. The DHO domain forms homodimers in solution. Crystallization experiments yielded small crystals that were suitable for X-ray diffraction studies. A diffraction data set was collected to 1.75 Šresolution using synchrotron radiation at the SLS, Villigen, Switzerland. The crystals belonged to the orthorhombic space group C222(1), with unit-cell parameters a=82.1, b=159.3, c=61.5 Å. The Matthews coefficient calculation suggested the presence of one protein molecule per asymmetric unit, with a solvent content of 48%.

Enzymes, embryos, and ancestors.

In the 1950s, cellular regulatory mechanisms were newly recognized; with Arthur Pardee I investigated the initial enzyme of pyrimidine biosynthesis, which he discovered is controlled by feedback inhibition. The protein proved unusual in having separate but interacting sites for substrates and regulators. Howard Schachman and I dissociated the protein into different subunits, one binding regulators and one substrates. The enzyme became an early prime example of allostery. In developmental biology I studied the egg of the frog, Xenopus laevis, characterizing early processes of axis formation. My excellent students and I described cortical rotation, a 30° movement of the egg's cortex over tracks of parallel microtubules anchored to the underlying cytoplasmic core, and we perturbed it to alter Spemann's organizer and effect spectacular phenotypes. The entire sequence of events has been elucidated by others at the molecular level, making Xenopus a prime example of vertebrate axis formation. Marc Kirschner, Christopher Lowe, and I then compared hemichordate (half-chordate) and chordate early development. Despite anatomical-physiological differences, these groups share numerous steps of axis formation, ones that were probably already in use in their pre-Cambrian ancestor. I've thoroughly enjoyed exploring these areas during a 50-year period of great advances in biological sciences by the worldwide research community.

A cooperative Escherichia coli aspartate transcarbamoylase without regulatory subunits .

Here we report the isolation, kinetic characterization, and X-ray structure determination of a cooperative Escherichia coli aspartate transcarbamoylase (ATCase) without regulatory subunits. The native ATCase holoenzyme consists of six catalytic chains organized as two trimers bridged noncovalently by six regulatory chains organized as three dimers, c(6)r(6). Dissociation of the native holoenzyme produces catalytically active trimers, c(3), and nucleotide-binding regulatory dimers, r(2). By introducing specific disulfide bonds linking the catalytic chains from the upper trimer site specifically to their corresponding chains in the lower trimer prior to dissociation, a new catalytic unit, c(6), was isolated consisting of two catalytic trimers linked by disulfide bonds. Not only does the c(6) species display enhanced enzymatic activity compared to the wild-type enzyme, but the disulfide bonds also impart homotropic cooperativity, never observed in the wild-type c(3). The c(6) ATCase was crystallized in the presence of phosphate and its X-ray structure determined to 2.10 A resolution. The structure of c(6) ATCase liganded with phosphate exists in a nearly identical conformation as other R-state structures with similar values calculated for the vertical separation and planar angles. The disulfide bonds linking upper and lower catalytic trimers predispose the active site into a more active conformation by locking the 240s loop into the position characteristic of the high-affinity R state. Furthermore, the elimination of the structural constraints imposed by the regulatory subunits within the holoenzyme provides increased flexibility to the c(6) enzyme, enhancing its activity over the wild-type holoenzyme (c(6)r(6)) and c(3). The covalent linkage between upper and lower catalytic trimers restores homotropic cooperativity so that a binding event at one or so active sites stimulates binding at the other sites. Reduction of the disulfide bonds in the c(6) ATCase results in c(3) catalytic subunits that display kinetic parameters similar to those of wild-type c(3). This is the first report of an active c(6) catalytic unit that displays enhanced activity and homotropic cooperativity.

Asymmetric allosteric signaling in aspartate transcarbamoylase.

Here we use the fluorescence from a genetically encoded unnatural amino acid, l-(7-hydroxycoumarin-4-yl)ethylglycine (HCE-Gly), replacing an amino acid in the regulatory site of Escherichia coli aspartate transcarbamoylase (ATCase) to decipher the molecular details of regulation of this allosteric enzyme. The fluorescence of HCE-Gly is exquisitely sensitive to the binding of all four nucleotide effectors. Although ATP and CTP are primarily responsible for influencing enzyme activity, the results of our fluorescent binding studies indicate that UTP and GTP bind with similar affinities, suggesting a dissociation between nucleotide binding and control of enzyme activity. Furthermore, while CTP is the strongest regulator of enzyme activity, it binds selectively to only a fraction of regulatory sites, allowing UTP to effectively fill the residual ones. Our results suggest that CTP and UTP are not competing for the same binding sites, but instead reveal an asymmetry between the two allosteric sites on the regulatory subunit of the enzyme. Correlation of binding and activity measurements explain how ATCase uses asymmetric allosteric sites to achieve regulatory sensitivity over a broad range of heterotropic effector concentrations.

Assignment of Ile, Leu, and Val methyl correlations in supra-molecular systems: an application to aspartate transcarbamoylase.

A number of complementary approaches for the assignment of Ile, Leu, and Val methyl groups in Methyl-TROSY spectra of supra-molecular protein complexes are presented and compared. This includes the transfer of assignments from smaller fragments to the complex using a "divide-and-conquer" approach, assignment transfer via exchange spectroscopy, or, alternatively, generating assignments of the complex through the measurement of pseudocontact shifts, facilitated by the introduction of paramagnetic probes. The methodology is applied to the assignment of the regulatory chains in the 300 kDa enzyme aspartate transcarbamoylase, ATCase. The "divide-and-conquer" method that has proven to be very powerful in applications to other systems produced assignments for approximately 60% of the observed methyl groups in TROSY maps of ATCase. By contrast, the combination of all approaches led to assignments for 86% of the methyls, providing a large number of probes of structure and dynamics. The derived assignments were used to interpret chemical shift changes of ATCase upon titration with the nucleotide ATP. Large shift changes in the N-terminal tails of the regulatory chain provide the first evidence for structural perturbations in a region that is known to play a critical role on the effect of nucleotide binding on distal catalytic sites of this allosteric enzyme.

Expression, purification, crystallization and preliminary X-ray crystallographic studies of a cold-adapted aspartate carbamoyltransferase from Moritella profunda.

Aspartate carbamoyltransferase (ATCase) catalyzes the carbamoylation of the alpha-amino group of L-aspartate by carbamoyl phosphate (CP) to yield N-carbamoyl-L-aspartate and orthophosphate in the first step of de novo pyrimidine biosynthesis. Apart from its key role in nucleotide metabolism, the enzyme is generally regarded as a model system in the study of proteins exhibiting allosteric behaviour. Here, the successful preparation, crystallization and diffraction data collection of the ATCase from the psychrophilic bacterium Moritella profunda are reported. To date, there is no structural representative of a cold-adapted ATCase. The structure of M. profunda ATCase is thus expected to provide important insights into the molecular basis of allosteric activity at low temperatures. Furthermore, through comparisons with the recently reported structure of an extremely thermostable ATCase from Sulfolobus acidocaldarius, it is hoped to contribute to general principles governing protein adaptation to extreme environments. A complete native data to 2.85 A resolution showed that the crystal belongs to space group P3(2)21, with unit-cell parameters a = 129.25, b = 129.25, c = 207.23 A, alpha = beta = 90, gamma = 120 degrees, and that it contains three catalytic and three regulatory subunits per asymmetric unit. The three-dimensional structure of the Escherichia coli ATCase was sufficient to solve the structure of the M. profunda ATCase via the molecular-replacement method and to obtain electron density of good quality.

The role of intersubunit interactions for the stabilization of the T state of Escherichia coli aspartate transcarbamoylase.

Homotropic cooperativity in Escherichia coli aspartate transcarbamoylase results from the substrate-induced transition from the T to the R state. These two alternate states are stabilized by a series of interdomain and intersubunit interactions. The salt link between Lys-143 of the regulatory chain and Asp-236 of the catalytic chain is only observed in the T state. When Asp-236 is replaced by alanine the resulting enzyme exhibits full activity, enhanced affinity for aspartate, no cooperativity, and no heterotropic interactions. These characteristics are consistent with an enzyme locked in the functional R state. Using small angle x-ray scattering, the structural consequences of the D236A mutant were characterized. The unliganded D236A holoenzyme appears to be in a new structural state that is neither T, R, nor a mixture of T and R states. The structure of the native D236A holoenzyme is similar to that previously reported for another mutant holoenzyme (E239Q) that also lacks intersubunit interactions. A hybrid version of aspartate transcarbamoylase in which one catalytic subunit was wild-type and the other had the D236A mutation was also investigated. The hybrid holoenzyme, with three of the six possible interactions involving Asp-236, exhibited homotropic cooperativity, and heterotropic interactions consistent with an enzyme with both T and R functional states. Small angle x-ray scattering analysis of the unligated hybrid indicated that the enzyme was in a new structural state more similar to the T than to the R state of the wild-type enzyme. These data suggest that three of the six intersubunit interactions involving D236A are sufficient to stabilize a T-like state of the enzyme and allow for an allosteric transition.

Molecular cloning, recombinant expression and partial characterization of the aspartate transcarbamoylase from Toxoplasma gondii.

A cDNA coding for a monofunctional aspartate transcarbamoylase (ATCase) was isolated from a Toxoplasma gondii tachyzoite cDNA library using a complementation method. The calculated molecular mass of the deduced amino acid sequence was 46.8 kDa, with a predicted pI of 7.1. Size exclusion chromatography/laser-light scattering showed a single, monodisperse peak with molecular mass of 144 kDa. Amino acid sequence alignments revealed that active site residues of the Escherichia coli ATCase catalytic chain were conserved in the T. gondii sequence, and the latter shared 26-33% overall sequence identity with other ATCases. A recombinant enzyme was overexpressed in E. coli, and was purified with a yield of approximately 0.8 mg l(-1) culture. The temperature dependence of the recombinant enzyme was similar to that of native ATCase in T. gondii extracts. The K(m)'s for aspartate and carbamoyl phosphate were 7.82 mM, and 67.6 microM, respectively. The V(max) was 23900 micromol h(-1) mg(-1). Pyrimidine nucleotides had no significant effect on the enzyme's activity. N-phosphonoacetyl-L-aspartate (PALA) inhibited the enzyme with K(i)=0.38 microM. The T. gondii ATCases contained two additional sequences of approximately 24 residues each, which are not found in other ATCases. One of these sequences was susceptible to proteolysis by elastase.

Random circular permutation leading to chain disruption within and near alpha helices in the catalytic chains of aspartate transcarbamoylase: effects on assembly, stability, and function.

A collection of circularly permuted catalytic chains of aspartate transcarbamoylase (ATCase) has been generated by random circular permutation of the pyrB gene. From the library of ATCases containing permuted polypeptide chains, we have chosen for further investigation nine ATCase variants whose catalytic chains have termini located within or close to an alpha helix. All of the variants fold and assemble into dodecameric holoenzymes with similar sedimentation coefficients and slightly reduced thermal stabilities. Those variants disrupted within three different helical regions in the wild-type structure show no detectable enzyme activity and no apparent binding of the bisubstrate analog N:-phosphonacetyl-L-aspartate. In contrast, two variants whose termini are just within or adjacent to other alpha helices are catalytically active and allosteric. As expected, helical disruptions are more destabilizing than loop disruptions. Nonetheless, some catalytic chains lacking continuity within helical regions can assemble into stable holoenzymes comprising six catalytic and six regulatory chains. For seven of the variants, continuity within the helices in the catalytic chains is important for enzyme activity but not necessary for proper folding, assembly, and stability of the holoenzyme.