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.

Three of the six possible intersubunit stabilizing interactions involving Glu-239 are sufficient for restoration of the homotropic and heterotropic properties of Escherichia coli aspartate transcarbamoylase.

A hybrid version of Escherichia coli aspartate transcarbamoylase was investigated in which one catalytic subunit has the wild-type sequence, and the other catalytic subunit has Glu-239 replaced by Gln. Since Glu-239 is involved in intersubunit interactions, this hybrid could be used to evaluate the extent to which T state stabilization is required for homotropic cooperativity and for heterotropic effects. Reconstitution of the hybrid holoenzyme (two different catalytic subunits with three wild-type regulatory subunits) was followed by separation of the mixture by anion-exchange chromatography. To make possible the resolution of the three holoenzyme species formed by the reconstitution, the charge of one of the catalytic subunits was altered by the addition of six aspartic acid residues to the C terminus of each of the catalytic chains (AT-C catalytic subunit). Control experiments indicated that the AT-C catalytic subunit as well as the holoenzyme formed with AT-C and wild-type regulatory subunits had essentially the same homotropic and heterotropic properties as the native catalytic subunit and holoenzyme, indicating that the addition of the aspartate tail did not influence the function of either enzyme. The control reconstituted holoenzyme, in which both catalytic subunits have Glu-239 replaced by Gln, exhibited no cooperativity, an enhanced affinity for aspartate, and essentially no heterotropic response identical to the enzyme isolated without reconstitution. The hybrid containing one normal and one mutant catalytic subunit exhibited homotropic cooperativity with a Hill coefficient of 1.4 and responded to the nucleotide effectors at about 50% of the level of the wild-type enzyme. Small angle x-ray scattering experiments with the hybrid enzyme indicated that in the absence of ligands it was structurally similar, but not identical, to the T state of the wild-type enzyme. In contrast to the wild-type enzyme, addition of carbamoyl phosphate induced a significant alteration in the scattering pattern, whereas the bisubstrate analog N-phosphonoacetyl-L-aspartate induced a significant change in the scattering pattern indicating the transition to the R-structural state. These data indicate that in the hybrid enzyme only three of the usual six interchain interactions involving Glu-239 are sufficient to stabilize the enzyme in a low affinity, low activity state and allow an allosteric transition to occur.

Heterologous gene expression in a membrane-protein-specific system.

We have constructed an expression system for heterologous proteins which uses the molecular machinery responsible for the high level production of bacteriorhodopsin in Halobacterium salinarum. Cloning vectors were assembled that fused sequences of the bacterio-opsin gene (bop) to coding sequences of heterologous genes and generated DNA fragments with cloning sites that permitted transfer of fused genes into H. salinarum expression vectors. Gene fusions include: (i) carboxyl-terminal-tagged bacterio-opsin; (ii) a carboxyl-terminal fusion with the catalytic subunit of the Escherichia coli aspartate transcarbamylase; (iii) the human muscarinic receptor, subtype M1; (iv) the human serotonin receptor, type 5HT2c; and (v) the yeast alpha mating factor receptor, Ste2. Characterization of the expression of these fusions revealed that the bop gene coding region contains previously undescribed molecular determinants which are critical for high level expression. For example, introduction of immunogenic and purification tag sequences into the C-terminal coding region significantly decreased bop gene mRNA and protein accumulation. The bacteriorhodopsin-aspartate transcarbamylase fusion protein was expressed at 7 mg per liter of culture, demonstrating that E. coli codon usage bias did not limit the system's potential for high level expression. The work presented describes initial efforts in the development of a novel heterologous protein expression system, which may have unique advantages for producing multiple milligram quantities of membrane-associated proteins.

Streptomyces aspartate transcarbamoylase is a dodecamer with dihydroorotase activity.

Aspartate transcarbamoylase (ATCase) was purified from Streptomyces griseus. The enzyme is a dodecamer with a molecular mass of approximately 450 kDa. The holoenzyme is a complex of ATCase and active dihydroorotase (DHOase) subunits. The ATCase and DHOase activities co-purify after gel filtration and ion-exchange chromatography. Denaturing gel electrophoresis separates the holoenzyme into a 38-kDa ATCase polypeptide and a 47-kDa DHOase polypeptide. The holoenzyme retained ATCase and DHOase activity after being heated to 65 degrees C for 5 min, but after storage at 4 degrees C for 24 hours lost ATCase activity. Previously, the Pseudomonas putida Class A ATCase was defined by Schurr et al. (J Bacteriol 177, 1751-1759) as requiring an inactive DHOase to be functional. Here, we show that an active DHOase is part of the dodecameric ATCase/DHOase complex in Streptomyces. To distinguish those Class A ATCases with active DHOases from those with degenerate DHOases, we suggest the subdivision, Class A(1), for the former and Class A(2) for the latter.

Aspartate carbamoyltransferase from the thermoacidophilic archaeon Sulfolobus acidocaldarius. Cloning, sequence analysis, enzyme purification and characterization.

The genes coding for aspartate carbamoyltransferase (ATCase) in the extremely thermophilic archaeon Sulfolobus acidocaldarius have been cloned by complementation of a pyrBI deletion mutant of Escherichia coli. Sequencing revealed the existence of an enterobacterial-like pyrBI operon encoding a catalytic chain of 299 amino acids (34 kDa) and a regulatory chain of 170 amino acids (17.9 kDa). The deduced amino acid sequences of the pyrB and pyrI genes showed 27.6-50% identity with archaeal and enterobacterial ATCases. The recombinant S. acidocaldarius ATCase was purified to homogeneity, allowing the first detailed studies of an ATCase isolated from a thermophilic organism. The recombinant enzyme displayed the same properties as the ATCase synthesized in the native host. It is highly thermostable and exhibits Michaelian saturation kinetics for carbamoylphosphate (CP) and positive homotropic cooperative interactions for the binding of L-aspartate. Moreover, it is activated by nucleoside triphosphates whereas the catalytic subunits alone are inhibited. The holoenzyme purified from recombinant E. coli cells or present in crude extract of the native host have an Mr of 340 000 as estimated by gel filtration, suggesting that it has a quaternary structure similar to that of E. coli ATCase. Only monomers could be found in extracts of recombinant E. coli or Saccharomyces cerevisiae cells expressing the pyrB gene alone. In the presence of CP these monomers assembled into trimers. The stability of S. acidocaldarius ATCase and the allosteric properties of the enzyme are discussed in function of a modeling study.

Wheat-germ aspartate transcarbamoylase: revised purification, stability and re-evaluation of regulatory kinetics in terms of the Monod-Wyman-Changeux model.

A revised and simplified purification scheme for aspartate transcarbamoylase (ATCase) from wheat-germ is reported, with an eightfold increase in scale (yielding approximately 10 mg of the pure protein from 4 kg of wheat-germ), and improved characteristics of stability and regulatory kinetics. The ATCase obtained is greater than 96% pure, as judged by polyacrylamide gel electrophoresis. The long-term stability (i.e. on a time-scale of several hours to weeks) of the activity of the purified enzyme, under various storage conditions, was investigated. At 4 degreesC and pH 7.5, stability was found to be strongly dependent on protein concentration (increased stability at high concentration), buffer concentration (decreased stability at high buffer concentration) and the inclusion of glycerol (increased stability with increasing glycerol concentration). The enzyme is routinely stored at 4 degreesC, in 0. 05 m Tris/HCl buffer containing 25% glycerol and at high protein concentration (approximately 1 mg.mL-1, or 10 microm in trimers). Under these conditions, the half-life of the enzyme activity is greater than 300 days. Over the time-scale of kinetic experiments (up to 20 min), the diluted activity (at around 1 nm of ATCase, in the presence of ligands) is completely stable. The specific activity remains constant in the range 0.1-10 nm, in the absence and presence of ligands, showing that dissociation of the trimeric enzyme into its subunits is negligible. Steady-state kinetics were examined using the enzyme at a concentration of 1.3 nm. Initial-rate curves for both allosteric ligands, carbamoylphosphate (CP) and uridine 5'-monophosphate (UMP), showed pronounced sigmoidicity, each in the presence of the other. In the absence of UMP, initial-rate curves for CP are hyperbolic. The initial rate data fit reasonably well to a trimeric Monod-Wyman-Changeux model, suggesting a two-state conformational mechanism, greatly favouring the active (R) state when both ligands are absent, in which the R-state binds CP exclusively (dissociation constant = 23.2 microm), and the T-state binds UMP exclusively (dissociation constant = 0.49 microm). This regulatory behaviour was found to be quite stable, and was indistinguishable from that of the enzyme in a freshly made crude extract, even after storage of the pure sample for 5 months. This enzyme preparation is therefore free of the anomalous allosteric kinetics produced by a previous purification scheme, in which the affinity for UMP was markedly reduced, CP rate curves showed no sigmoidicity, while UMP rate curves had sigmoidicity exaggerated by a low maximum.

Life-cycle-dependent changes of aspartate carbamoyltransferase localization in membranes of Saccharomyces cerevisiae--centrifugal elutriation and ultracytochemical study.

Exponential culture of a Saccharomyces cerevisiae strain with overexpressed aspartate carbamoyltransferase activity (ACTase) was chilled in ice and fractionated by centrifugal elutriation to several cell populations of increasing cell mass. The enzyme activity which belongs to the pyrimidine biosynthesis pathway, was detected in situ by a specific ultracytochemical reaction: the ACTase byproduct, monophosphate, was precipitated by cerium ions to cerium phosphate. During the outgrowth of nonbudding daughter cells (zero cells) the label appeared first in membranes of nuclear envelope and of mitochondria. In larger zero cells, this label appeared also in the endoplasmic reticulum, microvesicles and plasmalemma. In budding mother cells, the label was conspicuous in the whole cell-membrane complex. In most aged cells the ACTase activity was not detectable. The presence of ACTase activity in membranes of compartments conveying glycoproteins via the secretory pathway remains to be explained. To confirm the in situ detection of ACTase activity in membranes, we assayed the enzyme activity in both the 10,000 g sediment and supernatant prepared from yeast homogenate precentrifuged at 3000 g. From 23 to 43% of ACTase activity was detected in the sediments including membranes of wild-type and ACTase-overexpressing strains.

Properties of aspartate transcarbamylase from TAD1, a psychrophilic bacterial strain isolated from Antarctica.

TAD1 is a psychrophilic strain isolated from continental frozen water in Antarctica. Study of aspartate transcarbamylase in the bacterium shows an impressive activity of this enzyme at low temperature. At 0 degree C, its activity is up to 26% of its maximal activity observed at 30 degrees C. In comparison with the Escherichia coli enzyme, some of its kinetic properties suggest that this high activity at low temperature results from an increased catalytic efficiency. This property might result from a discrete modification localized at the catalytic site, since this psychrophilic enzyme is as stable as its Escherichia coli homologue at high temperature.

Conversion of the allosteric regulatory patterns of aspartate transcarbamoylase by exchange of a single beta-strand between diverged regulatory chains.

Although structurally very similar, the aspartate transcarbamoylases (ATCase) of Serratia marcescens and Escherichia coli differ in both regulatory and catalytic characteristics. Most notably, CTP stimulates the catalytic activity of the S. marcescens ATCase and CTP/UTP inhibitory synergism has been lost. These allosteric characteristics contradict the traditional logic developed from the E. coli enzyme in which CTP and UTP function together as end products of the pyrimidine pathway to allosterically control the catalytic activity. In this study, five divergent residues (r93-r97) of the regulatory polypeptide of the S. marcescens enzyme have been replaced with their E. coli counterparts. These residues correspond to the S5' beta-strand of the allosteric effector binding domain at the junction of the allosteric and zinc domains of the regulatory polypeptide. In spite of the fact that the chimeric ATCase (SM:rS5'ec) retained 455 out of 460 amino acids of the S. marcescens enzyme, it possessed characteristics similar to those of the E. coli enzyme: (1) the [Asp]0.5 decreased from 40 to 5 mM; (2) ATP activation of the enzyme was greatly reduced; (3) CTP was converted from a strong activator to a strong inhibitor; and (4) the synergistic inhibition by CTP and UTP was restored. The S5' beta-strand is located at the outer surface of a five-stranded beta-sheet of the allosteric domain, providing a potential structural mechanism defining the allostery of this enzyme.

A reappraisal of the diversity and class distribution of aspartate transcarbamoylases in gram-negative bacteria.

Recently, the subunit composition of class A aspartate transcarbamoylases (ATCases) in fluorescent pseudomonads has been clarified. We present evidence that distribution of this type of ATCase may be more widespread than at first suspected. Bacterial ATCases exist in three forms: class A (molecular mass approximately 450-500 kDA); class B, typified by Escherichia coli ATCase (approximately 300 kDa); and class C, typified by Bacillus subtilis ATCase (approximately 100 kDa). Using gradient gel electrophoresis with activity-staining to scan bacterial sonicates, we report the existence of six more class ATCases. We have purified one of these, Acinetobacter calcoaceticus ATACase, and found its subunit composition to be similar to that of the pseudomonad ATCases. Two of these ATCases come from bacteria outside the gamma-subgroup of the Proteobacteria, one from the alpha-subgroup and one from Deinococcus radiophilus, a species phylogenetically remote from the Proteobacteria. Unexpectedly, three bacterial species, closely related to the fluorescent pseudomonads and acinetobacters, have ATCases of 100 kDa (class C). One of these, Stenotrophomonas (formerly Xanthomonas) maltophilia has been purified and found to be a homotrimer of 35 kDa polypeptide chains. We believe this is the first time that class C ATCases have been reported in Gram-negative bacteria. A distinctive cluster in the gamma-3 subgroup of the Proteobacteria is formed by the enteric bacteria and their relatives. So far only class B ATCases have been reported in this group. The evolutionary implications of these findings are discussed.