This study shows that vascular smooth muscle cells express significantly higher levels of gamma interferoninducible indoleamine 2,3-dioxygenase (IDO) activity than endothelium or mononuclear cells. Since IDO activity is linked to persistent Chlamydophila pneumoniae infection, our results suggest that smooth muscle cells may be an important reservoir of that organism in atherosclerosis.Ample evidence indicates that T cells respond to local antigens and produce gamma interferon (IFN-␥) in atheroma (16). Recent attention has focused on the association of Chlamydophila pneumoniae with lesion progression (5,19,25). C. pneumoniae localizes in human atherosclerotic lesions as assessed by electron microscopy (30) and by PCR and immunostaining (17,18). Furthermore, C. pneumoniae can infect and proliferate within endothelial cells, smooth muscle cells, and macrophages in vitro (14,15).The ability of C. pneumoniae to survive chronically in vascular host tissue may derive from its capacity to establish a persistent infection. The persistent form of C. pneumoniae, characterized by enlarged abnormal forms, differs structurally and metabolically from the elementary and reticulate bodies (3,10,22). This obligate intracellular pathogen, like other chlamydial species, requires tryptophan for growth (7,27). The mammalian enzyme indoleamine 2,3-dioxygenase (IDO) catabolizes tryptophan and can lower the concentration of this amino acid in host cells. Interestingly, IFN-␥, a cytokine present in atherosclerotic plaques, can regulate IDO expression in some cells. Numerous studies have investigated the effects of various cytokines on IDO regulation in multiple cell lines (4,8,11,23,24,27,33). IFN-␥-induced IDO activity can induce the persistent form of C. pneumoniae infection in cultured cells (26). This study compared the effects of IFN-␥ on IDO activity in different types of human vascular wall cells, including saphenous vein endothelial cells (SVEC), saphenous vein smooth muscle cells (SVSMC), aortic smooth muscle cells (ASMC), and peripheral blood mononuclear cells (PBMC). SVEC, SVSMC, and ASMC were isolated as previously described (20). PBMC were isolated from healthy donors by plateletpheresis (6), followed by adherence to plastic culture flasks (2 h at 37°C) (21).SVEC, SVSMC, ASMC, and PBMC were plated at a cell density of 1.5 ϫ 10 5 cells/cm 2 in 96-well plates. Confluent monolayers were overlaid with media containing IFN-␥ (Endogen) from 0 to 800 U/ml. In some experiments, tumor necrosis factor alpha (TNF-␣) (0 to 1,000 U/ml) (Endogen) was added 24 h later. After 72 h of incubation at 37°C, the medium was replaced with [ 3 H]tryptophan pulse media containing 0.05 mM L-tryptophan (Sigma) and 1 Ci of L-5-[ 3 H]tryptophan (New England Nuclides)/ml in Hanks balanced salt solution (Gibco BRL). Plates were incubated an additional 4 h at 37°C, after which the supernatants and cell lysates prepared by 10% trichloroacetic acid extraction were collected and frozen until analysis. Each data point was determined in triplicate for two dif...

The genes from the thermophilic archaeabacterium Methanococcus jannaschii that code for the putative catalytic and regulatory chains of aspartate transcarbamoylase were expressed at high levels in Escherichia coli. Only the M. jannaschii PyrB (Mj-PyrB) gene product exhibited catalytic activity. A purification protocol was devised for the Mj-PyrB and M. jannaschii PyrI (Mj-PyrI) gene products. Molecular weight measurements of the Mj-PyrB and Mj-PyrI gene products revealed that the Mj-PyrB gene product is a trimer and the Mj-PyrI gene product is a dimer. Preliminary characterization of the aspartate transcarbamoylase from M. jannaschii cell-free extract revealed that the enzyme has a similar molecular weight to that of the E. coli holoenzyme. Kinetic analysis of the M. jannaschii aspartate transcarbamoylase from the cell-free extract indicates that the enzyme exhibited limited homotropic cooperativity and little if any regulatory properties. The purified Mj-catalytic trimer exhibited hyperbolic kinetics, with an activation energy similar to that observed for the E. coli catalytic trimer. Homology models of the Mj-PyrB and Mj-PyrI gene products were constructed based on the three-dimensional structures of the homologous E. coli proteins. The residues known to be critical for catalysis, regulation, and formation of the quaternary structure from the well characterized E. coli aspartate transcarbamoylase were compared.Organisms from the archaea, prokarya, and eukarya kingdoms all produce aspartate transcarbamoylase, the enzyme that catalyzes the committed step of the pyrimidine biosynthetic pathway, the reaction of carbamoyl phosphate and Laspartate to form N-carbamoyl-L-aspartate and inorganic phosphate (1). There are four major classes or forms of quaternary structures known for aspartate transcarbamoylases. In prokaryotes, aspartate transcarbamoylase is known to exist in three classes. The simplest is class C, a homotrimer of catalytic chains each with a molecular mass of approximately 34 kDa. The aspartate transcarbamoylase from Bacillus subtilis, which lacks both homotropic and heterotropic properties, is an example of this class (2). A second form of aspartate transcarbamoylase, class A, is a dodecamer of six 34-kDa and six 45-kDa polypeptides. Catalytic and regulatory functions of this enzyme are both located on the 34-kDa polypeptides, whereas the function of the 45-kDa polypeptides is unknown. There are several species of Pseudomonas that produce this type of aspartate transcarbamoylase, including Pseudomonas fluorescens (3, 4). The third and best characterized class of aspartate transcarbamoylase is class B, comprised of two trimeric catalytic subunits of 34-kDa polypeptides and three dimeric regulatory subunits of 17-kDa polypeptides. The class B form is an allosteric enzyme, exhibiting both homotropic and heterotropic interactions. Escherichia coli, Salmonella typhimurium, Erwinia herbicola, Serratia marcescens, and other members of the family Enterobacteriaceae produce class B aspartate transcarbamoylase ...

Stabilization of the T and R allosteric states of Escherichia coli aspartate transcarbamoylase is governed by specific intra-and interchain interactions. The six interchain interactions between Glu-239 in one catalytic chain of one catalytic trimer with both Lys-164 and Tyr-165 of a different catalytic chain in the other catalytic trimer have been shown to be involved in the stabilization of the T state. In this study a series of hybrid versions of aspartate transcarbamoylase was studied to determine the minimum number of these Glu-239 interactions necessary to maintain homotropic cooperativity and the T allosteric state. Hybrids with zero, one, and two Glu-239 stabilizing interactions do not exhibit cooperativity, whereas the hybrids with three or more Glu-239 stabilizing interactions exhibit cooperativity. The hybrid enzymes with one or more of the Glu-239 stabilizing interactions also exhibit heterotropic interactions. Two hybrids with three Glu-239 stabilizing interactions, in different geometric relationships, had identical properties. From this and previous studies, it is concluded that the 239 stabilizing interactions play a critical role in the manifestation of homotropic cooperativity in aspartate transcarbamoylase by the stabilization of the T state of the enzyme. As substrate binding energy is utilized, more and more of the T state stabilizing interactions are relaxed, and finally the enzyme shifts to the R state. In the case of the Glu-239 stabilizing interactions more than three of the interactions must be broken before the enzyme shifts to the R state. The interactions between the catalytic and regulatory chains and between the two catalytic trimers of aspartate transcarbamoylase provide a global set of interlocking interactions that stabilize the T and R states of the enzyme. The substrate-induced local conformational changes observed in the structure of the isolated catalytic subunit drive the quaternary T to R transition of aspartate transcarbamoylase and functionally induced homotropic cooperativity.Allosteric regulation is modulated by molecular transitions within a polymeric enzyme between a low activity low affinity T state and a high activity high affinity R state (1). The two states may differ on the quaternary level due to differences in the relative positions of subunits with respect to each other. Interface contacts may undergo significant changes during the T to R state transition, and these contacts often contribute to the relative stabilization of the two states of the enzyme.Escherichia coli aspartate transcarbamoylase (EC 2.1.3.2), which catalyzes the committed step of pyrimidine biosynthesis, the condensation of carbamoyl phosphate and L-aspartate to form N-carbamoyl-L-aspartate and inorganic phosphate (2), has become a model system for the study of allosteric regulation. The enzyme shows homotropic cooperativity for the substrate L-aspartate and is heterotropically regulated by ATP, CTP (2), and UTP in the presence of CTP (3). The holoenzyme from E. coli is a dodecamer composed o...

X-ray crystallographic studies indicate that the N-terminal region of the regulatory chain in Escherichia coli aspartate transcarbamoylase resides close to the effector binding site. The proximity of the N-terminal region to the binding site suggests it may be important for nucleotide binding and, therefore, the heterotropic mechanism. The N-terminal region of the structure is not well-defined since the electron density in this region is weak, indicating a flexible and mobile region. Furthermore, alanine scanning mutagenesis of residues 2-7 indicated that the N-terminal region may be involved in nucleotide binding and the heterotropic mechanism, especially, UTP recognition [Dembowski, N., and Kantrowitz, E. R. (1994) Protein Eng. 7, 673-679]. In order to investigate further the role of the N-terminal region in the heterotropic mechanism, the first 10 N-terminal residues of the regulatory chain were deleted using site-specific mutagenesis. This mutant enzyme was compared to the wild-type enzyme, and both solubility and functional differences were observed. The mutant enzyme forms an insoluble aggregate which can be solubilized by the addition of nucleotides, such as CTP, suggesting that the exposed nucleotide binding site is involved in aggregate formation. Kinetic analyses of the mutant enzyme showed a lower maximal velocity and slightly lower aspartate affinity. Apparent binding constants determined for CTP, ATP, UTP, and CTP in the presence of UTP suggest the heterotropic response is also altered. This study suggests that the N-terminal region of the regulatory subunit is important for controlling nucleotide binding, creating the high-affinity and low-affinity effector binding sites, and coupling the binding sites within the regulatory dimer.

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.Escherichia coli aspartate transcarbamoylase (EC 2.1.3.2) catalyzes the committed step of pyrimidine biosynthesis, the condensation of carbamoyl phosphate and L-aspartate to form N-carbamoyl-L-aspartate and inorganic phosphate (2). The enzyme shows homotropic cooperativity for the substrate L-aspartate and is heterotropically regulated by ATP, CTP (2), and UTP in the presence of CTP (3). The enzyme from E. coli is a dodecamer composed of six catalytic chains of M r 34,000 and six regulatory chains of M r 17,000. The catalytic chains are organized as two trimeric subunits (C), 1 whereas the regulatory chains are organized as three dimeric subunits (R). The active sites are located at the interfaces between adjacent catalytic chains, whereas the nucleotide effectors bind to the same site on each of the regulatory chains (4 -8).Two functionally and structurally different states of aspartate transcarbamoylase have been characterized. The low affinity, low activity conformation of the enzyme is described as the T state and the high affinity, high activity conformation of the enzyme is described as the R state. The conversion from the T to the R state occurs upon aspartate binding to the enzyme in the presence of carbamoyl phosphate. Structurally, the enzyme elongates by at least 11 Å along the 3-fold ax...