Immunochemical analysis of the domain structure of CAD, the multifunctional protein that initiates pyrimidine biosynthesis in mammalian cells

CAD, is a multidomain polypeptide, with a molecular weight of over 200,000, that has glutamine-dependent carbamyl-phosphate synthetase, aspartate transcarbamylase, and dihydroorotase activity as well as regulatory sites that bind UTP and 5-phosphoribosyl 1-pyrophosphate. The protein thus catalyzes the first three steps of de novo pyrimidine biosynthesis and controls the activity of the pathway in higher eukaryotes. Controlled proteolysis of CAD isolated from Syrian hamster cells, cleaves the molecule into seven major proteolytic fragments that contain one or more of the functional domains. The two smallest fragments, which had molecular weights of 44,000 and 40,000, corresponded to the fully active dihydroorotase (DHO) and aspartate transcarbamylase (ATC) domains, respectively, but the larger fragments have not been previously characterized. In this study, enzymatic assays of partially fractionated digests and immunoblotting with antibodies specifically directed against the purified ATC domain, the purified dihydroorotase domain and an 80-kDa fragment of the putative carbamyl-phosphate synthetase domain established the precursor-product relationships among all of the major proteolytic fragments of CAD. These results indicate that 1) only the intact molecule had all of the functional domains, 2) a species with a molecular weight of 200,000 was produced in the first step of proteolysis which had glutamine-dependent carbamyl-phosphate synthetase and dihydroorotase activity, but neither aspartate transcarbamylase activity nor the antigenic determinants present on the isolated ATC domain, and 3) cleavage of the 200-kDa species produced a species, with a molecular mass of 150,000 which lacked both aspartate transcarbamylase and dihydroorotase domains. This 150-kDa species, containing the postulated carbamyl-phosphate synthetase, glutamine, and regulatory (UTP, 5-phosphoribosyl 1-pyrophosphate) domains, had two elastase-sensitive sites that divided this region of the polypeptide chain into 10-, 65-, and 80-kDa segments. The location of the functional sites on these segments has not yet been established. The immunochemical analysis also revealed the existence of possible precursors of the stable aspartate transcarbamylase and dihydroorotase domains, suggesting that the chain segments connecting the functional domains of CAD are extensive and that the overall size of the intact polypeptide chain has been underestimated. On the basis of these studies we have proposed a model of the domain structure of CAD.     

Dihydroorotase from Escherichia coli. Purification and characterization

Dihydroorotase (4,5-L-dihydroorotate amidohydrolase (EC 3.5.2.3], which catalyzes the reversible cyclization of N-carbamyl-L-aspartate to dihydro-L-orotate, has been purified to homogeneity from an over-producing strain of Escherichia coli. Treatment of 70 g of frozen cell paste produces about 7 mg of pure enzyme, a yield of about 35%. The native molecular weight, determined by equilibrium sedimentation, is 80,900 +/- 4,300. The subunit molecular weight, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 38,400 +/- 2,600, and by amino acid analysis is 41,000. The enzyme is thus a dimer and contains 0.95 +/- 0.08 tightly bound zinc atoms per subunit when isolated by the described procedure, which would remove any loosely bound metal ions. Isoelectric focusing under native conditions yields a major species at isoelectric point 4.97 +/- 0.27 and a minor species at 5.26 +/- 0.27; dihydroorotase activity is proportionately associated with both bands. The enzyme has a partial specific volume of 0.737 ml/g calculated from the amino acid composition and a specific absorption at 278 nm of 0.638 for a 1 mg/ml solution. At 30 degrees C, the Michaelis constant and kcat for dihydro-DL-orotate (at pH 8.0) are 0.0756 mM and 127 s-1, respectively; for N-carbamyl-DL-aspartate (at pH 5.80), they are 1.07 mM and 195 s-1.     

The structural organization of the hamster multifunctional protein CAD. Controlled proteolysis, domains, and linkers.

CAD is a multidomain protein that catalyzes the first three steps in mammalian de novo pyrimidine biosynthesis. The 243-kDa polypeptide consists of four functional domains; glutamine amidotransferase (GLNase), carbamyl phosphate synthetase (CPSase), aspartate transcarbamylase (ATCase), and dihydroorotase (DHOase). Controlled proteolysis of hamster CAD was found to cleave the molecule into 18 fragments which successively accumulate and disappear during the course of digestion. Each fragment was isolated and partially sequenced to determine its location in the polypeptide chain. Proteolysis was found to usually occur at the junctions between the domains and sub-domains identified by sequence homology. All proteases of low to moderate specificity cleaved the molecule in a similar fashion. The rate of proteolysis widely varied and the interdomain regions were not always accessible to proteases. Each of the major functional domains is postulated to consist of subdomains. The duplicated halves of the CPSase domain (116 kDa) have a homologous structure consisting of 11-, 25-26-, and 21-22-kDa subdomains. Prolonged digestion cleaved the DHOase domain (36.6 kDa) into two stable species suggesting that this region is comprised of 11.5- and 15.0-kDa subdomains. Similarly, proteolysis of the 21-kDa catalytic subdomain of the GLNase domain (40 kDa) indicated a bilobal structure consisting of 12.3- and 8.5-kDa chain segments. The connecting region between the two ATCase subdomains (16.4 and 18 kDa) was not cleaved. Copurification of many of the domains showed that they remain associated by noncovalent interactions even after the connecting segments have been cleaved. The chain segments, the linkers, which connect the domains and subdomains were conserved in length but not in sequence, were predicted to be relatively hydrophilic and flexible but did not show a tendency to assume a particular secondary structure. These studies provide a more detailed map of the structural organization of the CAD polypeptide.     

PHYLOGENETIC IMPLICATIONS OF THE CAD COMPLEX FROM THE PRIMITIVE RED ALGA CYANIDIOSCHYZON MEROLAE (CYANIDIALES,RHODOPHYTA)1

The de novo pyrimidine biosynthetic pathway consists of six enzymes: carbamoyl‐phosphate synthetase II (CPS II), aspartate carbamoyltransferase (ACT), dihydroorotase (DHO), dihydroorotate dehydrogenase, orotate phosphoribosyltransferase, and orotidine‐5′‐monophosphate decarboxylase. The origin and organization of the first three enzymes differ markedly between Opisthokonta (Metazoa and Fungi) and the Amoebozoa and green plants. However, no information has been available regarding the characteristics of such genes in other photosynthetic eukaryotes. In this study, we examined the pyrimidine biosynthetic cluster in the primitive red alga Cyanidioschyzon merolae P. DeLuca et al. isolate 10D. Unlike the situation in green plants, the CPS II, ACT, and DHO of C. merolae were fused to form a single open reading frame (the CAD complex), as in the Opisthokonta and Amoebozoa. Phylogenetic analysis of the CPS domain sequences suggested that this red algal CAD complex did not result from a recent lateral gene transfer from Metazoa or Fungi but that the fusion of the three genes occurred before the divergence of Opisthokonta, Amoebozoa, and the red algae. These results cast doubt on the recent hypothesis that the Opisthokonta and Amoebozoa form a monophyletic group, based on the presence in both of the CAD complex.     

Intersubunit communication in the dihydroorotase–aspartate transcarbamoylase complex of Aquifex aeolicus

Aspartate transcarbamoylase and dihydroorotase, enzymes that catalyze the second and third step in de novo pyrimidine biosynthesis, are associated in dodecameric complexes in Aquifex aeolicus and many other organisms. The architecture of the dodecamer is ideally suited to channel the intermediate, carbamoyl aspartate from its site of synthesis on the ATC subunit to the active site of DHO, which catalyzes the next step in the pathway, because both reactions occur within a large, internal solvent‐filled cavity. Channeling usually requires that the reactions of the enzymes are coordinated so that the rate of synthesis of the intermediate matches its rate of utilization. The linkage between the ATC and DHO subunits was demonstrated by showing that the binding of the bisubstrate analog, N‐phosphonacetyl‐L ‐aspartate to the ATC subunit inhibits the activity of the distal DHO subunit. Structural studies identified a DHO loop, loop A, interdigitating between the ATC domains that would be expected to interfere with domain closure essential for ATC catalysis. Mutation of the DHO residues in loop A that penetrate deeply between the two ATC domains inhibits the ATC activity by interfering with the normal reciprocal linkage between the two enzymes. Moreover, a synthetic peptide that mimics that part of the DHO loop that binds between the two ATC domains was found to be an allosteric or noncompletive ATC inhibitor (K_i = 22 μM). A model is proposed suggesting that loop A is an important component of the functional linkage between the enzymes.     

Organization of the functional domains of anthranilate synthase from Neurospora crassa. Limited proteolysis studies

Treatment of the multifunctional alpha 2 beta 2 anthranilate synthase complex of Neurospora crassa with elastase produced two fragments of the complex, one possessing anthranilate synthase activity and the other having both indole-3-glycerol phosphate (InGP) synthase and N-(5'-phosphoribosyl)anthranilate (PRA) isomerase activities. Sequencing the NH2 terminus of the InGP synthase-PRA isomerase fragment revealed that cleavage was between positions 237 and 238 of the beta-subunit within a segment of the polypeptide chain which links the glutamine-binding (G) domain with the InGP synthase-PRA isomerase domains. The fragment containing anthranilate synthase activity has a molecular weight of 98,000, as estimated by gel filtration, and is composed of an apparently intact alpha-subunit (70 kDa) associated with the G-domain fragment (29 kDa) derived from the beta-subunit. The alpha X G-domain complex was resistant to further degradation by elastase. When either the alpha 2 beta 2 complex or the alpha X G-domain complex was incubated with trypsin, the alpha-subunit was degraded to a 66-kDa alpha-fragment with reduced enzymatic activity, which was resistant to further cleavage. In contrast, incubation of alpha-subunit alone with either elastase or trypsin resulted in its complete degradation, indicating that association of the alpha-subunit with either G-domain or beta-subunit protected the alpha-subunit from this extensive degradation. A model for the anthranilate synthase complex is proposed in which the trifunctional beta-subunit forms a dimer by the self-association of the InGP synthase-PRA isomerase domains; the G-domain is connected to the InGP synthase-PRA isomerase domain by a relatively disordered region of the peptide chain which, in the alpha 2 beta 2 complex, remains susceptible to proteases; and neither alpha-subunit nor G-domain significantly self-associates.     

Purification and characterization of a plasminogen activator inhibitor from the histiocytic lymphoma cell line U-937

We report the production, purification, characterization, and partial amino acid sequence of a plasminogen inhibitor (PA-I). The starting material is culture fluid from phorbol myristate 13-acetate-treated U-937 cells and the isolation steps consist of preparative isoelectric focusing followed by affinity chromatography on Cibacron Blue-Sepharose. PA-I migrates as a closely spaced doublet of 47-kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and forms covalent complexes with urokinase and two-chain tissue-type plasminogen activator, displaying second order rate constants of 0.9 X 10(6) M-1 s-1 and 0.2 X 10(6) M-1 s-1, respectively. Upon treatment with 1 M NH4OH, the covalent complexes were hydrolyzed, yielding a 35-kDa inhibitor fragment. A partial amino acid sequence of PA-I showed that it belongs to the antithrombin III family of inhibitors. PA-I is immunologically related to a PA-inhibitor from human placenta. mRNA from phorbol myristate 13-acetate-treated U-937 cells directed, in a rabbit reticulocyte derived cell-free system, the biosynthesis of only one 47-kDa protein that could be immunoprecipitated with anti-PA-I IgG, indicating that the two molecular forms of PA-I are the products of post-translational processing.     

The crystal structure of a novel, latent dihydroorotase from Aquifex aeolicus at 1.7A resolution

Dihydroorotases (EC 3.5.2.3) catalyze the reversible cyclization of carbamoyl aspartate to form dihydroorotate in de novo pyrimidine biosynthesis. The X-ray structures of Aquifex aeolicus dihydroorotase in two space groups, C222(1) and C2, were determined at a resolution of 1.7A. These are the first structures of a type I dihydroorotase, a class of molecules that includes the dihydroorotase domain of mammalian CAD. The type I enzymes are more ancient and larger, at 45 kDa, than the type II enzymes exemplified by the 38 kDa Escherichia coli dihydroorotase. Both dihydroorotases are members of the metallo-dependent hydrolase superfamily, whose members have a distorted "TIM barrel" domain containing the active site. However, A.aeolicus dihydroorotase has a second, composite domain, which the E.coli enzyme lacks and has only one of the two zinc atoms present in the E.coli enzyme. A.aeolicus dihydroorotase is unique in exhibiting significant activity only when complexed with aspartate transcarbamoylase, whereas the E.coli dihydroorotase and the CAD dihydroorotase domain are active as free proteins. The latency of A.aeolicus dihydroorotase can be related to two differences between its structure and that of E.coli dihydroorotase: (1) the monoclinic structure has a novel cysteine ligand to the zinc that blocks the active site and possibly functions as a "cysteine switch"; and (2) active site residues that bind the substrate in E.coli dihydroorotase are located in disordered loops in both crystal structures of A.aeolicus dihydroorotase and may function as a disorder-to-order "entropy switch".     

Mutations of the anti-mullerian hormone gene in patients with persistent mullerian duct syndrome: biosynthesis, secretion, and processing of the abnormal proteins and analysis using a three-dimensional model

Anti-Müllerian hormone (AMH), a TGF-beta family member, determines whether an individual develops a uterus and Fallopian tubes. Mutations in the AMH gene lead to persistent Müllerian duct syndrome in males. The wild-type human AMH protein is synthesized as a disulfide-linked dimer of two identical 70-kDa polypeptides, which undergoes proteolytic processing to generate a 110-kDa N-terminal dimer and a bioactive 25-kDa TGF-beta-like C-terminal dimer. We have studied the biosynthesis and secretion of wild-type AMH and of seven persistent Müllerian duct syndrome proteins, containing mutations in either the N- or C-terminal domain. Mutant proteins lacking the C-terminal domain are secreted more rapidly than full-length AMH, whereas single amino acid changes in both domains can have profound effects on protein stability and folding. The addition of a cysteine in an N-terminal domain mutant, R194C, prevents proper folding, whereas the elimination of the cysteine involved in forming the interchain disulfide bond, in a C-terminal domain mutant, C525Y, leads to a truncation at the C terminus. A molecular model of the AMH C-terminal domain provides insights into how some mutations could affect biosynthesis and function.     

The evolutionary history of the first three enzymes in pyrimidine biosynthesis

Some metabolic pathways are nearly ubiquitous among organisms: the genes encoding the enzymes for such pathways must therefore be ancient and essential. De novo pyrimidine biosynthesis is an example of one such metabolic pathway. In animals a single protein called CAD

1 Abbreviations: CAD, trifunctional protein catalyzing the first three steps of de novo pyrimidine biosynthesis in higher eukaryotes; CPS, carbamyl phosphate synthetase domain; CPSase, carbamyl phosphate synthetase activity; ATC, aspartate transcarbamylase domain; ATCase, aspartate transcarbamylase activity; DHO, dihydroorotase domain; DHOase, dihydroorotase activity; GLN, glutaminase subdomain or subunit of carbamyl phosphate synthetase, GL Nase, glutaminase activity; SYN, synthetase subdomain or subunit of carbamyl phosphate synthetase; SYNase, synthetase activity.

carries the first three steps of this pathway. The same three enzymes in prokaryotes are associated with separate proteins. The CAD gene appears to have evolved through a process of gene duplication and DNA rearrangement, leading to an in-frame gene fusion encoding a chimeric protein. A driving force for the creation of eukaryotic genes encoding multienzymatic proteins such as CAD may be the advantage of coordinate expression of enzymes catalyzing steps in a biosynthetic pathway. The analogous structure in bacteria is the operon. Differences in the translational mechanisms of eukaryotes and prokaryotes may have dictated the different strategies used by organisms to evolve coordinately regulated genes.     

Physical properties, limited proteolysis, and acetylation of tyrosine aminotransferase from rat liver

The native and one of the modified forms of tyrosine aminotransferase were purified from rat liver and characterized. Several hydrodynamic properties of the native enzyme are: Stokes radius, 46 A; subunit isoelectric point, 5.6; sedimentation coefficient, 5.6 S, frictional ratio, 1.44; diffusion coefficient, 4.65 X 10(-7) cm2 s-1; extinction coefficient of a 1% solution (w:v) at 280 nm, 10.5 cm-1. The molecular weight of the dimeric protein is 110,500 as calculated from the Stokes radius and sedimentation coefficient. The subunit of the modified form is of lower molecular weight than the subunit of the native enzyme and has a pI of about 5.9. During isoelectric focusing, both forms of the enzyme separate into two components. The more acidic component that is resolved from the native enzyme is phosphorylated, but the other component is not. The amino acid composition of native tyrosine aminotransferase differs from values reported for mixtures of the three forms of this enzyme. Neither the native nor the modified forms of the enzyme possess a free alpha-amino group as judged by dansylation, nor can they be digested with leucine aminopeptidase, implying that the NH2-terminus is blocked. The possibility that tyrosine aminotransferase is acetylated was examined by translating poly(A)+RNA from hepatoma cells in a cell-free translational system in the presence and absence of inhibitors of protein acetylation. [35S]Tyrosine aminotransferase synthesized in the presence of the inhibitors has a more basic isoelectric point than the native enzyme as determined by isoelectric focusing, suggesting that the enzyme is acetylated either at the NH2-terminal or the epsilon-amino group of an internal lysine. When digested by either of two lysosomal proteases, tyrosine aminotransferase is cleaved to a smaller size. These data show that tyrosine aminotransferase is susceptible to several post-translational modifications.     

Effect of 6-azauridine on de novo pyrimidine biosynthesis in cultured Ehrlich ascites cells. Orotate inhibition of dihydroorotase and dihydroorotate dehydrogenase.

The inhibition of dihydro-orotase (E 3.5.2.3) and dihydroorotate (DHO) dehydrogenase (dihydro-orotate oxidase, EC 1.3.3.1) by cellular orotate (OA) in Ehrlich ascites cells was studied by measuring the accumulation of the intermediates of de novo pyrimidine biosynthesis at various times after the addition of 6-azauridine to the culture medium. The addition of 6-azauridine resulted in the accumulation of orotidine, OA, DHO, and carbamyl aspartate (CAA). The use of the observed ratios of [CCA]/[OA] and [DHO]/[OA] and other known constants allowed us to calculate that the increased cellular OA concentration caused primarily an inhibition of DHO dehydrogenase rather than an inhibition of dihydroorotase. A constant ratio of [CAA]/[DHO] was observed which probably indicates that the interconversion of these two intermediates catalyzed by dihydroorotase is near equilibrium in these cells as has been observed in vitro (Christopherson, R.I., Matsuura, T., and Jones, M.E. (1978) Anal. Biochem. 89, 225-234). It is suggested that the probable intracellular accumulation of CAA in patients with oroticaciduria may have significant secondary effects.     

Organization of a multifunctional protein in pyrimidine biosynthesis. Analyses of active, tryptic fragments

The multifunctional protein which catalyzes the first three steps of pyrimidine biosynthesis in hamster cells can be cleaved by trypsin into enzymatically active fragments. When the fragments are separated by nondenaturing polyacrylamide gel electrophoresis, three major polypeptide bands appear. Carbamyl phosphate synthetase (EC 2.7.2.9), aspartate transcarbamylase (EC 2.1.3.2), and dihydroorotase (EC 3.5.2.3) activities are associated with 129,000-, 660,000-, and 94,000-dalton bands, respectively. Further analysis of these fragments by denaturing polyacrylamide gel electrophoresis has shown that the aspartate transcarbamylase band seen on the nondenaturing gel is actually a large aggregate of 39,000-dalton fragments and the dihydroorotase band is a dimer of 44,000-dalton fragments. The data reported here indicate that (i) this multifunctional protein is composed of three enzymatically independent domains, (ii) the sum of the molecular weights of these three domains (129,000 + 39,000 + 44,000 = 212,000) is similar to that of the undigested monomer (220,000 daltons), and (iii) a site important to the formation of the native multimeric protein is probably near the aspartate transcarbamylase domain.     

Location of the dihydroorotase domain within trifunctional hamster dihydroorotate synthetase

Mammalian dihydroorotase (DHOase, EC 3.5.2.3) is part of a trifunctional protein, dihydroorotate synthetase which catalyzes the first three reactions of de novo pyrimidine biosynthesis. We have subcloned a portion of the cDNA from the plasmid pCAD142 and obtained a nucleotide sequence which extends 2.1 kb in the 5' direction from the sequence encoding the aspartate transcarbamoylase (ATCase) domain at the 3'-end of the cDNA. The DHOase and ATCase domains have been purified from an elastase digest of the trifunctional protein and subjected to amino acid (aa) sequencing from their N termini. The sequence of the N-terminal 24 aa of the DHOase domain has been obtained and aligned with the cDNA sequence. The C-terminal residues of the DHOase domain have been identified as Leu followed by Val which, when taken with partial sequences of the CNBr fragments of this domain, defines the coding sequence of the active, globular DHOase domain released by proteolysis. Prediction of protein secondary structure from the deduced aa sequence showed that the DHOase domain (Mr 37,751) is separated from the C-terminal ATCase domain (Mr 34,323) by a bridging sequence (Mr 12,532) consisting of multiple beta-turns.     

Characterization of kinectin, a kinesin-binding protein: primary sequence and N-terminal topogenic signal analysis.

Kinectin is a kinesin-binding protein (Toyoshima et al., 1992) that is required for kinesin-based motility (Kumar et al., 1995). A kinectin cDNA clone containing a 4.7-kilobase insert was isolated from an embryonic chick brain cDNA library by immunoscreening with a panel of monoclonal antibodies. The cDNA contained an open reading frame of 1364 amino acids encoding a protein of 156 kDa. A bacterially expressed product of the full length cDNA bound purified kinesin. Transient expression in CV-1 cells gave an endoplasmic reticulum distribution that depended upon the N-terminal domain. Analysis of the predicted amino acid sequence indicated a highly hydrophobic near N-terminal stretch of 28 amino acids and a large portion (326-1248) of predicted alpha helical coiled coils. The 30-kDa fragment containing the N-terminal hydrophobic region was produced by cell-free in vitro translation and found to assemble with canine pancreas rough microsomes. Cleavage of the N terminus was not observed confirming its role as a potential transmembrane domain. Thus, the kinectin cDNA encodes a cytoplasmic-oriented integral membrane protein that binds kinesin and is likely to be a coiled-coil dimer.     

Kinetic studies on the interaction of eglin c with human leukocyte elastase and cathepsin G

We have investigated the inhibition of human leukocyte elastase and cathepsin G by recombinant Eglin c under near physiological conditions. The association rate constants k on of Eglin c for elastase and cathepsin G were 1.3 X 10(7) M-1 s-1 and 2 X 10(6) M-1 s-1, respectively. Under identical conditions, the k on for the association of human plasma alpha 1-proteinase inhibitor with the two leukocproteinases were 2.4 X 10(7) M-1 s-1 and 10(6) M-1 s-1, respectively. The consistency of these data could be verified using a set of competition experiments. The elastase-Eglin c interaction was studied in greater detail. The dissociation rate constant k off was determined by trapping of free elastase from an equilibrium mixture of elastase and Eglin c with alpha 1-proteinase inhibitor or alpha 2-macroglobulin. The rate of dissociation was very low (k off = 3.5 X 10(-5) s-1). The calculated equilibrium dissociation constant of the complex, Ki(calc) = k off/k on, was found to be 2.7 X 10(-12) M. Ki was also measured by adding elastase to mixtures of Eglin c and substrate and determining the steady-state rates of substrate hydrolysis. The Ki determined from these experiments (7.5 X 10(-11) M) was significantly higher than Ki(calc). This discrepancy might be explained by assuming that the interaction of Eglin c with elastase involves two steps: a fast binding reaction followed by a slow isomerization step. From the above kinetic constants it may be inferred that at a therapeutic concentration of 5 X 10(-7) M, Eglin c will inhibit leukocyte elastase in one second and will bind this enzyme in a "pseudo-irreversible" manner.     

N2-methylation of guanosine at position 10 in tRNA is catalyzed by a THUMP domain-containing, S-adenosylmethionine-dependent methyltransferase, conserved in Archaea and Eukaryota

In sequenced genomes, genes belonging to the cluster of orthologous group COG1041 are exclusively, and almost ubiquitously, found in Eukaryota and Archaea but never in Bacteria. The corresponding gene products exhibit a characteristic Rossmann fold, S-adenosylmethionine-dependent methyltransferase domain in the C terminus and a predicted RNA-binding THUMP (thiouridine synthases, RNA methyltransferases, and pseudouridine synthases) domain in the N terminus. Recombinant PAB1283 protein from the archaeon Pyrococcus abyssi GE5, a member of COG1041, was purified and shown to behave as a monomeric 39-kDa entity. This protein (EC 2.1.1.32), now renamed (Pab)Trm-G10, which is extremely thermostable, forms a 1:1 complex with tRNA and catalyzes the adenosylmethionine-dependent methylation of the exocyclic amino group (N(2)) of guanosine located at position 10. Depending on the experimental conditions used, as well as the tRNA substrate tested, the enzymatic reaction leads to the formation of either N(2)-monomethyl (m(2)G) or N(2)-dimethylguanosine (m(2)(2)G). Interestingly, (Pab)Trm-G10 exhibits different domain organization and different catalytic site architecture from another, earlier characterized, tRNA-dimethyltransferase from Pyrococcus furiosus ((Pfu)Trm-G26, also known as (Pfu)Trm1, a member of COG1867) that catalyzes an identical two-step dimethylation of guanosine but at position 26 in tRNAs and is also conserved among all sequenced Eukaryota and Archaea. The co-occurrence of these two guanosine dimethyltransferases in both Archaea and Eukaryota but not in Bacteria is a hallmark of distinct tRNAs maturation strategies between these domains of life.     

Molecular interaction of the first 3 enzymes of the de novo pyrimidine biosynthetic pathway of Trypanosoma cruzi

The first 3 reaction steps of the de novo pyrimidine biosynthetic pathway are catalyzed by carbamoyl-phosphate synthetase II (CPSII), aspartate transcarbamoylase (ATC), and dihydroorotase (DHO), respectively. In eukaryotes, these enzymes are structurally classified into 2 types: (1) a CPSII-DHO-ATC fusion enzyme (CAD) found in animals, fungi, and amoebozoa, and (2) stand-alone enzymes found in plants and the protist groups. In the present study, we demonstrate direct intermolecular interactions between CPSII, ATC, and DHO of the parasitic protist Trypanosoma cruzi, which is the causative agent of Chagas disease. The 3 enzymes were expressed in a bacterial expression system and their interactions were examined. Immunoprecipitation using an antibody specific for each enzyme coupled with Western blotting-based detection using antibodies for the counterpart enzymes showed co-precipitation of all 3 enzymes. From an evolutionary viewpoint, the formation of a functional tri-enzyme complex may have preceded-and led to-gene fusion to produce the CAD protein. This is the first report to demonstrate the structural basis of these 3 enzymes as a model of CAD. Moreover, in conjunction with the essentiality of de novo pyrimidine biosynthesis in the parasite, our findings provide a rationale for new strategies for developing drugs for Chagas disease, which target the intermolecular interactions of these 3 enzymes.     

Identification and distribution of a novel, collagen-binding protein in the developing subepicardium and endomysium.

A protein doublet (M(r) = 68,000) that copurifies with chicken cardiac collagen types I and III is purified and characterized in the present study. Peptide mapping and amino terminus sequencing for both 68-kDa polypeptides show they have similar structures. This is supported by amino terminus sequencing of a 39-kDa proteolytic fragment of each polypeptide. The 68-kDa polypeptides appear at pI 6.7-6.8 in two-dimensional gels. Under nonreducing, electrophoretic conditions, the doublet appears as a large multimer or aggregate. Amino acid sequencing of the protein shows that its amino terminus contains a heptapeptide (VCLXXGK) that appears in the heparin/fibrin-binding domain of fibronectin and the collagen-binding domain of laminin. Cardiac myocytes synthesize and secrete the protein in vitro onto cell surfaces and onto the substratum. Indirect immunofluorescence shows the protein first appears in the chicken subepicardium at approximately 10 days following fertilization. As collagen accumulates in the subepicardium and the volume of the subepicardial space increases, the 68-kDa protein is found predominantly at the interface between myocardial cells and the connective tissue and between epicardial cells and the connective tissue. In adult hearts, the protein is also present at lower concentrations in endomysial connective tissue. The 68-kDa protein is also present in the skeletal muscle endomysium of embryonic chickens. Electron microscopic immunocytochemistry shows the 68-kDa protein is located at the surface of subepicardial collagen fibers. In addition, a direct interaction between the 68-kDa protein and collagen are indicated by: 1) equilibrium gel filtration of the 68-kDa protein in the presence of gelatin, 2) gelatin affinity chromatography of the 68-kDa protein, and 3) comigration of type I collagen and the 68-kDa protein during gel filtration under reducing conditions. The 68-kDa protein exhibits no collagenase activity under native conditions or in zymograms. Together, the data indicate that the 68-kDa protein is a novel collagen-associated protein appearing in late epicardial development.