High-level expression, purification, and characterization of Staphylococcus aureus dihydroorotase (PyrC) as a cleavable His-SUMO fusion.

Staphylococcus aureus is a pathogenic bacterium that causes a variety of mild to lethal human diseases. The rapid spread of multidrug-resistant strains makes the discovery of new antimicrobial agents critical. Dihydroorotase (PyrC), the third enzyme in the bacterial pyrimidine biosynthesis pathway, is structurally and mechanistically distinct from its mammalian counterpart. It has been confirmed to be essential in S. aureus making it an attractive antibacterial drug target. No protocol to express and purify S. aureus PyrC (SaPyrC) has been reported. To obtain the SaPyrC enzyme and overcome anticipated solubility problems, the SaPyrC gene was cloned into the pET-SUMO vector. The N-terminal His-SUMO fused SaPyrC was expressed in Escherichia coli BL21 (DE3) with an HRV 3C protease recognition site inserted between the SUMO tag and SaPyrC to allow for improved cleavage by HRV protease. Purification of cleaved protein using HisTrap affinity and gel filtration columns resulted in native SaPyrC with estimated 95% purity and 40% yield. Both His-SUMO tagged and native SaPyrC form dimers, and enzyme characterization studies have shown that the His-SUMO tag affects enzyme activity slightly. Forward and reverse kinetic rate constants for both tagged and native SaPyrC were determined, and pH profiling studies revealed the optimal pH values for forward and reverse reactions.

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%.

Transcriptional repression of human cad gene by hypoxia inducible factor-1alpha.

De novo biosynthesis of pyrimidine nucleotides provides essential precursors for DNA synthesis and cell proliferation. The first three steps of de novo pyrimidine biosynthesis are catalyzed by a multifunctional enzyme known as CAD (carbamoyl phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase). In this work, a decrease in CAD expression is detected in numerous cell lines and primary culture human stromal cells incubated under hypoxia or desferrioxamine (DFO)-induced HIF-1alpha accumulation. A putative hypoxia response element (HRE) binding matrix is identified by analyzing human cad-gene promoter using a bioinformatic approach. Promoter activity assays, using constructs harboring the cad promoter (-710/+122) and the -67/HRE fragment (25-bases), respectively, demonstrate the suppression of reporter-gene expression under hypoxia. Suppression of cad-promoter activity is substantiated by forced expression of wild-type HIF-1alpha but abolished by overexpression of dominant-negative HIF-1alpha. A chromatin immunoprecipitation assay provides further evidence that HIF-1alpha binds to the cad promoter in vivo. These data demonstrate that the cad-gene expression is repressed by HIF-1alpha, which represents a functional link between hypoxia and cell-cycle arrest.

Overexpression of cyclin D1 enhances gene amplification.

Defects in cell cycle control and increased genomic instability, including gene amplification, often occur during cancer development. Cyclin D1 plays a pivotal role in G1, and this gene is frequently amplified and overexpressed in several types of human cancer. This study demonstrates that ectopic overexpression of cyclin D1 in a rat liver epithelial cell line markedly increased the yield of cells containing amplified copies of the CAD gene. This effect was associated with a loss of G1-S checkpoint control, although the cyclin D1-overexpressing cells had a normal p53 gene. The capacity of cyclin D1 to enhance gene amplification may contribute to the process of genomic instability during tumor development.

Magnesium cations are required for the association of U small nuclear ribonucleoproteins and SR proteins with pre-mRNA in 200 S large nuclear ribonucleoprotein particles.

In previous studies we have shown that specific nuclear pre-mRNAs and their splicing products, as well as the general population of nuclear poly(A)+ RNA, are found packaged in 200 S large nuclear ribonucleoprotein (lnRNP) particles that represent the splicing machinery in vivo. The lnRNP particles contain all U small nuclear ribonucleoproteins (snRNPs) required for splicing, as well as several proteins including non-snRNP splicing factors. Here we show that upon addition of EDTA to sucrose gradient-fractionated 200 S particles, part of their components (e.g. part of the U snRNPs) are no longer associated with pre-mRNAs, which are now packaged in 70 S particles. This 200 S to 70 S transition makes the pre-mRNA more susceptible to digestion by RNase. The effect of EDTA is reversible, as back addition of Mg2+ results in the reconstitution into 200 S lnRNP particles of: (1) all five snRNPs required for splicing; (2) the SR proteins; and (3) CAD mRNA, as a representative of nuclear RNA polymerase II transcripts. Remarkably, electron microscopy of the reconstituted particles shows a compact structure, 50 nm in diameter, that is indistinguishable from the original undissociated particles. We conclude that Mg2+ is required for the integrity of the 200 S lnRNP particles.

Protein preparation, crystallization and preliminary X-ray crystallographic studies of dihydroorotase from Bacillus subtilis.

B. subtilis dihydroorotase is an important enzyme in de novo pyrimidine biosynthesis pathway and encoded by pyrC gene in pyr operon. pyrC was amplified from B. subtilis genomic DNA and cloned into expression vector pET21-DEST. Dihydroorotase was expressed soluble form in E. coli and purified. The protein was crystallized and diffracted to 2.2 A. The crystal belongs to P2(1)2(1)2(1) space-group, with unit cell parameters a = 48.864 A, b = 84.99 A, c = 203.05 A. There are 2 molecules per asymmetry unit.

Synthesis of the nonconserved dihydroorotase domain of the multifunctional hamster CAD protein in Escherichia coli.

CAD is the multifunctional protein of higher eukaryotes which catalyzes the first three steps of pyrimidine biosynthesis. Its enzymatic activities exist as independent domains in the order: N terminus-carbamylphosphate synthetase II(CPSase)-dihydroorotase(DHOase)-aspartate transcarbamylase(ATCase)-C terminus. To functionally define the minimum hamster cDNA region required to encode an active DHOase, expression constructs were generated. Many such constructs complement Escherichia coli mutants defective not only in DHOase but also in ATCase. Constructs deleted for most of the sequence encoding the ATCase domain continue to complement E. coli mutants defective in DHOase. All of these smaller constructs also lack the region encoding CPSase. Therefore, a 'genetic cassette', containing information for neither the CPSase nor the ATCase domain, can direct the synthesis of a polypeptide with DHOase activity. Interestingly, inclusion of a portion of the DHOase-ATCase interdomain bridge appears to be required for optimum activity.

Synthesis and enzymic evaluation of 4-mercapto-6-oxo-1, 4-azaphosphinane-2-carboxylic acid 4-oxide as an inhibitor of mammalian dihydroorotase.

The design, synthesis, and enzymic evaluation of cis- and trans-4-mercapto-6-oxo-1,4-azaphosphinane-2-carboxylic acid 4-oxide 5 against mammalian dihydroorotase is presented. The design strategy for 5 was based on the strong affinity of phosphinothioic acids for zinc and that 5 also resembles the postulated tetrahedral transition state for the enzyme-catalyzed reaction. The synthesis of 5 utilized a novel protection/deprotection sequence upon 4-hydroxy-6-oxo-1, 4-azaphosphinane-2-carboxylic acid 4-oxide 4, followed by incorporation of alpha-phenyl benzenemethanethiol and exhaustive deprotection to afford 5 in 40% overall yield from 4. The activities of both isomers of 5 as inhibitors of mammalian dihydroorotase were marginally greater than that of the parent phosphinic acid 4, indicating a weak binding enhancement due to the phosphinothioic acid moiety.

Selection of N-(phosphonacetyl)-L-aspartate resistant Chinese hamster mutants in the presence of the uridine uptake inhibitor dipyridamole.

In mammalian cells selected in culture for resistance to PALA the CAD gene is amplified and these cells are a widely used model system to study gene amplification. Selection of resistant mutants is routinely performed in medium supplemented with dialyzed serum, because the cytotoxic effect of PALA is reversed by uridine, which is contained in serum. We have shown that in Chinese hamster cells dipyridamole reduced uridine uptake to less than 5% with limited effect on cell survival. Moreover, in medium supplemented with complete serum and 10 microM dipyridamole the toxicity of PALA was similar to that obtained in medium containing dialyzed serum. We then used 10 microM dipyridamole to inhibit uridine uptake during selection of PALA resistant colonies and found that both the frequency and the type of mutants were as those obtained in the presence of dialyzed serum. In particular, in the five mutants tested, the mechanism of resistance to PALA was amplification of the CAD gene.

Coordinate synthesis of the enzymes of pyrimidine biosynthesis in Bacillus subtilis.

Strains of Bacillus subtilis that were resistant to repression of pyrimidine nucleotide biosynthetic enzymes were selected by isolating spontaneous uracil-tolerant derivatives of a uracil-sensitive strain, which lacks arginine-repressible carbamyl phosphate synthetase. The relative content of all six enzymes of uridylic acid biosynthesis de novo in these strains was in a constant ratio over a 10-fold range of derepression, which indicates that synthesis of these enzymes is coordinately regulated.

CAD gene expression in serum-starved and serum-stimulated hamster cells.

The enzymes in the pathway for de novo pyrimidine biosynthesis, including those associated with the tri-functional CAD protein, show a marked increase in activity in rapidly growing cells and tissues. To learn more about the relationship of this pathway to cellular proliferation, we have studied changes in levels of CAD RNA, rates of CAD protein synthesis, and levels of aspartate transcarbamylase activity in Syrian hamster ts13 cells in response to serum starvation and serum stimulation. The steady-state level of CAD RNA and the synthetic rate of CAD protein decrease by 12- to 15-fold following 24 hr of serum starvation, as compared to exponentially growing cells. Upon serum stimulation of quiescent cells, steady-state CAD RNA levels increase substantially (13-fold), peaking during mid to late G1. Parallel increases occur in the synthesis of new CAD protein and in aspartate transcarbamylase activity. At the same time, the rate of CAD transcription increases only about twofold. These findings indicate that regulation of CAD expression in this system is primarily at the post-transcriptional level. This is in contrast to the transcriptional regulation of CAD previously reported in terminally differentiating HL60 cells (Rao et al., Mol. Cell. Biol. 7, 1961-1966, 1987). While both systems indicate that CAD gene expression is dependent on cell growth, there apparently are alternative mechanisms that can produce the same effect. Evidence is also presented that indicates that the accumulation of CAD transcripts during serum stimulation requires the synthesis of new proteins.

The genes for and regulation of the enzyme activities of two multifunctional proteins required for the de novo pathway for UMP biosynthesis in mammals.

UMP biosynthesis requires six enzyme activities. Five of these enzyme centers are clustered into two multienzymatic proteins which are known to, or appear to, sequester the intermediates carbamyl approximately P, carbamyl aspartate and orotidylic acid. The advantages of sequestering these intermediates appear to be a conservation of energy, since two intermediates, carbamyl approximately P and orotidylate, might otherwise be rapidly degraded in mammalian cells. Carbamyl-aspartate appears not to be degraded rapidly in mammalian cells but it can pass into the blood and could possible disrupt brain metabolism by action as an acetylaspartate analog, if it passes the blood-brain barrier. For this, and possible for other reasons, there may be advantages to the fact that these intermediates are not other reasons, there may be advantages to the fact that these intermediates are not readily released from Complex A and U. In addition, these multienzymatic proteins may have other kinetic advantages, some of which have been discussed above. Studies with intact cells illustrate that azauridine, a chemical designed originally as an antineoplastic drug, produces a "ripple" effect when it inhibits the last enzyme of this pathway which leads to a sequential accumulation of pools of the various intermediates or their metabolites. This same agent increases the amount of some of the enzymes of this biosynthetic pathway in cells exposed to this drug. Both of these effects can negate the effectiveness of this potential antineoplastic drug. Sophisticated drug design may depend on whole-cell studies, such as those discussed here, in addition to the classic studies on the inhibition of a single enzyme center to select drugs that may be without significant side effects when they are finally tested in animals.

Molecular cloning of a human cDNA encoding a trifunctional enzyme of carbamoyl-phosphate synthetase-aspartate transcarbamoylase-dihydroorotase in de Novo pyrimidine synthesis.

A human CAD cDNA encoding a trifunctional enzyme of carbamoylphosphate synthetase-aspartate transcarbamoylase-dihydroorotase, which catalyzes the first three steps of de novo pyrimidine nucleotide biosynthesis, was cloned from a human fibroblast cell line of TIG-1-20 by polymerase chain reaction (PCR). The predicted open reading frame encodes a protein of 2,225 amino acids with a deduced molecular weight (Mr) OF 242,913. The deduced amino acid sequence exhibits 95.3 and 76.1% identity with the CAD sequences of hamster and Squalus acanthias. The DNA fragment of 6,679 bp containing the full-length coding sequence was amplified by nested PCR using the first-strand cDNA of human cell lines of TIG-1-20 and COLO205 as a template. Southern blot analysis suggested that the CAD gene exists as a single copy in the human genome.

Identification of the Shine-Dalgarno sequence required for expression and translational control of the pyrC gene in Escherichia coli K-12.

Expression of the pyrC gene in Escherichia coli K-12 is regulated by a translational control mechanism in which CTP (and perhaps GTP) pool sizes determine the selection of alternative transcriptional start sites at the pyrC promoter. High CTP levels cause transcription to start primarily at a site that directs the synthesis of untranslatable pyrC transcripts. These transcripts form a hairpin at their 5' ends that blocks ribosome binding to the Shine-Dalgarno (SD) sequence. The pyrC ribosome binding site is unusual in that it contains two potential SD sequences, designated SD1 and SD2, which are located 11 and 4 nucleotides upstream of the translational initiation codon, respectively. In this study, we examined the functions of these two SD sequences in translational initiation. Mutations that inactivate either SD1 or SD2 were constructed and incorporated separately into a pyrC::lacZ protein fusion. The effects of the mutations on pyrC::lacZ expression, regulation, and transcript levels were determined. The results indicate that SD1 is the only functional pyrC SD sequence. The SD2 mutation did cause a small reduction in expression, but this effect appeared to be due to a decrease in transcript stability. In addition, we constructed a mutation that introduces a long spacer region between the hairpin at the 5' end of the pyrC transcript and a new pyrC SD sequence. As predicted by the model for translational control, this mutation caused constitutive expression of a pyrC::lacZ protein fusion.

Stable mutants of mammalian cells that overproduce the first three enzymes of pyrimidine nucleotide biosynthesis.

Upon exposure to 0.1 mM N-phosphonacetyl-L-aspartate (PALA), a transition state analog inhibitor of aspartate transcarbamylase, most cells of a simian virus 40 (SV40)-transformed Syrian hamster line are killed within a few days, but resistant mutants form spontaneously with frequency 2-5 X 10(-5) in a stochastic process not dependent upon the presence of the inhibitor. The resistant phenotype is stable for many months in the absence of PALA. Other cell lines also give resistant mutants, but with substantially lower frequencies. Serial selection with PALA at concentrations up to 25 mM has yielded clones with more than 100 times the original aspartate transcarbamylase activity. The activities of carbamyl-P synthetase and dihydroorotase, which co-purify with aspartate transcarbamylase as a three-enzyme complex, increase in parallel with aspartate transcarbamylase activity in each resistant clone tested, but there is no substantial change in the activities of the last three enzymes of the de novo pathway, which are not in this complex. In each of the three resistant clones tested, there is an increase in the number of aspartate transcarbamylase active sites, determined by titration with 3H-PALA, which closely parallels the increase in enzyme activity. In one resistant clone tested, there is no change in the Ki for PALA or the Km for carbamyl-P. The only mechanism detected for achieving resistance to PALA is an increase in the steady state amount of the three enzyme complex.