Cloning, overexpression, and characterization of the functional dihydroorotase domain of the mammalian multifunctional protein CAD.

Mammalian dihydroorotase (DHOase) is part of a large multidomain protein called CAD, which initiates the first three steps in the de novo pyrimidine biosynthetic pathway. DHOase activity is carried out by a 44-kDa structural domain which could be isolated in active form from elastase digests. A core domain the same size as monofunctional dihydroorotases was defined, although the domain borders were uncertain. Two recombinants were overexpressed in Escherichia coli. The first encoded the core domain with 55 and 13 residues added to the amino and carboxyl ends, respectively, and was expressed in insoluble form. The recombinant protein was refolded from urea into a soluble form which was resistant to protease digestion but was catalytically inactive. In contrast, the proteolytic fragment from CAD could be unfolded and refolded with recovery of 40-100% catalytic activity. The second construct, which approximated the proteolytic fragment, had 21 residues on the amino end and 65 residues on the carboxyl end of the core domain. A 46-kDa soluble protein was expressed at 4% of the total soluble protein. The recombinant protein was catalytically active and had the expected amino-terminal sequence. The protein was purified to homogeneity. Dihydroorotase saturation curves gave a Km = 41.9 +/- 3.5 microM and a kcat = 2.79 +/- 0.06 s-1, parameters that were similar to those obtained for the proteolytic fragment. The Km was 6-fold higher and the kcat 2-fold lower than the values obtained for the parent protein, which suggests that interdomain interactions stabilize the active conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Proton chemical shift imaging.

Proton resonance spectra contain two broad classes of hydrogen-containing compounds: those that are fat-like and those that are water-like. Proton chemical shift imaging is a means to produce water or fat proton images. Three methods of proton chemical shift imaging are discussed.

Influence of magnetic field strength on image contrast.

In magnetic resonance imaging (MRI) S/N increases with magnetic field strength. The curve, however, flattens out at higher fields, resulting in diminishing gains as the field strength is increased.

Function of conserved histidine residues in mammalian dihydroorotase.

Dihydroorotase (DHOase, EC 3.5.2.3) catalyzes the reversible cyclization of carbamyl aspartate to form dihydroorotate, the third step in de novo pyrimidine biosynthesis. In mammals this activity is carried by the zinc-containing domain of the 243 kDa multifunctional protein CAD. We have replaced conserved residues in the cloned 46 kDa DHOase domain by site-directed mutagenesis. Mutants His1471Ala and His1473Ala lacked catalytic activity, judging by their failure to complement a DHOase-deficient Escherichia coli strain, and were unable to coordinate the active site zinc ion in zinc blotting experiments. This result confirmed earlier predictions. A mutant protein in which the third suspected zinc ligand was changed, Glu1512Asn, had a kcat similar to that of the intact CAD molecule and a Km similar to that of the wild-type recombinant DHOase, observations that argue against a role for glutamate 1512 in catalysis. Mutant His1590Asn had no measurable catalytic activity. This histidine residue was tentatively identified as the third zinc ligand by the failure of the mutant to bind the full complement of zinc in atomic absorption measurements. Mutant His1690Asn had a kcat 34-fold lower and a Km 9-fold higher than those of wild-type recombinant. The kinetic parameters of the mutant His1642Asn were also altered, but to a lesser extent. Diethyl pyrocarbonate (DEPC) was shown previously to inactivate mammalian DHOase. Spectroscopic studies and [14C]DEPC incorporation demonstrated that the loss of activity is associated with the modification of approximately two histidine residues located at or near the active site.(ABSTRACT TRUNCATED AT 250 WORDS)

Properties of a copper-containing cytochrome ba3: a second terminal oxidase from the extreme thermophile Thermus thermophilus.

We describe an alternate terminal oxidase found in the plasma membrane of Thermus thermophilus and designate it cytochrome ba3. The enzyme consists of a single approximately equal to 35-kDa polypeptide that binds one heme B molecule, one heme A molecule, and two Cu ions. Optical spectra suggest the presence of cytochrome b, cytochrome a3, and CuA in this protein. Quantitative EPR and Mössbauer studies of the oxidized protein indicate the presence of one low-spin ferric heme, which is assigned to cytochrome b. Mössbauer studies of the reduced protein show the presence of one low-spin ferrous heme, assigned to cytochrome b, and a predominant high-spin ferrous heme that reacts quantitatively with CO to yield an additional low-spin ferrous heme. The latter Fe atom is associated with the heme A and is designated cytochrome a3. The EPR spectrum of the oxidized protein also reveals the presence of a CuA-type center that accounts for half the total Cu. The remainder of the Cu would appear to be present as CuB that is magnetically coupled to the heme A. Amino acid analyses of cytochrome ba3 show the presence of eight to nine histidine residues and one cysteine residue.

Recombinant Cry3Aa has insecticidal activity against the Andean potato weevil, Premnotrypes vorax.

The Andean potato weevil, Premnotrypes vorax, an insect of the order Coleoptera, is a major cause of damage to potato crops in the Andean regions of South America. The insecticidal Cry proteins from Bacillus thuringiensis are useful biological pesticides, and some are toxic to Coleopteran insects. We overexpressed recombinant, histidine-tagged Cry3Aa protein in Escherichia coli host cells. The recombinant protein was solubilized at high pH with urea, purified using Ni(2+)-nitrilo-triacetic acid affinity resin, and dialysed to lower pH and remove urea. Bioassays were performed with an insect media whose surface was spread with 70 microgram/mL purified native or recombinant toxins. First instar larvae exposed to toxin treated media for 5 days exhibited mortalities from 57% (native Cry3Aa) to 52% (recombinant Cry3Aa). Purified native and recombinant Cry3Aa proteins appeared to be equally toxic to the Andean potato weevil.

Evidence for structural heterogeneities and a study of exchange coupling. Mössbauer studies of cytochrome c1aa3 from Thermus thermophilus.

We have studied samples of oxidized (as isolated) cytochrome c1aa3 from Thermus thermophilus in the pH range 5.7 to 9.3 with Mössbauer spectroscopy. In this pH range, the spectra of cytochromes c1 and a are independent of pH, whereas the spectra of cytochrome a3 are not. Most importantly, spectra taken in applied fields up to 6.0 T reveal the presence of multiple ferric forms of cytochrome a3. At any given pH value, at least two high-spin ferric cytochrome a3 species can be distinguished; in addition, most samples contain a low-spin ferric cytochrome a3 species (less than 20% of cytochrome a3). The Mössbauer spectra show clearly that all forms of cytochrome a3 are spin coupled (to CuB). We have analyzed the high field (H greater than or equal to 1.5 tesla) spectra of a sample at pH 6.5 in the framework of a model that considers isotropic exchange-coupling, JS1.S2, between a high-spin ferric (S1 = 5/2) cytochrome a3 and cupric CuB (S2 = 1/2). In strong applied fields, the spectra can be fitted with any value for J greater than or equal to 0.5 cm-1. In the strong coupling case (J/D1 approximately greater than 3), a zero field splitting parameter D1 approximately 2.5 cm-1 is required for cytochrome a3; this value is distinctly smaller than those observed for high-spin ferric hemes (4-20 cm-1). A model assuming weak coupling magnitude of J approximately 1 cm-1, yields D1 approximately 8 cm-1 and a parameter set for cytochrome a3 quite similar to that reported for metmyoglobin. A J-value of approximately 1 cm-1 does not demand the presence of a ligand bridging between cytochrome a3 and CuB.

Mammalian dihydroorotase: nucleotide sequence, peptide sequences, and evolution of the dihydroorotase domain of the multifunctional protein CAD.

Mammalian DHOase (S-dihydroorotate amidohydrolase, EC 3.5.2.3) is part of a large multifunctional protein called CAD, which also has a carbamoyl-phosphate synthetase [carbon-dioxide: L-glutamine amido-ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.5.5] and aspartate transcarbamoylase (carbamoyl-phosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2) activities. We sequenced selected restriction fragments of a Syrian hamster CAD cDNA. The deduced amino acid sequence agreed with the sequence of tryptic peptides and the amino acid composition of the DHOase domain isolated by controlled proteolysis of CAD. Escherichia coli transformed with a recombinant plasmid containing the cDNA segment 5' to the aspartate transcarbamoylase coding region expressed a polypeptide recognized by DHOase domain-specific antibodies. Thus, the order of domains within the polypeptide is NH2-carbamoyl-phosphate synthetase-DHO-aspartate transcarbamoylase-COOH. The 334-residue DHOase domain has a molecular weight of 36,733 and a pI of 6.1. A fragment of CAD having DHOase activity that was isolated after trypsin digestion has extensions on both the NH2 (18 residues) and COOH (47-65 residues) termini of this core domain. Three of five conserved histidines are within short, highly conserved regions that may participate in zinc binding. Phylogenetic analysis clustered the monofunctional and fused DHOases separately. Although these families may have arisen by convergent evolution, we favor a model involving DHOase gene duplication and insertion into an ancestral bifunctional locus.

Molecular genetic and protein chemical characterization of the cytochrome ba3 from Thermus thermophilus HB8.

Thermus thermophilus HB8 cells grown under reduced dioxygen tensions contain a substantially increased amount of heme A, much of which appears to be due to the presence of the terminal oxidase, cytochrome ba3. We describe a purification procedure for this enzyme that yields approximately 100 mg of pure protein from 2 kg of wet mass of cells grown in < or = 50 microM O2. Examination of the protein by SDS-polyacrylamide gel electrophoresis followed by staining with Coomassie Blue reveals one strongly staining band at approximately 35 kDa and one very weakly staining band at approximately 18 kDa as reported earlier (Zimmermann, B.H., Nitsche, C.I., Fee, J. A., Rusnak, F., and Münck, E. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5779-5783). By contrast, treatment of the gels with AgNO3 reveals that the larger polypeptide stains quite weakly while the smaller polypeptide stains very strongly. These results suggested the presence of two polypeptides in this protein. Using partial amino acid sequences from both proteins to obtain DNA sequence information, we isolated and sequenced a portion of the Thermus chromosome containing the genes encoding the larger protein, subunit I (cbaA), and the smaller protein, subunit II (cbaB). The two polypeptides were isolated using reversed phase liquid chromatography, and their mole percent amino acid compositions are consistent with the proposed translation of their respective genes. The two genes appear to be part of a larger operon, but we have not extended the sequencing to identify initiation and termination sequences. The deduced amino acid sequence of subunit I includes the six canonical histidine residues involved in binding the low spin heme B and the binuclear center Cu(B)/heme A. These and other conserved amino acids are placed along the polypeptide among alternating hydrophobic and hydrophilic segments in a pattern that shows clear homology to other members of the heme- and copper-requiring terminal oxidases. The deduced amino acid sequence of the subunit II contains the CuA binding motif, including two cysteines, two histidines, and a methionine, but, in contrast to most other subunits II, it has only one region of hydrophobic sequence near its N terminus. Alignment of these two polypeptides with other cytochrome c and quinol oxidases, combined with secondary structure analysis and previous spectral studies, clearly establish cytochrome ba3 as a bona fide member of the superfamily of heme- and copper-requiring oxidases. The alignments further indicate that cytochrome ba3 is phylogenetically distant from other cytochrome c and quinol oxidases, and they substantially decrease the number of conserved amino acid residues.