Strategies for the use of computational SAR methods in assessing genotoxicity

The relationship between computational SAR studies and relevant data gathering and generation activities is complex. First, the chemical class to be studied is selected on the basis of information requirements for hazard identification and assessment. Membership in the class is determined by consideration of chemical structure and reactivity. Compilation of the existing bioassay data for this chemical class follows immediately from the specification of the class. Bioassay data, qualitative knowledge of general chemical reactivities in this class, and knowledge concerning potential interactions with biomolecular targets all contribute to the derivation of possible mechanisms for biological activity. Computational studies based on modeling the proposed mechanism of action and/or the existing data base can provide a quantitative basis for the differentiation between chemicals. There is the opportunity for continuing feedback between the quantitative computational studies and the development of a relevant bioassay data base for this chemical class. The qualitative and quantitative information on the potential biological responses obtained will provide a rational basis for extrapolation from the extant data base to the chemicals of interest, and to biological responses significant to the assessment for which complete data are unavailable. Knowledge concerning possible mechanisms of action and preexisting data determine the type of computational study that will be most useful.     

Site-directed mutagenesis of yeast C1-tetrahydrofolate synthase: analysis of an overlapping active site in a multifunctional enzyme

C1-tetrahydrofolate (THF) synthase is a trifunctional protein possessing the activities 10-formyl-THF synthetase, 5,10-methenyl-THF cyclohydrolase, and 5,10-methylene-THF dehydrogenase. The current model divides this protein into two functionally independent domains with dehydrogenase/cyclohydrolase activities sharing an overlapping active site on the N-terminal domain and synthetase activity associated with the C-terminal domain. Previous chemical modification studies on C1-THF synthase from the yeast Saccharomyces cerevisiae indicated at least two cysteinyl residues involved in the dehydrogenase/cyclohydrolase reactions [Appling, D. R., & Rabinowitz, J. C. (1985) Biochemistry 24, 3540-3547]. In the present work, site-directed mutagenesis of the S. cerevisiae ADE3 gene, which encodes C1-THF synthase, was used to individually change each cysteine contained within the dehydrogenase/cyclohydrolase domain (Cys-11, Cys-144, and Cys-257) to serine. The resulting proteins were overexpressed in yeast and purified for kinetic analysis. Site-specific mutations in the dehydrogenase/cyclohydrolase domain did not affect synthetase activity, consistent with the proposed domain structure. The C144S and C257S mutations result in 7- and 2-fold increases, respectively, in the dehydrogenase Km for NADP+. C144S lowers the dehydrogenase maximal velocity roughly 50% while C257S has a maximal velocity similar to that of the wild type. Cyclohydrolase catalytic activity is reduced 20-fold by the C144S mutation but increased 2-fold by the C257S mutation. Conversion of Cys-11 to serine has a negligible effect on dehydrogenase/cyclohydrolase activity. A double mutant, C144S/C257S, results in catalytic properties roughly multiplicative of the individual mutations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleotide sequence of the Clostridium acidiurici ("Clostridium acidi-urici") gene for 10-formyltetrahydrofolate synthetase shows extensive amino acid homology with the trifunctional enzyme C1-tetrahydrofolate synthase from Saccharomyces cerevisiae

The nucleotide sequence of the gene for 10-formyltetrahydrofolate synthetase (EC 6.3.4.3) from Clostridium acidiurici ("Clostridium acidi-urici") was determined. The synthetase mRNA initiation and termination regions were determined by primer extension and S1 nuclease mapping. Two potential -10 and -35 promoter regions were identified upstream of mRNA initiation. The terminator region was found to be in a large region of dyad symmetry. A comparison of the amino acid sequences of the monofunctional synthetase and the eucaryotic trifunctional enzyme, C1-tetrahydrofolate synthase, from Saccharomyces cerevisiae demonstrated a region of strong homology.     

Isolation and characterization of the Saccharomyces cerevisiae MIS1 gene encoding mitochondrial C1-tetrahydrofolate synthase

C1-Tetrahydrofolate synthase is a trifunctional polypeptide found in eukaryotic organisms that catalyzes 10-formyltetrahydrofolate synthetase (EC 6.3.4.3), 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9), and 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) activities. In Saccharomyces cerevisiae, C1-tetrahydrofolate synthase is found in both the cytoplasm and the mitochondria. The gene encoding yeast mitochondrial C1-tetrahydrofolate synthase was isolated using synthetic oligonucleotide probes based on the amino-terminal sequence of the purified protein. Hybridization analysis shows that the gene (designated MIS1) has a single copy in the yeast genome. The predicted amino acid sequence of mitochondrial C1-tetrahydrofolate synthase shares 71% identity with yeast C1-tetrahydrofolate synthase and shares 39% identity with clostridial 10-formyltetrahydrofolate synthetase. Chromosomal deletions of the mitochondrial C1-tetrahydrofolate synthase gene were generated using the cloned MIS1 gene. Mutant strains which lack a functional MIS1 gene are viable and can grow in medium containing a nonfermentable carbon source. In fact, deletion of the MIS1 locus has no detectable effect on cell growth.     

Purification and characterization of a mitochondrial isozyme of C1-tetrahydrofolate synthase from Saccharomyces cerevisiae

C1-Tetrahydrofolate synthase is a trifunctional polypeptide found in eukaryotic organisms that catalyzes 10-formyltetrahydrofolate synthetase (EC 6.3.4.3), 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9), and 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) activities. In Saccharomyces cerevisiae, C1-tetrahydrofolate synthase is encoded by the ADE3 locus, yet ade3 mutants have low but detectable levels of these enzyme activities. Synthetase, cyclohydrolase, and dehydrogenase activities in an ade3 deletion strain co-purify 4,000-fold to yield a single protein species as seen on sodium dodecyl sulfate-polyacrylamide gels. The native molecular weight of the isozyme (Mr = 200,000 by gel exclusion chromatography) and the size of its subunits (Mr = 100,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) are similar to those of C1-tetrahydrofolate synthase. Cell fractionation experiments show that the isozyme, but not C1-tetrahydrofolate synthase, is localized in the mitochondria. Genetic studies indicate that the isozyme is encoded in the nuclear genome. Peptide mapping experiments show that C1-tetrahydrofolate synthase and the isozyme are not structurally identical. However, immunotitration experiments and amino acid sequence analysis suggest that C1-tetrahydrofolate synthase and the isozyme are structurally related. We propose to call the isozyme "mitochondrial C1-tetrahydrofolate synthase."     

Analysis of cloned mRNA sequences encoding subfragment 2 and part of subfragment 1 of alpha- and beta-myosin heavy chains of rabbit heart

Two cardiac myosin heavy chain cDNA clones, pMHC alpha 252 and pMHC beta 174, were constructed using rabbit ventricular mRNA isolated from adult thyrotoxic and normal hearts, respectively. The complete DNA sequences of the 2.2- and 1.4-kilobase inserts of pMHC beta 174 and pMHC alpha 252, respectively, were obtained. The 736 amino acids specified by pMHC beta 174 begin 439 (1.3 kilobases) residues from the heavy chain NH2 terminus and include a 400-amino acid segment of subfragment 1 and the entire subfragment 2 region. Clone pMHC alpha 252 encodes 465 amino acids encompassing all of subfragment 2 and a portion of light meromyosin. Comparison of these two clones revealed extensive sequence overlap which included 1107 nucleotides specifying a 369-amino acid segment corresponding to subfragment 2. Within this region 78 (7%) base and 32 (8.7%) amino acid mismatches were noted. These differences were clustered within discrete regions, with the subfragment 1/subfragment 2 junctional region being particularly divergent. Structural differences between pMHC alpha 252 and pMHC beta 174 indicate that these two clones represent two similar but distinct myosin heavy chain genes whose expression is responsible for ventricular myosin heavy chain isoforms alpha and beta, respectively. The derived amino acid sequences of both clones exhibit extensive homology (greater than 81%) with sequences obtained by direct analysis of adult rabbit skeletal muscle myosin heavy chain protein. The sequences corresponding to the subfragment 2 region are consistent with an alpha-helical conformation with a characteristic 7-residue periodicity in the linear distribution of nonpolar amino acids. Conversely, subfragment 1 sequences specified by pMHC beta 174 suggest a folded highly irregular structure.