Two biochemically distinct classes of fumarase in Escherichia coli

Biochemical studies with strains of Escherichia coli that are amplified for the products of the three fumarase genes, fumA (FUMA), fumB (FUMB) and fumC (FUMC), have shown that there are two distinct classes of fumarase. The Class I enzymes include FUMA, FUMB, and the immunologically related fumarase of Euglena gracilis. These are characteristically thermolabile dimeric enzymes containing identical subunits of Mr 60,000. FUMA and FUMB are differentially regulated enzymes that function in the citric acid cycle (FUMA) or to provide fumarate as an anaerobic electron acceptor (FUMB), and their affinities for fumarate and L-malate are consistent with these roles. The Class II enzymes include FUMC, and the fumarases of Bacillus subtilis, Saccharomyces cerevisiae and mammalian sources. They are thermostable tetrameric enzymes containing identical subunits Mr 48,000-50,000. The Class II fumarases share a high degree of sequence identity with each other (approx. 60%) and with aspartase (approx. 38%) and argininosuccinase (approx. 15%), and it would appear that these are all members of a family of structurally related enzymes. It is also suggested that the Class I enzymes may belong to a wider family of iron-dependent carboxylic acid hydro-lyases that includes maleate dehydratase and aconitase. Apart from one region containing a Gly-Ser-X-X-Met-X-X-Lys-X-Asn consensus sequence, no significant homology was detected between the Class I and Class II fumarases.     

Studies on mutants affecting amidophosphoribosyltransferase activity in Saccharomyces cerevisiae

Mutants at the ade4 locus of yeast were isolated following mutagenesis of ade+ and ade2 with ultraviolet light (UV), ethylmethane sulphonate, and the acridine half mustard ICR-170. Tests for interallelic complementation, osmotic remediality, temperature sensitivity, and mutagen-specific reversion were carried out on 19 mutants. Six mutants showed interallelic complementation and fell into four groups, defining three complons. Three mutants were osmotic remedial and the same three were temperature sensitive. Three mutants induced by ICR-170 gave purine-excreting revertants, designated Pur6 or ade4.RCF, after exposure to UV. Activity of amidophosphoribosyltransferase (PRPPAT) was assayed in the ade4 mutants and other alleles at this locus. The ade4 mutants lacked activity of the enzyme; the alleles su-pur+, su-pur, PUR6, and Pur6, showed different levels of activity. The enzyme was subject to feedback inhibition by AMP and IMP in su-pur+ and PUR6; su-pur was hypersensitive to inhibition by AMP, whereas Pur6 was slightly resistant. Purine synthesis de novo was shown to be repressible in su-pur+ and constitutive in PUR6 and Pur6 by following the accumulation of aminoimidazole ribotide in the presence and absence of cycloheximide. These observations were confirmed by direct assay of enzyme activity.     

Transformation of Clostridium acetobutylicum Protoplasts with Bacteriophage DNA

Techniques for the transformation of Clostridium acetobutylicum protoplasts with bacteriophage DNA are described. Transformation required regeneration of protoplasts and a 2-h eclipse period.     

Hypoxanthine: Guanine phosphoribosyltransferase mutants in Saccharomyces cerevisiae

Summary Yeast mutants lacking activity of the enzyme hypoxanthine: guanine phosphoribosyltransferase (H:GPRT) have been isolated by selecting for resistance to 8-azaguanine in a strain carrying the wild type allele, ade4 ⁺ of the gene coding for amidophosphoribosyltransferase (PRPPAT), the first enzyme of de novo purine synthesis. The mutants excrete purines and are cross-resistant to 8-azaadenine. They are recessive and represent a single complementation group, designated hpt1. Ade4-su, a prototrophic allele of ade4 with reduced activity of PRPPAT, is epistatic to hpt1, suppressing purine excretion and resistance to azaadenine but not resistance to azaguanine. The genotype ade2 hpt1 does not respond to hypoxanthine. Hpt1 complements and is not closely linked to the purine excreting mutants pur1 to pur5. Hpt1 and pur6, a regulatory mutant of PRPPAT, are also unlinked but do not complement, suggesting a protein-protein interaction between H:G-PRT and PRPPAT. Mycophenolic acid (MPA), an inhibitor of de novo guanine nucleotide synthesis, inhibits the growth of hpt1 and hpt1 ⁺. Xanthine allows both genotypes to grow in the presence of MPA whereas guanine only allows growth of hpt1 ⁺. Activity of A-PRT, X-PRT and H:G-PRT is present in hpt ⁺. Hpt1 lacks activity of H:G-PRT but has normal A-PRT and X-PRT.