Nucleotide sequence and deduced amino acid sequence of a coleopteran-active delta-endotoxin gene from Bacillus thuringiensis subsp. san diego.

A gene from Bacillus thuringiensis subsp. san diego that is responsible for a delta-endotoxin active against Colorado potato beetle and some other Coleoptera was sequenced and shown to have surprising regional homology with both lepidopteran and dipteran active delta-endotoxins from other strains of B. thuringiensis. Unlike the lepidopteran active toxins from B. thuringiensis subsp. kurstaki that exist as approx. 130-kDa protoxins and form bipyramidal crystalline inclusions, the coleopteran toxic protein forms a square-shaped crystal composed of an approx. 65-kDa protein. Comparisons of the gene sequences encoding the active portions of these protoxins indicate conservation of N-terminal hydrophilic and hydrophobic regions, and suggest a distant ancestral origin for these insecticidal proteins.

Isolation of the ARO1 cluster gene of Saccharomyces cerevisiae.

The AROl cluster gene was isolated by complementation in Saccharomyces cerevisiae after transformation with a comprehensive yeast DNA library of BamHI restriction fragments inserted into the shuttle vector YEp13. Most of the transformants exhibited the expected episomal inheritance of the ARO+ phenotype; however, one stable transformant has been shown to be an integration of the AROl fragment and the vector YEp13 at the arol locus. The insert containing AROl is a 17.2-kilobase pair (kbp) BamHI fragment which complements both nonsense and missense alleles of arol. Subcloning by Sau3AI partial digestion further locates the AROl segment to a 6.2-kbp region. An autonomously replicating sequence (ars) was found on the 17.2-kbp fragment. Yeast arol mutants transformed with the AROl episome express 5 to 12 times the normal level of the five AROl enzyme activities and possess elevated amounts of the AROl protein. The yeast AROl fragment also complemented aroA, aroB, aroD, and aroE mutants of Escherichia coli. The expression of AROl in both S. cerevisiae and E. coli was independent of the orientation of the fragment with respect to the vector.

The functional unit of the arom conjugate in Neurospora.

The functional unit of arom polyenzyme conjugate of Neurospora crassa was determined by analysis of radiation inactivation of each of the five activities in the conjugate. The functional targets for all five enzymes were in close agreement with the value of 300,000 obtained by conventional hydrodynamic procedure for the native dimeric structure. These data indicate that at least 95% of the functional enzyme system in crude extracts exists in a dimeric form and that both polypeptide chains of the homodimer are required for full activity of each of the five enzymes.

Genetics of formamidase-5 (brain formamidase) in the mouse: localization of the structural gene on chromosome 14.

A single formamidase, which is different from the formamidases found in other tissues occurs in the brains of mice. This enzyme is here called formamidase-5 and the gene symbol is designated For-5. Two alleles are recognized on the basis of their differential heat sensitivity:For-5b is relatively heat stable and is present in strain C57BL/6J, while For-5d is relatively heat sensitive and is present in strain DBA/2J. The heat sensitivity of formamidase-5 in 44 other inbred strains and substrains was tested and found to resemble that of C57BL/6J or DBA/2J. Thirty-six recombinant inbred strains derived from progenitors that differed at For-5 were studies to test for single-gene inheritance and linkage with other loci. Complete concordance was found with the esterase-10 locus (Es-10), indicating close linkage. The 99% upper confidence limit of the distance between For-5 and Es-10 is 3.7 centimorgans (cM). Es-10 is located on chromosome 14 about 19 cM from the centromere. An independent demonstration of linkage of For-5 with Es-10 and another chromosome 14 marker, hairless (hr), is provided by the finding that the HRS/J strain, which has been sibmated for 60 generations with forced heterozygosity at the hr locus, is cosegregating at For-5 and Es-10. A survey of 32 inbred strains and substrains revealed that the For-5d allele is associated with the Es-10b allele, and that the For-5b allele is associated with Es-10a and Es-10c. Formamidase-5 segregates as expected in the F2 generation of crosses between strains bearing For-5b and For-5d alleles. It is possible that this unique formamidase of the brain is involved in the metabolism of a neurotransmitter substance.

Kynureninase-type enzymes and the evolution of the aerobic tryptophan-to-nicotinamide adenine dinucleotide pathway.

Kynureninase-type (L-kynurenine hydrolase, EC 3.7.1.3) activity has been found to be present in the livers of fish, amphibia, reptiles, and birds. In addition to past information concerning this enzyme activity in mammalian liver, it is now clear that all the major classes of vertebrates carry a highly specialized kynureninase-type enzyme, which we have termed a hydroxykynureninase. To compare the reactivities of these enzymes with L-kynurenine and L-3-hydroxykynurenine, ratios of tau values (Km/V) were used. Based on this comparison, the bacterium Pseudomonas fluorescens carries the most efficient kynureninase, whereas the amphibian Xenopus laevis has the most efficient hydroxykynureniase. In these two cases, the ratio of tau values differs by a factor of 38 000. It is hypothesized that the tryptophan-to-nicotinamide adenine dinucleotide biosynthetic pathway evolved from a catabolic system of enzymes, and that the differences observed in the kynureninase-type enzymes between lower and higher organisms reflect the specialization of the function of these enzymes from a strictly catabolic role to an anabolic one during the course of evolution.

Influence of an aggregated multienzyme system on transient time: kinetic evidence for compartmentation by an aromatic-amino-acid synthesizing complex of Neurospora crassa.

The aromatic complex of Neurospora crassa is an aggregated multienzyme system which catalyzes five consecutive reactions in the central pathway leading to the biosynthesis of the aromatic amino acids. In an attempt to understand the physiological importance of this complex in particular, as well as the importance of cellular organization of enzyme systems in general, we have isolated the complex and have begun to characterize its catalytic properties. Optimum conditions for the assay of the overall 5-step reaction catalyzed by the partially purified complex have been determined. An analog computer was programmed to represent an unaggregated system of five enzymes with rate constants identical to those found for the constituent enzymes of the complex. By direct comparison, it was shown that the lags (transient times) obtained for the overall reaction were 10-15 times longer for the hypothetical unaggregated system than for the complex. We conclude from these data that the aggregated multienzyme system compartmentalizes intermediate substrates during the course of the overall reaction. We suggest that, in addition to "channeling" intermediates of competing pathways, reduction of the transient time may be an important consequence of the containment of intermediates within a physically associated enzyme sequence. The fact that the aromatic complex exhibits a second catalytic property unique to aggregated enzyme systems, "coordinate activation" [Welch, G.R. & Gaertner, F.H. (1975) Arch. Biochem. Biophys., in press] indicates that the physical association of these enzymes may have more than one physiological function.

Kynureninase-Type enzymes of Penicillum roqueforti, Aspergillus niger, Rhizopus stolonifer, and Pseudomonas fluorescens: further evidence for distinct kynureninase and hydroxykynureninase activities.

The kynureninase-type enzymes of three fungi and one bacterium were isolated and examined kinetically for their ability to catalyze the hydrolysis of L-kynurenine and L-3-hydroxykynurenine. The phycomycete Rhizopus stolonifer was found to contain a single, constitutive enzyme with Km for L-3-hydroxykynurenine and L-kynurenine of 6.67 times 10-minus 6 and 2.5 times 10-minus 4 M, respectively. The ascomycetes Aspergillus niger and Penicillium roqueforti each contain an enzyme, induced by L-tryptophan, with similar Km for L-3-hydroxykynurenine and L-kynurenine ranging from 5.9 times 10-minus 5 to 14.3 times 10-minus 5 M, as well as a constitutive enzyme with Km for the two substrates of similar to 4 times 10-minus 6 M and 10-minus 4 M. The bacterium Pseudomonas fluorescens has a single, inducible enzyme with Km for L-3-hydroxykynurenine and L-kynurenine of 5 times 10-minus 4 and 7 times 10-minus 5 M. In addition, significant differences in maximal velocities (Vmax) were observed in two cases. The Vmax of the inducible activity from P. fluorescens was 4.5 times greater for L-kynurenine than L-3-hydroxykynurenine, whereas the Vmax of the constitutive activity from R. stolonifer was 2.5 times greater for L-3-hydroxykynurenine. It is concluded (i) that the constitutive activities are hydroxykynureninases involved in the biosynthesis of nicotinamide adenine dinucleotide from L-tryptophan, (ii) that the inducible activities are kynureninases involved in the catabolism of L-tryptophan to anthranilate, and (iii) that R. stolonifer and P. fluorescens, respectively, carry the most specific examples of each type of enzyme.

Hydroxykynureninase and the excretion of 3-hydroxyanthranilate by yeast.

A comparative analysis of the kynureninase-type activity found in various organisms has demonstrated two forms of enzyme. One, inducible by tryptophan, has a relatively low Km for L-kynurenine, and is found in microorganisms such as Pseudomonas fluorescens and Neurospora crassa. The other, unaffected by tryptophan, has a low Km for L-3-hydroxykynurenine and is found in a wide variety of organisms, including molds, amphibia, birds and mammals. The yeast Saccharomyces cerevisiae lacks the inducible kynureninase but contains the constitutive enzyme termed an hydroxykynureninase. As a result, the yeast, unlike M. crassa, excretes 3-hydroxyanthranilate in response to L-tryptophan. However, initial studies indicate that little if any 3-hydroxyanthranilate accumulates in beer, wine or bread. Although 3-hydroxyanthranilate is suspect as an endogenous carcinogen, its carcinogenicity as an exogenous agent has not been shown.