Catherine O'Reilly

The nitrilase family of CN hydrolysing enzymes - a comparative study.

The enzymes nitrilase, cyanide dihydratase and cyanide hydratase are a group of closely related proteins. The proteins show significant similarities at the amino acid and protein structure level but the enzymes show many differences in catalytic capability. Nitrilases, while catalysing the hydration of nitrile to the corresponding acid, vary widely in substrate specificity. Cyanide dihydratase and cyanide hydratase use HCN as the only efficient substrate but produce acid and amide products, respectively. The similarities of all these enzymes at the amino acid level but the functional differences between them provide a rich source of material for the study of structure/function relationships in this biotechnologically important group of enzymes. This review provides an overview of current understanding of the genetics and biochemistry of this interesting group of enzymes.

Molecular cloning of the a1 locus of Zea mays using the transposable elements En and Mu1.

The a1 locus of Zea mays has been cloned using transposable elements as gene tags. The strategy was to make genomic libraries from maize stocks with a1 mutations induced either by En(Spm) or by Robertsons Mutator‐system. These libraries were then screened with either Spm‐I8 and En1, for the En‐containing mutant, or with Mu1 for the Mu‐induced mutation. There are many En and Mu1 hybridizing sequences present in the maize genome, however, by a process of cross‐screening of the positives from the two libraries and by molecular analysis of the En‐positive clones it was possible to identify clones in both libraries carrying all or part of the a1 gene.

Purification and properties of cyanide hydratase from Fusarium lateritium and analysis of the corresponding chy1 gene

The filamentous fungus Fusarium lateritium is cyanide tolerant, due, at least in part, to the induction by cyanide of the enzyme formamide hydrolyase (EC 4.2.1.66). This enzyme, more commonly known as cyanide hydratase, catalyses the hydration of cyanide to formamide. The enzyme was purified from F. lateritium and showed a subunit molecular mass of 43 kDa (as judged by SDS-PAGE), while the native protein appeared to form aggregates of up to 1217 kDa (as judged by gel-filtration and non-denaturing PAGE). mRNA samples from cultures grown with and without cyanide were in vitro translated and immunoprecipitated. This demonstrated that, in this species, the gene encoding the enzyme designated chy1, is cyanide inducible. Differential screening was used to isolate a cyanide hydratase cDNA clone which was subsequently used to obtain the corresponding genomic clone. A fragment of the cDNA clone encoding all but the first seven amino acids of the protein was expressed in E. coli using the expression vector pGEX-2T. Features of F. lateritium cyanide hydratase together with an analysis of the nucleotide sequence encoding this enzyme are presented.

The cyanide hydratase enzyme of Fusarium lateritium also has nitrilase activity

The filamentous fungus Fusarium lateritium produces cyanide hydratase when grown in the presence of cyanide. The cyanide hydratase protein produced at a high level in Escherichia coli shows a low but significant nitrilase activity with acetonitrile, propionitrile and benzonitrile. The nitrilase activity is sufficient for growth of the recombinant strain on acetonitrile, propionitrile or benzonitrile as the sole source of nitrogen. The recombinant enzyme shows highest nitrilase activity with benzonitrile. Site-directed mutagenesis of the F. lateritium cyanide hydratase gene indicates that mutations leading to a loss of cyanide hydratase activity also lead to a loss of nitrilase activity. This suggests that the active site for cyanide hydratase and nitrilase activity in the protein is the same. This is the first evidence of cyanide hydratase having nitrilase activity.

DNA sequence of the cut A, B and C genes, encoding the molybdenum containing hydroxylase carbon monoxide dehydrogenase, from Pseudomonas thermocarboxydovorans strain C2.

Pseudomonas thermocarboxydovorans strain C2 is capable of using carbon monoxide as the sole source of carbon and energy. The key enzyme for CO utilisation is the molybdenum containing iron-flavoprotein carbon monoxide dehydrogenase (CODH). This paper reports the DNA sequencing of a 4.7 kb region of the C2 genome which appears to encode the CODH enzyme. The genes for the three subunits of CODH, which we have named cut A, B and C, have been identified and they appear to form an operon. The predicted protein sequences of the three subunits have homology to the structurally related protein, xanthine dehydrogenase, from Drosophila melanogaster. By comparison with xanthine dehydrogenase it can be predicted that the molybdenum cofactor binds to the large subunit of CODH, the small subunit of CODH contains the iron-sulphur centers and the medium subunit binds FAD/NAD+.

Efficient expression in E-coli of an enantioselective nitrile hydratase from Rhodococcus erythropolis

The genes encoding an enantioselective nitrile hydratase (NHase) from Rhodococcus erythropolis AJ270 have been cloned and an active NHase has been produced in Escherichia coli. Maximal activity was found when the genes encoding the α- and β-subunits were transcribed as one unit and the gene encoding the P44k activator protein as a separate ORF on a single replicon. Addition of n-butyric acid and FeSO4 could improve NHase activity. Coexpression of the GroEL-GroES chaperone proteins increased activity in the absence of P44k protein but had no effect in the presence of P44k. The recombinant enzyme was highly enantioselective in the synthesis of S-(+)-3-benzoyloxy- 4-cyanobutyramide from the prochiral substrate 3-benzoyloxyglutaronitrile.

Development of novel real-time TaqMan(®) PCR assays for the species and sex identification of otter (Lutra lutra) and their application to noninvasive genetic monitoring.

Developing strategies to maintain biodiversity requires baseline information on the current status of each individual species. The development of genetic techniques and their application to noninvasively collected samples have the potential to yield information on the structure of elusive animal populations and so are important tools in conservation management. Using DNA isolated from faecal samples can be challenging owing to low quantity and quality. This study, however, presents the development of novel real‐time polymerase chain reaction assays using fluorescently labelled TaqMan® MGB probes enabling species and sex identification of Eurasian otter (Lutra lutra) spraints (faeces). These assays can also be used in determining an optimum microsatellite panel and can be employed as cost‐saving screening tools for downstream genetic testing including microsatellite genotyping and haplotype analysis. The techniques are shown to work efficiently with L. lutra DNA isolated from tissue, hair, spraint, blood and anal jelly samples.

Molecular cloning of genes involved in purine biosynthetic and salvage pathways of Salmonella typhimurium.

SummaryGenes have been cloned from Salmonella typhimurium which when present on the multicopy plasmid pBR322 in the E. coli strain NT31 confer a Gua+ phenotype on this strain. NT31 is a purE gpt double mutant and it was expected that a Gua+ phenotype could be conferred on it by the cloning of either gpt or purE. It was, however found that in addition to these two loci the molecular cloning of another gene, which has been identified as hpt, in pBR322 confers a Gua+ phenotype on NT31. This result is explained by the overproduction of the hpt gene product, hypoxanthine phosphoribosyl transferase, which compensates for the lack of the gpt product guanine-xanthine phosphoribosyl transferase. Restriction analysis of the three loci, gpt, hpt and purE is also presented.