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274 12 Mining Genomes for Nitrilases

dependent on the structure of the substrate, bulky and electron-withdrawing groups
supporting the formation of amide by some nitrilases [16].
There are many examples of nitrilase-catalyzed reactions in which amides form
a considerable amount of the reaction products, such as the transformations of
acrylonitrile analogs and α-fluoroarylacetonitriles by nitrilase 1 from Arabidopsis
thaliana [17], the conversion of β-cyano-l-alanine into a mixture of l-asparagine and
l-aspartic acid by nitrilase 4 from the same organism [18] or the transformations
of mandelonitrile by nitrilase from Pseudomonas fluorescens [19] or some fungi [8].
Moreover, formamide is the only product of the cyanide transformation by cyanide
hydratase. Therefore, this enzyme was classified as a lyase (EC 4.2.1.66), although
it is closely related to nitrilases, as far as its aa sequence and reaction mechanism
are concerned [3].
Cyanide dihydratases also act on cyanide but convert it into formiate and
ammonia, and can be therefore classified as hydrolases.

12.2.1
Nitrilases in Bacteria

The occurrence of nitrilases in bacteria seems to be limited to specific taxa, as
indicated by a sequential analysis [20] of a set of biochemically characterized
nitrilases from cultivated species and hypothetical nitrilases identified in over 150
bacterial genomes that had been sequenced at the time (2005), of which less than
7% contained nitrilase genes. The enzymes from known species (18 in total) were
classified, according to sequence similarities, into subfamilies 1 and 2. All the
nitrilases in actinobacteria, among them the well characterized enzymes in R.
rhodochrous strains J1 and K22, belonged to subfamily 2, together with the known
nitrilases in Acidovorax, Alcaligenes, Bradyrhizobium, Bacillus, and so on, and the
cyanide dihydratase in Pseudomonas stutzeri. Subfamily 1 contained enzymes from
cyanobacteria, verrucomicrobia, and so on. Sequences flanking the nitrilase genes
were identified in some of the bacterial operons, making it possible to hypothesize
the various biological roles of nitrilases in various bacterial species.

12.2.2
Nitrilases in Fungi
Using known bacterial and fungal nitrilases as templates, homologous genes can
be found in the classes of Eurotiomycetes (Arthroderma, Aspergillus, Penicillium,
etc.), Ascomycetes (Neurospora), Dothideomycetes (Leptosphaeria, Pyrenophora, etc.),
Leotiomycetes (Botrytis, Sclerotinia), and Sordariomycetes (Fusarium, Gibberella, Tri-
choderma, etc.). The identity levels between fungal nitrilases are in some cases as
low as about 30%. Biochemical characterization of a few of them demonstrated
that they belong to different substrate specificity types (see following text).
Cyanide hydratases are more closely related to each other, with over 50% identi-
ties. It was hypothesized [20] that their ancestor genes were acquired by fungi from
bacteria via horizontal gene transfer, which was followed by neofunctualization of