A novel dehydratase that catalyzes the stoichiometric dehydration of Z-phenylacetaldoxime to phenylacetonitrile has been purified 483-fold to homogeneity from a cell-free extract of Bacillus sp. strain OxB-1 isolated from soil. It has a M(r) of about 40 000 and is composed of a single polypeptide chain with a loosely bound protoheme IX. The enzyme is inactive unless FMN is added to the assay, but low activity is also observed when sulfite replaces FMN. The activity in the presence of FMN is enhanced 5-fold under anaerobic conditions compared to the activity measured in air. The enzyme has maximum activity at pH 7.0 and 30 degrees C, and it is stable at up to 45 degrees C at around neutral pH. The aerobically measured activity in the presence of FMN is also enhanced by Fe(2+), Sn(2+), SO(3)(2)(-), and NaN(3). Metal-chelating reagents, carbonyl reagents, electron donors, and ferri- and ferrocyanides strongly inhibit the enzyme with K(i) values in the micromolar range. The enzyme is active with arylalkylaldoximes and to a lesser extent with alkylaldoximes. The enzyme prefers the Z-form of phenylacetaldoxime over its E-isomer. On the basis of its substrate specificity, the enzyme has been tentatively named phenylacetaldoxime dehydratase. The gene coding for the enzyme was cloned into plasmid pUC18, and a 1053 base-pair open reading frame that codes for 351 amino acid residues was identified as the oxd gene. A nitrilase, which participates in aldoxime metabolism in the organism, was found to be coded by the region just upstream from the oxd gene. In addition an open reading frame (orf2), whose gene product is similar to bacterial regulatory (DNA-binding) proteins, was found just upstream from the coding region of the nitrilase. These findings provide genetic evidence for a novel gene cluster that is responsible for aldoxime metabolism in this microorganism.

An enzyme "alkylaldoxime dehydratase (OxdRG)" was purified and characterized from Rhodococcus globerulus A-4, in which nitrile hydratase (NHase) and amidase coexisted with the enzyme. The enzyme contains heme b as a prosthetic group, requires reducing reagents for the reaction, and is most active at a neutral pH and at around 30°C, similar to the phenylacetaldoxime dehydratase from Bacillus sp. OxB-1 (OxdB). However, some differences were seen in subunit structure, substrate specificity, and effects of activators and inhibitors. The corresponding gene, oxd, encoding a 1059-base pair ORF consisting of 353 codons, was cloned, sequenced, and overexpressed in Escherichia coli. The predicted polypeptide showed 30.3% identity to OxdB. The gene is mapped just upstream of the gene cluster encoding the enzymes involved in the metabolism of aliphatic nitriles, i.e., NHase and amidase, and their regulatory and activator proteins. We report here the existence of an aldoxime dehydratase genetically linked with NHase and amidase, and responsible for the metabolism of alkylaldoxime in R. globerulus.Aldoximes derived from amino acids are considered to be intermediates in the biosynthesis of cyanogenic glucosides and glucosinolates in plants (1). However, information on aldoxime metabolism is quite limited and the genetics and enzymology have not been well characterized. One oximemetabolizing enzyme (cytochrome P450 CYP71E1) has been reported to catalyze the conversion of aldoxime to R-hydroxynitrile in the pathway for biosynthesis of cyanogenic glucoside dhurrin in Sorghum bicolor (2-4). Other cytochrome P450s, namely, CYP83 homologues (A1 and B1), have also been identified as oxime-metabolizing enzymes, which catalyze the conversion of indoleacetaldoxime to the corresponding aci-nitro compound, the first step in the biosynthesis of indole glucosinolates in Arabidopsis thaliana. However, the level of activity is quite low, and the mechanisms involved have not been studied. Indoleacetaldoxime is known to be a metabolic branch point between the production of indoleacetic acid and indole glucosinolates in A. thaliana (5-7), but the enzymes responsible for the metabolism have yet to be purified and characterized.Asano et al. have isolated various nitrile-degrading microorganisms, e.g., Rhodococcus rhodochrous (formerly Arthrobacter sp.) strains J-1 and I-9 (K22, AKU 629) (8) and Pseudomonas chlororaphis B23 (9). They first purified, characterized, and named nitrile hydratase (NHase, EC. 4.2.1.84) from R. rhodochrous J-1 (10-12). They also found that P. chlororaphis B23 accumulates large quantities of amides from nitriles and is suitable for the industrial production of acrylamide from acrylonitrile (9, 12). Moreover, nicotinamide and 5-cyanovaleramide are also industrially produced by NHase (13,14). Despite its important uses, the physiological function of NHase in nature remains unclear.We have studied the metabolism of aldoximes from a physiological as well as an applicative perspective, and have isolated Bacillus sp. O...

Hydroxynitrile lyase (HNL) catalyzes the degradation of cyanohydrins and causes the release of hydrogen cyanide (cyanogenesis). HNL can enantioselectively produce cyanohydrins, which are valuable building blocks for the synthesis of fine chemicals and pharmaceuticals, and is used as an important biocatalyst in industrial biotechnology. Currently, HNLs are isolated from plants and bacteria. Because industrial biotechnology requires more efficient and stable enzymes for sustainable development, we must continuously explore other potential enzyme sources for the desired HNLs. Despite the abundance of cyanogenic millipedes in the world, there has been no precise study of the HNLs from these arthropods. Here we report the isolation of HNL from the cyanide-emitting invasive millipede Chamberlinius hualienensis, along with its molecular properties and application in biocatalysis. The purified enzyme displays a very high specific activity in the synthesis of mandelonitrile. It is a glycosylated homodimer protein and shows no apparent sequence identity or homology with proteins in the known databases. It shows biocatalytic activity for the condensation of various aromatic aldehydes with potassium cyanide to produce cyanohydrins and has high stability over a wide range of temperatures and pH values. It catalyzes the synthesis of (R)-mandelonitrile from benzaldehyde with a 99% enantiomeric excess, without using any organic solvents. Arthropod fauna comprise 80% of terrestrial animals. We propose that these animals can be valuable resources for exploring not only HNLs but also diverse, efficient, and stable biocatalysts in industrial biotechnology.millipede hydroxynitrile lyase | bioresource exploration | biocatalysis | white biotechnology | arthropod

Hydroxynitrile lyases are valuable enzymes for asymmetric synthesis of cyanohydrins. These hydroxyl and nitrileÀcontaining compounds are being used in production of very useful pharmaceuticals, agrochemicals, and other biologically active compounds using chemical or chemoenzymatic follow-up reactions in industry. Although a huge amount of information exists on the reaction parameters of these enzymes, including stability to pH and organic solvents, yield, reaction time, and valuable data on the enantiopurity of their products, cyanohydrins, there is a lack of update on the biochemistry, discovery, and engineering of the HNLs. Therefore, in the Introduction, we will have a look into these enzymes, cyanohydrins, and aldoxime-nitrile pathways. A brief view of functional groups and several examples of cyanohydrin-based chemicals and pharmaceuticals will also be described. Then we will present characteristics of many S-and R-selective HNLs with comparative tables for several enzymatic properties under biochemistry section. The methods of screening and discovery of these enzymes both from nature and a library of mutants will be described as well as their potential in the synthesis of chemicals. Cloning and expression of new HNLs will also be described under the discovery section. A pool of successful applications of protein engineering methods and the subsequent improvement in the properties of mutant HNLs will be reviewed in detail afterward.

Methylaspartate ammonia lyase (MAL) catalyzes the magnesium-dependent reversible alpha,beta-elimination of ammonia from L-threo-(2S,3S)-3-methylaspartic acid to mesaconic acid. The 1.3 A MAD crystal structure of the dimeric Citrobacter amalonaticus MAL shows that each subunit comprises two domains, one of which adopts the classical TIM barrel fold, with the active site at the C-terminal end of the barrel. Despite very low sequence similarity, the structure of MAL is closely related to those of representative members of the enolase superfamily, indicating that the mechanism of MAL involves the initial abstraction of a proton alpha to the 3-carboxyl of (2S,3S)-3-methylasparic acid to yield an enolic intermediate. This analysis resolves the conflict that had linked MAL to the histidine and phenylalanine ammonia lyase family of enzymes.

Aldoxime dehydratase (Oxd) catalyzes the dehydration of aldoximes (R-CH‫؍‬N-OH) to their corresponding nitrile (R-C'N). Oxd is a heme-containing enzyme that catalyzes the dehydration reaction as its physiological function. We have determined the first two structures of Oxd: the substrate-free OxdRE at 1.8 Å resolution and the n-butyraldoxime-and propionaldoxime-bound OxdREs at 1.8 and 1.6 Å resolutions, respectively. Unlike other heme enzymes, the organic substrate is directly bound to the heme iron in OxdRE. We determined the structure of the Michaelis complex of OxdRE by using the unique substrate binding and activity regulation properties of Oxd. The Michaelis complex was prepared by x-ray cryoradiolytic reduction of the ferric dead-end complex in which Oxd contains a Fe 3؉ heme form. The crystal structures reveal the mechanism of substrate recognition and the catalysis of OxdRE.

A cyanide-free platform technology for the synthesis of chiral nitriles by biocatalytic enantioselective dehydration of a wide range of aldoximes is reported. The nitriles were obtained with high enantiomeric excess of >90 % ee (and up to 99 % ee) in many cases, and a "privileged substrate structure" with respect to high enantioselectivity was identified. Furthermore, a surprising phenomenon was observed for the enantiospecificity that is usually not observed in enzyme catalysis. Depending on whether the E or Z isomer of the racemic aldoxime substrate was employed, one or the other enantiomer of the corresponding nitrile was formed preferentially with the same enzyme.

The distribution of phenylacetaldoxime-degrading and pyridine-3-aldoxime-degrading ability was examined with intact cells of 975 microorganisms, including 45 genera of bacteria, 11 genera of actinomyces, 22 genera of yeasts, and 37 genera of fungi, by monitoring the decrease of the aldoximes by high-pressure liquid chromatography. The abilities were found to be widely distributed in bacteria, actinomyces, fungi, and some yeasts: 98 and 107 strains degraded phenylacetaldoxime and pyridine-3-aldoxime, respectively. All of the active strains exhibited not only the aldoxime-dehydration activity to form nitrile but also nitrile-hydrolyzing activity. On the other hand, all of 19 nitrile-degrading microorganisms (13 species, 7 genera) were found to exhibit aldoxime dehydration activity. It is shown that aldoxime dehydratase and nitrile-hydrolyzing activities are widely distributed among 188 aldoxime and 19 nitrile degraders and that the enzymes were induced by aldoximes or nitriles.Nitrile compounds are discharged into the environment as industrial waste water, agricultural chemicals, etc. (23). Many microorganisms can use nitriles as a source of carbon and/or nitrogen for growth. Asano et al. have isolated various nitriledegrading microorganisms from soil (2, 5, 24) and clarified that nitriles are converted to carboxylic acids either by a combination of nitrile hydratase (1, 3, 6) and amidase (4) or by nitrilase (8). These enzymes have been extensively evaluated from the viewpoint of chemical industry: the industrial production of acrylamide, nicotinamide, and 5-cyanovalelamide are typical examples (5,6,16,26). Despite the importance of the nitrilehydrolyzing enzymes in industry, information about their distribution in microorganisms is quite limited, and their physiological role has never been well understood since they have been screened only from nitrile-degrading microorganisms.We have been studying aldoxime-degrading enzymes and isolated various aldoxime-degrading microorganisms, e.g., Bacillus sp. strain OxB-1 (7) and Rhodococcus sp. strain YH3-3 (12), from soil. The isolated strains metabolized aldoximes through nitriles into the corresponding carboxylic acid by a combination of a novel aldoxime dehydratase and nitrile-hydrolyzing enzymes (7, 12) ( Fig. 1). The novel aldoxime dehydratase was purified and characterized from Bacillus sp. strain OxB-1 (7, 15). The enzyme from Rhodococcus sp. strain YH3-3 was applied to the enzymatic synthesis of nitriles from aldoximes under a mild condition (12, 13). A nitrile hydratase responsible for aldoxime metabolism was purified and characterized from Rhodococcus sp. strain YH3-3, and its properties were compared with the known nitrile hydratases (14).To elucidate the generality of the relationship of aldoxime dehydratase and nitrile-hydrolyzing enzymes in microorganisms, we examined the distribution of both enzyme activities with the intact cells of a variety of aldoxime-or nitrile-degrading microorganisms, monitoring the decrease of the aldoximes, and we studied the relat...