Primary or secondary? Versatile nitrilases in plant metabolism

The potential of plant nitrilases to convert indole-3-acetonitrile into the plant growth hormone indole-3-acetic acid has earned them the interim title of "key enzyme in auxin biosynthesis". Although not widely recognized, this view has changed considerably in the last few years. Recent work on plant nitrilases has shown them to be involved in the process of cyanide detoxification, in the catabolism of cyanogenic glycosides and presumably in the catabolism of glucosinolates. All plants possess at least one nitrilase that is homologous to the nitrilase 4 isoform of Arabidopsis thaliana. The general function of these nitrilases lies in the process of cyanide detoxification, in which they convert the intermediate detoxification product beta-cyanoalanine into asparagine, aspartic acid and ammonia. Cyanide is a metabolic by-product in biosynthesis of the plant hormone ethylene, but it may also be released from cyanogenic glycosides, which are present in a large number of plants. In Sorghum bicolor, an additional nitrilase isoform has been identified, which can directly use a catabolic intermediate of the cyanogenic glycoside dhurrin, thus enabling the plant to metabolize its cyanogenic glycoside without releasing cyanide. In the Brassicaceae, a family of nitrilases has evolved, the members of which are able to hydrolyze catabolic products of glucosinolates, the predominant secondary metabolites of these plants. Thus, the general theme of nitrilase function in plants is detoxification and nitrogen recycling, since the valuable nitrogen of the nitrile group is recovered in the useful metabolites asparagine or ammonia. Taken together, a picture emerges in which plant nitrilases have versatile functions in plant metabolism, whereas their importance for auxin biosynthesis seems to be minor.     

Cyanide detoxification in an insect herbivore: Molecular identification of β-cyanoalanine synthases from Pieris rapae

Cyanogenic compounds occur widely in the plant kingdom. Therefore, many herbivores are adapted to the presence of these compounds in their diet by either avoiding cyanide release or by efficient cyanide detoxification mechanisms. The mechanisms of adaptation are not fully understood. Larvae of Pieris rapae (Lepidoptera: Pieridae) are specialist herbivores on glucosinolate-containing plants. They are exposed to cyanide during metabolism of phenylacetonitrile, a product of benzylglucosinolate breakdown catalyzed by plant myrosinases and larval nitrile-specifier protein (NSP) in the gut. Cyanide is metabolized to β-cyanoalanine and thiocyanate in the larvae. Here, we demonstrate that larvae of P. rapae possess β-cyanoalanine activity in their gut. We have identified three gut-expressed cDNAs designated PrBSAS1-PrBSAS3 which encode proteins with similarity to β-substituted alanine synthases (BSAS). Characterization of recombinant PrBSAS1-PrBSAS3 shows that they possess β-cyanoalanine activity. In phylogenetic trees, PrBSAS1-PrBSAS3, the first characterized insect BSAS, group together with a characterized mite β-cyanoalanine synthase and bacterial enzymes indicating a similar evolutionary history.     

The Arabidopsis thaliana isogene NIT4 and its orthologs in tobacco encode beta-cyano-L-alanine hydratase/nitrilase

Nitrilases (nitrile aminohydrolases, EC ) are enzymes that catalyze the hydrolysis of nitriles to the corresponding carbon acids. Among the four known nitrilases of Arabidopsis thaliana, the isoform NIT4 is the most divergent one, and homologs of NIT4 are also known from species not belonging to the Brassicaceae like Nicotiana tabacum and Oryza sativa. We expressed A. thaliana NIT4 as hexahistidine tag fusion protein in Escherichia coli. The purified enzyme showed a strong substrate specificity for beta-cyano-l-alanine (Ala(CN)), an intermediate product of cyanide detoxification in higher plants. Interestingly, not only aspartic acid but also asparagine were identified as products of NIT4-catalyzed Ala(CN) hydrolysis. Asn itself was no substrate for NIT4, indicating that it is not an intermediate but one of two reaction products. NIT4 therefore has both nitrilase and nitrile hydratase activity. Several lines of evidence indicate that the catalytic center for both reactions is the same. The NIT4 homologs of N. tabacum were found to catalyze the same reactions and protein extracts of A. thaliana, N. tabacum and Lupinus angustifolius also converted Ala(CN) to Asp and Asn in vitro. NIT4 may play a role in cyanide detoxification during ethylene biosynthesis because extracts from senescent leaves of A. thaliana showed higher Ala(CN) hydratase/nitrilase activities than extracts from nonsenescent tissue.     

A conserved mechanism for nitrile metabolism in bacteria and plants

Pseudomonas fluorescens SBW25 is a plant growth-promoting bacterium that efficiently colonises the leaf surfaces and rhizosphere of a range of plants. Previous studies have identified a putative plant-induced nitrilase gene ( pinA ) in P. fluorescens SBW25 that is expressed in the rhizosphere of sugar beet plants. Nitrilase enzymes have been characterised in plants, bacteria and fungi and are thought to be important in detoxification of nitriles, utilisation of nitrogen and synthesis of plant hormones. We reveal that pinA is a NIT4-type nitrilase that catalyses the hydrolysis of β-cyano- l -alanine, a nitrile common in the plant environment and an intermediate in the cyanide detoxification pathway in plants. In plants cyanide is converted to β-cyano- l -alanine, which is subsequently detoxified to aspartic acid and ammonia by NIT4. In P. fluorescens SBW25 pinA is induced in the presence of β-cyano- l -alanine, and the β-cyano- l -alanine precursors cyanide and cysteine. pinA allows P. fluorescens SBW25 to use β-cyano- l -alanine as a nitrogen source and to tolerate toxic concentrations of this nitrile. In addition, pinA is shown to complement a NIT4 mutation in Arabidopsis thaliana , enabling plants to grow in concentrations of β-cyano- l -alanine that would otherwise prove lethal. Interestingly, over-expression of pinA in wild-type A. thaliana not only resulted in increased growth in high concentrations of β-cyano- l -alanine, but also resulted in increased root elongation in the absence of exogenous β-cyano- l -alanine, demonstrating that β-cyano- l -alanine nitrilase activity can have a significant effect on root physiology and root development.     

Assessing functional diversity in the soybean β-substituted alanine synthase enzyme family

In plants, proteins of the β-substituted alanine synthase (BSAS) enzyme family perform a diverse range of reactions, including formation of cysteine from O-acetylserine and sulfide, detoxification of cyanide by its addition to cysteine, the breakdown of cysteine into pyruvate, ammonia, and sulfide, and the synthesis of S-sulfocysteine. With the completed genome sequence of soybean (Glycine max (L.) Merr. cv. Williams 82), the functional diversity of the BSAS in this highly duplicated plant species was examined to determine whether soybean BSAS enzymes catalyze the various reactions connected to cysteine metabolism. The 16 soybean BSAS can be grouped into clades that are similar to those observed in Arabidopsis. Biochemical analysis of soybean BSAS proteins demonstrate that enzymes of clades I and III function as O-acetylserine sulfhydrylases for cysteine synthesis, clade II encodes cysteine desulfhydrase activity, and that clade V proteins function as β-cyanoalanine synthase for cyanide detoxification. Although clade IV is similar to Arabidopsis S-sulfocysteine synthase, this activity was not detected in the soybean homolog. Overall, our results show that bioinformatics approach provides a useful method to assess the biochemical properties of BSAS enzymes in plant species.     

Cyanide Metabolism in Higher Plants: Cyanoalanine Hydratase is a NIT4 Homolog

Cyanoalanine hydratase (E.C. 4.2.1.65) is an enzyme involved in the cyanide detoxification pathway of higher plants and catalyzes the hydrolysis of β-cyano-l-alanine to asparagine. We have isolated the enzyme from seedlings of blue lupine (Lupinus angustifolius) to obtain protein sequence information for molecular cloning. In contrast to earlier reports, extracts of blue lupine cotyledons were found also to contain cyanoalanine-nitrilase (E.C. 3.5.5.4) activity, resulting in aspartic acid production. Both activities co-elute during isolation of cyanoalanine hydratase and are co-precipitated by an antibody directed against Arabidopsis thaliana nitrilase 4 (NIT4). The isolated cyanoalanine hydratase was sequenced by nanospray-MS/MS and shown to be a homolog of Arabidopsis thaliana and Nicotiana tabacum NIT4. Full-length cDNA sequences for two NIT4 homologs from blue lupine were obtained by PCR using degenerate primers and RACE-experiments. The recombinant LaNIT4 enzymes, like Arabidopsis NIT4, hydrolyze cyanoalanine to asparagine and aspartic acid but show a much higher cyanoalanine-hydratase activity. The two nitrilase genes displayed differential but overlapping expression. Taken together these data show that the so-called ‘cyanoalanine hydratase’ of plants is not a bacterial type nitrile hydratase enzyme but a nitrilase enzyme which can have a remarkably high nitrile-hydratase activity.     

Structure of soybean β-cyanoalanine synthase and the molecular basis for cyanide detoxification in plants

Plants produce cyanide (CN-) during ethylene biosynthesis in the mitochondria and require β-cyanoalanine synthase (CAS) for CN- detoxification. Recent studies show that CAS is a member of the β-substituted alanine synthase (BSAS) family, which also includes the Cys biosynthesis enzyme O-acetylserine sulfhydrylase (OASS), but how the BSAS evolved distinct metabolic functions is not understood. Here we show that soybean (Glycine max) CAS and OASS form α-aminoacrylate reaction intermediates from Cys and O-acetylserine, respectively. To understand the molecular evolution of CAS and OASS in the BSAS enzyme family, the crystal structures of Gm-CAS and the Gm-CAS K95A mutant with a linked pyridoxal phosphate (PLP)-Cys molecule in the active site were determined. These structures establish a common fold for the plant BSAS family and reveal a substrate-induced conformational change that encloses the active site for catalysis. Comparison of CAS and OASS identified residues that covary in the PLP binding site. The Gm-OASS T81M, S181M, and T185S mutants altered the ratio of OASS:CAS activity but did not convert substrate preference to that of a CAS. Generation of a triple mutant Gm-OASS successfully switched reaction chemistry to that of a CAS. This study provides new molecular insight into the evolution of diverse enzyme functions across the BSAS family in plants.     

植物的不同组织对氰化物降解的可行性研究

氰化物是目前世界范围内最常使用的提取黄金等贵重金属的沥滤剂,其对自然生态环境的污染和破坏以及对人畜的毒性作用是众所周知的,氰化物污染的治理也就成为了环境工作者关注的焦点课题。植物细胞的悬浮液通常用于研究污染物的降解及在植物体内的生化反应机制。本实验直接用三种杨柳科植物的不同组织(根须、嫩茎、老茎和叶片)来观察和测定植物细胞线粒体中的氰丙氨酸合成酶(β-cyanoalanine synthase)转化氰化物的可行性。实验是在一封闭的玻璃器皿(100mL)中进行的(100mL的氰化钾溶液中加入1.0g(鲜重)的植物组织,氰化钾溶液的浓度0.444～0.457 CN mg·L~(-1))。在为期28 h的时间内,水溶液中的氰化物22.13～67.04％被植物的不同组织去除。最快的氰化物去除率发现在美洲黑杨(Populus deltoides)的叶片实验组(1.9449 mg CN/kg(鲜重)·h),苏柳(Salix matssudana Koidz×Salix alba L)的叶片实验组次之(1.7259 mg CN/kg(鲜重)·h),最慢的氰化物去除率发现在苏柳的嫩茎实验组(0.4934 mg CN/kg(鲜重)·h)。实验结果表明,选用植物的组织同样可以观察和测定污染物在植物体内的转化,特别是植物的叶片表现的尤其敏感。     

Cytosolic r{beta}-Cyanoalanine Synthase Activity Attributed to Cysteine Synthases in Cocklebur Seeds. Purification and Characterization of Cytosolic Cysteine Synthases

The activity of rß-cyanoalanine synthase (CAS, EC4.4.1.9 [EC] ) in cotyledons of cocklebur seeds (Xanthium penn-sylvanicumWallr.) was detected both in the soluble and particulate fractions.The CAS activity of the soluble fraction (cytosolic CAS activity)was 10 times higher than that of the particulate fraction. TheCAS activity of the particulate fraction was confirmed to belocalized in the mitochondria. Both enzymatic activities wereclearly separated by non-denaturing PAGE. The enzyme with cytosolicCAS activity has been extensively purified and separated intothree different forms designated as cyt-1, cyt-2, and cyt-3.According to the SDS-PAGE analysis, the three enzymes are estimatedto be a homodimer composed of 35-kDa sub-units. The purifiedenzymes showed CS activity. Partial amino acid sequences ofcyt-1 were determined and had a high homology with cysteinesynthases (CS, EC 4.2.99.8 [EC] ) from other plant sources. The catalyticaction of the purified CSs in converting cyanide and cysteineinto H₂S and rß-cyanoalanine was confirmed by thedetection of significant ¹⁴CN incorporation into rß-cyanoalanine.These results indicated that cytosolic CAS activity is due tocytosolic CS and suggested that the CAS activity of CS is likelyto be involved in cyanide metabolism in plant tissues. (Received January 7, 1998; Accepted March 16, 1998)     

Maize nitrilases have a dual role in auxin homeostasis and beta-cyanoalanine hydrolysis

The auxin indole-3-acetic acid (IAA), which is essential for plant growth and development, is suggested to be synthesized via several redundant pathways. In maize (Zea mays), the nitrilase ZmNIT2 is expressed in auxin-synthesizing tissues and efficiently hydrolyses indole-3-acetonitrile to IAA. Zmnit2 transposon insertion mutants were compromised in root growth in young seedlings and sensitivity to indole-3-acetonitrile, and accumulated lower quantities of IAA conjugates in kernels and root tips, suggesting a substantial contribution of ZmNIT2 to total IAA biosynthesis in maize. An additional enzymatic function, turnover of beta-cyanoalanine, is acquired when ZmNIT2 forms heteromers with the homologue ZmNIT1. In plants carrying an insertion mutation in either nitrilase gene this activity was strongly reduced. A dual role for ZmNIT2 in auxin biosynthesis and in cyanide detoxification as a heteromer with ZmNIT1 is therefore proposed.     

Formation of β-cyanoalanine and pyruvate by Acacia georginae

Pyruvate is formed on incubation of l-cysteine with acetone powder preparations of Acacia georginae but in the presence of cyanide, β-cyanoalanine is produced and pyruvate production is highly depressed. The pH optimum for pyruvate production is 8·5. In the presence of fluoride (1·5 mM), the pH profile is unchanged and in the presence of cyanide (1·5 mM), minimal pyruvate production occurs at pH 8·5. Although addition of pyridoxal phosphate had no influence on pyruvate or β-Cyanoalanine production, these processes were prevented by sodium borohydride, an inhibitor of pyridoxal enzymes. Neither l-serine nor O-acetyl-l-serine serve as alternative substrates for pyruvate production. β-Fluoroalanine was not detected on incubating fluoride with an enzyme preparation from A. georginae acetone powders.     

Induction of auxin biosynthetic enzymes by jasmonic acid and in clubroot diseased Chinese cabbage plants

Nitrilase (NIT) and myrosinase are important enzymes for auxin biosynthesis in Brassicaceae, which is increased during clubroot disease. Therefore, NIT and myrosinase levels during club development and possible regulation mechanisms were investigated. In addition, the occurrence of different nitrilase isoforms in Chinese cabbage has been shown. Nitrilase activity was enhanced in infected roots during later stages of club development (35–42 days after inoculation). However, no differences in nitrilase mRNA levels between infected and healthy roots were found during symptom development. Myrosinase expression was increased in clubbed roots at slightly earlier time points (28 days after inoculation) and also at later time points during infection. The activities of tryptophan oxidizing enzyme (TrpOxE), which catalyzes the first step in tryptophan-dependent auxin biosynthesis in Brassicaceae, and nitrilase were enhanced after treatment with jasmonic acid (JA) and methyl jasmonate. Similarly, the amount of myrosinase mRNA was increased by JA. During clubroot disease the endogenous concentration of JA increased in infected roots 3–5 weeks after inoculation. From our results it can be concluded that: (1) de novo indole-3-acetic acid (IAA) biosynthesis plays a role for symptom development of clubroot disease in Brassicaceae during later developmental stages; and (2) JA which increased during club development, may be involved in the up-regulation of three enzymes important for IAA synthesis.     

Isolation and Characterization of Novel β-Cyanoalanine Synthase and Cysteine Synthase Genes from Cassava

Cassava is an important staple food crop, feeding 600 million people worldwide, which produce cyanogenic glycosides. Cyanogenic glycosides in cassava are known to act as a deterrent for herbivores as well as serve as a mobile source of reduced nitrogen. Cassava is also equipped with a cyanide detoxification pathway, mediated by β-cyanoalanine synthase (β-CAS) which converts cyanide into asparagine. β-CAS, belonging to the Bsas family of enzymes, is multi functional and shares sequence homology with cysteine synthase (CS). Using rapid amplification of cDNA end-polymerase chain reaction (RACE-PCR), two cDNA sequences were isolated from cassava. The two clones named MANes;BsasA (accession no. EU350583) and MANes;BsasB (accession no. HQ257219), showed high homology to known β-CAS enzymes (80% and 75% amino acid similarity to Arabidopsis and 76% and 82% similarity to spinach, respectively). The kinetic properties of the two clones were characterized in a Escherichia coli NK3 mutant strain which lacks activity for any of the Bsas proteins. Kinetic studies showed that MANes;BsasB is a β-CAS with a CAS/CS activity ratio of 72 while MANes;BsasA is a CS showing bifunctional capabilities and with a CAS/CS activity ratio of 11. The isolation of cassava β-CAS and CS genes reported here paves the way for their utilization in genetically enhancing the cyanide detoxification potential of cassava and/or increase of the essential amino acid cysteine, which has been found to be low in nutritionally compromised individuals.     

Turning the 'mustard oil bomb' into a 'cyanide bomb': aromatic glucosinolate metabolism in a specialist insect herbivore

Plants have evolved a variety of mechanisms for dealing with insect herbivory among which chemical defense through secondary metabolites plays a prominent role. Physiological, behavioural and sensorical adaptations to these chemicals provide herbivores with selective advantages allowing them to diversify within the newly occupied ecological niche. In turn, this may influence the evolution of plant metabolism giving rise to e.g. new chemical defenses. The association of Pierid butterflies and plants of the Brassicales has been cited as an illustrative example of this adaptive process known as 'coevolutionary armsrace'. All plants of the Brassicales are defended by the glucosinolate-myrosinase system to which larvae of cabbage white butterflies and related species are biochemically adapted through a gut nitrile-specifier protein. Here, we provide evidence by metabolite profiling and enzyme assays that metabolism of benzylglucosinolate in Pieris rapae results in release of equimolar amounts of cyanide, a potent inhibitor of cellular respiration. We further demonstrate that P. rapae larvae develop on transgenic Arabidopsis plants with ectopic production of the cyanogenic glucoside dhurrin without ill effects. Metabolite analyses and fumigation experiments indicate that cyanide is detoxified by β-cyanoalanine synthase and rhodanese in the larvae. Based on these results as well as on the facts that benzylglucosinolate was one of the predominant glucosinolates in ancient Brassicales and that ancient Brassicales lack nitrilases involved in alternative pathways, we propose that the ability of Pierid species to safely handle cyanide contributed to the primary host shift from Fabales to Brassicales that occured about 75 million years ago and was followed by Pierid species diversification.     

Metabolism of hydrogen cyanide by higher plants

A survey has been made of the occurrence and distribution of three enzymes which metabolize cyanide in a variety of higher plants including both cyanogenic and non-cyanogenic species. The enzymes investigated were β-cyanoalanine synthase, rhodanese and formamide hydrolyase. β-Cyanoalanine synthase was found to be present in every higher plant tested whereas rhodanese was found to occur far less commonly in plants. Formamide hydrolyase activity was not detected in any of the higher plants tested.     

Expression of MdCAS1 and MdCAS2, encoding apple beta-cyanoalanine synthase homologs, is concomitantly induced during ripening and implicates MdCASs in the possible role of the cyanide detoxification in Fuji apple (Malus domestica Borkh.) fruits

Fruit ripening involves complex biochemical and physiological changes. Ethylene is an essential hormone for the ripening of climacteric fruits. In the process of ethylene biosynthesis, cyanide (HCN), an extremely toxic compound, is produced as a co-product. Thus, most cyanide produced during fruit ripening should be detoxified rapidly by fruit cells. In higher plants, the key enzyme involved in the detoxification of HCN is β-cyanoalanine synthase (β-CAS). As little is known about the molecular function of β-CAS genes in climacteric fruits, we identified two homologous genes, MdCAS1 and MdCAS2, encoding Fuji apple β-CAS homologs. The structural features of the predicted polypeptides as well as an in vitro enzyme activity assay with bacterially expressed recombinant proteins indicated that MdCAS1 and MdCAS2 may indeed function as β-CAS isozymes in apple fruits. RNA gel-blot studies revealed that both MdCAS1 and MdCAS2 mRNAs were coordinately induced during the ripening process of apple fruits in an expression pattern comparable with that of ACC oxidase and ethylene production. The MdCAS genes were also activated effectively by exogenous ethylene treatment and mechanical wounding. Thus, it seems like that, in ripening apple fruits, expression of MdCAS1 and MdCAS2 genes is intimately correlated with a climacteric ethylene production and ACC oxidase activity. In addition, β-CAS enzyme activity was also enhanced as the fruit ripened, although this increase was not as dramatic as the mRNA induction pattern. Overall, these results suggest that MdCAS may play a role in cyanide detoxification in ripening apple fruits.     

A role for ethylene in the metabolism of cyanide by higher plants

The action of ethylene on the capacity of plant tissues to metabolize cyanide to β-cyanoalanine was examined. Beta-cyanoalanine synthase (EC 4.4.1.9) catalyzes the reaction between cyanide and cysteine to form β-cyanoalanine and hydrogen sulfide. Levels of β-cyanoalanine synthase activity in tissues of 6 day old etiolated pea (Pisum sativum) seedlings were enhanced severalfold by 1 microliter per liter ethylene. The promotive effect of ethylene increased with increasing ethylene concentrations from 0.01 to 100 microliters per liter and with the period of exposure from 3 to 24 hours. Ethylene enhanced β-cyanoalanine synthase activity in all regions of the seedling (shoots and roots, internodal regions, cotyledons). The promotive effect was eliminated by norbornadiene, a competitive inhibitor of ethylene action. Levels of β-cyanoalanine synthase in seedlings of four other dicots (Phaseolus aureas, Glycine max, Lactuca sativa, Sinapis arvensis) and two monocots (Hordeum vulgares, Triticum aestivum) were also increased in response to ethylene. Our results suggest an important regulatory role for ethylene in the metabolism of cyanide by higher plants.     

Heterologous expression of mitochondria‐targeted microbial nitrilase enzymes increases cyanide tolerance in Arabidopsis

Anthropogenic activities have resulted in cyanide (CN) contamination of both soil and water in many areas of the globe. While plants possess a detoxification pathway that serves to degrade endogenously generated CN, this system is readily overwhelmed, limiting the use of plants in bioremediation. Genetic engineering of additional CN degradation pathways in plants is one potential strategy to increase their tolerance to CN. Here we show that heterologous expression of microbial nitrilase enzymes targeted to the mitochondria increases CN tolerance in Arabidopsis. Root length in seedlings expressing either a CN dihydratase from Bacillus pumilis or a CN hydratase from Neurospora crassa was increased by 45% relative in wild‐type plants in the presence of 50 μm KCN. We also demonstrate that in contrast to its strong inhibitory effects on seedling establishment, seed germination of the Col‐0 ecotype of Arabidopsis is unaffected by CN.