Key points are not available for this paper at this time.
Nitrilases (nitrile aminohydrolases, EC 3.5.5.1) are enzymes that catalyze the hydrolysis of nitriles to the corresponding carbon acids. Among the four known nitrilases ofArabidopsis 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 andOryza sativa. We expressed A. thaliana NIT4 as hexahistidine tag fusion protein in Escherichia coli. The purified enzyme showed a strong substrate specificity for β-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 andLupinus angustifolius also converted Ala(CN) to Asp and Asnin vitro. NIT4 may play a role in cyanide detoxification during ethylene biosynthesis because extracts from senescent leaves ofA. thaliana showed higher Ala(CN) hydratase/nitrilase activities than extracts from nonsenescent tissue. Nitrilases (nitrile aminohydrolases, EC 3.5.5.1) are enzymes that catalyze the hydrolysis of nitriles to the corresponding carbon acids. Among the four known nitrilases ofArabidopsis 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 andOryza sativa. We expressed A. thaliana NIT4 as hexahistidine tag fusion protein in Escherichia coli. The purified enzyme showed a strong substrate specificity for β-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 andLupinus angustifolius also converted Ala(CN) to Asp and Asnin vitro. NIT4 may play a role in cyanide detoxification during ethylene biosynthesis because extracts from senescent leaves ofA. thaliana showed higher Ala(CN) hydratase/nitrilase activities than extracts from nonsenescent tissue. indole-3-acetonitrile β-cyano-l-alanine indole-3-acetic acid liquid chromatography coupled to electrospray-ionization mass spectrometry nitrile hydratase pentadecafluorooctanoic acid 3-phenylpropionitrile dithiothreitol Among the nitrilases of Arabidopsis thaliana, the first higher plant nitrilases that have been cloned (1Bartling D. Seedorf M. Mithöfer A. Weiler E.W. Eur. J. Biochem. 1992; 205: 417-4424Crossref PubMed Scopus (106) Google Scholar, 2Bartling D. Seedorf M. Schmidt R.C. Weiler E.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6021-6025Crossref PubMed Scopus (103) Google Scholar, 3Bartel B. Fink G.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6649-6653Crossref PubMed Scopus (168) Google Scholar), isoform 4 is clearly divergent. The members of the NIT1 group (NIT1, NIT2, and NIT3) are highly similar and share a minimum of 82% identical amino acids. In A. thaliana, they occur clustered on chromosome 3, and although the patterns of expression are distinctly different for each isoform (2Bartling D. Seedorf M. Schmidt R.C. Weiler E.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6021-6025Crossref PubMed Scopus (103) Google Scholar, 3Bartel B. Fink G.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6649-6653Crossref PubMed Scopus (168) Google Scholar, 4Vorwerk, S., Biernacki, S., Hillebrand, H., Janzik, I., Müller, A., Weiler, E. W., and Piotrowski, M. (2000) Planta, in pressGoogle Scholar, 5Müller A. Hillebrand H. Weiler E.W. Planta. 1998; 206: 362-369Crossref PubMed Scopus (69) Google Scholar), their enzymatic characteristics are very similar, albeit not identical (4Vorwerk, S., Biernacki, S., Hillebrand, H., Janzik, I., Müller, A., Weiler, E. W., and Piotrowski, M. (2000) Planta, in pressGoogle Scholar). As shown previously (4Vorwerk, S., Biernacki, S., Hillebrand, H., Janzik, I., Müller, A., Weiler, E. W., and Piotrowski, M. (2000) Planta, in pressGoogle Scholar), a major role for these nitrilases appears to be in the metabolism of nitriles released by breakdown of glucosinolates. A further common feature is that all three isoenzymes can convert indole-3-acetonitrile (IAN)1 to indole-3-acetic acid (IAA), the plant growth hormone (1Bartling D. Seedorf M. Mithöfer A. Weiler E.W. Eur. J. Biochem. 1992; 205: 417-4424Crossref PubMed Scopus (106) Google Scholar, 2Bartling D. Seedorf M. Schmidt R.C. Weiler E.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6021-6025Crossref PubMed Scopus (103) Google Scholar, 3Bartel B. Fink G.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6649-6653Crossref PubMed Scopus (168) Google Scholar). A nitrilase (which isoform, is at present unknown) may be part of the IAA synthase enzyme complex of A. thaliana, a soluble 160-kDa complex catalyzing the conversion of l-tryptophan to IAA in vitro(6Müller A. Weiler E.W. Biol. Chem. Hoppe-Seyler. 2000; 381: 679-686PubMed Google Scholar). Nitrilase 4 is peculiar in that it occurs as a single gene at a different chromosomal location (chromosome 5) (3Bartel B. Fink G.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6649-6653Crossref PubMed Scopus (168) Google Scholar), does not accept IAN as a substrate (Ref. 7Schmidt R.C. Müller A. Hain R. Bartling D. Weiler E.W. Plant J. 1996; 9: 683-691Crossref PubMed Scopus (50) Google Scholar and this paper), and occurs in plants of different taxonomic position, such as tobacco (8Tsunoda H. Yamaguchi K. Plant Physiol. 1995; 109: 339Google Scholar) and rice (GenBankTM accession number AB027054). Nitrilases belonging to the NIT4 family thus may have a different and more general function and will likely not be associated with auxin production. A nitrile of widespread occurrence in higher plants is β-cyano-l-alanine (Ala(CN)), which is enzymatically produced by cyanoalanine synthase from cyanide and cysteine as substrates (9Blumenthal S.G. Hendrickson H.R. Conn E.E. J. Biol. Chem. 1968; 243: 5302-5307Abstract Full Text PDF PubMed Google Scholar). Depending on the plant species observed, Ala(CN) is subsequently converted to asparagine or to the dipeptide γ-Glu-(Ala(CN)). An enzyme converting Ala(CN) to Asp (cyanoalanine hydratase, EC 4.2.1.65) was characterized biochemically from lupine (Lupinus angustifolius) (10Castric P.A. Farnden K.J.F. Conn E.E. Arch. Biochem. Biophys. 1972; 152: 62-69Crossref PubMed Scopus (85) Google Scholar, 11Galoyan S.M. Tolosa E.A. Goryachenkova E.V. Biokhimiya. 1982; 47: 1949-1953PubMed Google Scholar), but the gene encoding this enzyme has not yet been cloned. In a previous study we reported about the enzymatic characterization of the A. thaliana nitrilase subfamily encoded by theNIT2/NIT1/NIT3 gene cluster (4Vorwerk, S., Biernacki, S., Hillebrand, H., Janzik, I., Müller, A., Weiler, E. W., and Piotrowski, M. (2000) Planta, in pressGoogle Scholar). During this work, we notified that Arabidopsis NIT4 has a quite different substrate specificity compared with the NIT1/NIT2/NIT3 group. Here, we report about the elucidation of the enzymatic function of the NIT4 enzyme family. A. thaliana ecotype C24 andNicotiana tabacum W38 were grown in a greenhouse in standard soil at 20 °C, 70% relative humidity, and 210 μmol photons m−2 s−1 for a 16-h photoperiod. Seeds of L. angustifolius were sown on Vermiculite and grown in a growth chamber under the following climatic conditions: 16-h photoperiod, 120 μmol photons m−2 s−1, 24 °C during photoperiod, 20 °C during night, 70% relative humidity. The following general procedures have been described elsewhere (4Vorwerk, S., Biernacki, S., Hillebrand, H., Janzik, I., Müller, A., Weiler, E. W., and Piotrowski, M. (2000) Planta, in pressGoogle Scholar): sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein determination. All basic molecular techniques were adapted from Ausubel et al. (12Ausubel F.A. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley 205: 417-4424Crossref PubMed Scopus (106) Google Scholar, 2Bartling D. Seedorf M. Schmidt R.C. Weiler E.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6021-6025Crossref PubMed Scopus (103) Google Scholar, 3Bartel B. Fink G.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6649-6653Crossref PubMed Scopus (168) Google Scholar), S. B. K. Fritsch M. J. Physiol. Scopus Google Scholar, M. R. S. T. J. J. Plant Physiol. 1995; Scopus Google Scholar), (GenBankTM accession number N. tabacum (8Tsunoda H. Yamaguchi K. Plant Physiol. 1995; 109: 339Google Scholar), and (GenBankTM accession number AB027054). the these nitrilases they can be into two The first to as NIT1 to be for nitrilases from species of the Brassicaceae are characterized by their and nitriles are the substrates for a function of these enzymes in metabolism has been (4Vorwerk, S., Biernacki, S., Hillebrand, H., Janzik, I., Müller, A., Weiler, E. W., and Piotrowski, M. (2000) Planta, in pressGoogle Scholar). Arabidopsis NIT4 to the further to as NIT4 group to in to the of the all known nitrilases of plants of the NIT4 also be by using the J. of of not The NIT4 homologs therefore have to be as which that the present share a common therefore likely that members of the NIT4 group may also have a using the same as described previously for NIT2, and (4Vorwerk, S., Biernacki, S., Hillebrand, H., Janzik, I., Müller, A., Weiler, E. W., and Piotrowski, M. (2000) Planta, in pressGoogle Scholar), NIT4 be expressed in E. from as a fusion protein with a hexahistidine tag to the amino acid of the enzyme a dipeptide as in the of the three nitrilases from A. thaliana, most of the expressed protein was found in the a of the nitrilase be purified from the soluble protein by chromatography on The was purified at to as by We more than substrates using the purified enzyme because in we and different the purified enzyme or a of E. was The that NIT4 is highly for β-cyano-l-alanine The of NIT4 3-phenylpropionitrile or which are the substrates for (4Vorwerk, S., Biernacki, S., Hillebrand, H., Janzik, I., Müller, A., Weiler, E. W., and Piotrowski, M. (2000) Planta, in pressGoogle Scholar), was very and indole-3-acetonitrile a of the plant hormone indole-3-acetic acid (IAA), was not converted by is in with in of Schmidt et al. R.C. Müller A. Hain R. Bartling D. Weiler E.W. Plant J. 1996; 9: 683-691Crossref PubMed Scopus (50) Google Scholar) and al. M. J. H. Yamaguchi K. PubMed Scopus Google Scholar) were to an auxin with IAN in tobacco (which at two NIT4 as as in tobacco M. J. H. Yamaguchi K. PubMed Scopus Google Scholar). tobacco plants R.C. Müller A. Hain R. Bartling D. Weiler E.W. Plant J. 1996; 9: 683-691Crossref PubMed Scopus (50) Google Scholar), NIT1 M. J. H. Yamaguchi K. PubMed Scopus Google Scholar), or M. J. H. Yamaguchi K. PubMed Scopus Google Scholar) ofA. thaliana converted this substrate to IAA and strong of auxin specificity of NIT4 of A. was purified as tag fusion protein from E. coli. from to were with the substrates in a of at °C for 20 to 4 Nitrilase was as described under A of to not and in a NIT4 was purified as tag fusion protein from E. coli. from to were with the substrates in a of at °C for 20 to 4 Nitrilase was as described under A of to not and the enzymatic was on the of released it was to that this the nitrile and not the amino we the reaction products by chromatography not and liquid chromatography coupled to mass spectrometry The of aspartic acid be shown by with Asp during not as as by and by Asn also be and in about higher than NIT4 not only has a nitrilase Ala(CN) to but also a nitrile hydratase Ala(CN) to Nitrilases nitriles by the of two of the substrate to the cysteine S. Arch. Biochem. Biophys. PubMed Scopus Google Scholar). that Asn may occur as a intermediate of the nitrilase reaction than this be by the that Asn is only converted to Asp by NIT4 of NIT4 for the of Asp that is at a of was not in and therefore most likely from of the with E. In showed that Asp and Asn are at the same with a with no of Asn Asn be an intermediate of the we it to Asp with the of Asp these that NIT4 Ala(CN) to Asp or Asn is no substrate of the and Asn is no intermediate of the nitrilase reaction of this of Ala(CN) hydrolysis by NIT4 ofA. Arabidopsis NIT4 was with Ala(CN) at °C for of the amino acid of were and to as described under The shown are of three The known reaction of nitrilases and are quite different 1998; PubMed Scopus (50) Google Scholar). the first their substrate to a cysteine the a for their activity. The of Asn from Ala(CN) by NIT4 may be the of a of Asn during the nitrilase reaction or it may occur at a with activity. The substrate of the two reactions 4 and that of Ala(CN) for both reactions is very similar not the is higher for the Asn that the catalytic center for both reactions be the same. reactions showed the same and and they were both by at The of a cysteine in both reactions was further by their at higher of at this the cysteine of NIT4 for the nitrilase reaction was mutated to The protein showed no nitrilase and a of the the of for both activities but also that the does not on this of nitrilase and of NIT4 from A. was purified as tag fusion protein from E. coli. to of purified protein were with Ala(CN) of a of were for 20 at of nitrilase and was done as described under in a NIT4 was purified as tag fusion protein from E. coli. to of purified protein were with Ala(CN) of a of were for 20 at of nitrilase and was done as described under In al. E. R. 1995; PubMed Scopus Google Scholar) showed that a single amino acid to in the cysteine in a of the mutated The ofA. thaliana NIT4 therefore be the of an be because the reported and cDNA of NIT4 from A. thaliana the same was with three different NIT4 homologs A may be the of the of the enzyme introduced with the tag that was in all NIT4 study the of the we expressed NIT4 using in E. and the protein by and gel NIT4 was found in the of a indicating that the molecular mass is than The showed no under the but both nitrilase and activities be a of like the protein not The is therefore an of the NIT4 As to NIT4 are also known from tobacco (8Tsunoda H. Yamaguchi K. Plant Physiol. 1995; 109: 339Google Scholar) and The cDNAs of the tobacco nitrilases TNIT4A and TNIT4B were kindly provided to by Dr. Kazuo Yamaguchi (Institute for Gene Research, Kanazawa University, Kanazawa, using the same cloning as for A. thaliana NIT4, both cDNAs be expressed in E. and the were purified using chromatography not enzymes Ala(CN) to Asp and Asn a of compared with thus more Interestingly, was a substrate for the tobacco enzymes than for Arabidopsis NIT4, the of Ala(CN) to was higher with the tobacco enzymes As A. thaliana has a of and nitriles are substrates for the Arabidopsis isoenzymes NIT2, and is therefore that Arabidopsis NIT4 during to hydrolysis of of Arabidopsis NIT4 with tobacco TNIT4A and nitrilase and TNIT4B were purified as tag fusion coli. of were with the substrates in a of at °C for 20 to was or by as described under are of three in a NIT4, and TNIT4B were purified as tag fusion coli. of were with the substrates in a of at °C for 20 to was or by as described under are of three In et al. (10Castric P.A. Farnden K.J.F. Conn E.E. Arch. Biochem. Biophys. 1972; 152: 62-69Crossref PubMed Scopus (85) Google Scholar) reported the characterization of a hydratase from lupine which Asn from is not from this the also for Asp from Ala(CN) by their We extracts from leaves of A. thaliana and tobacco and from of was from the extracts by All extracts showed Ala(CN) as as nitrilase the no be indicating that the Asp is by a nitrilase The tobacco produced Asp and Asn from Ala(CN) in similar the from lupine showed an higher activity. was to the described of a (10Castric P.A. Farnden K.J.F. Conn E.E. Arch. Biochem. Biophys. 1972; 152: 62-69Crossref PubMed Scopus (85) Google Scholar). We also Ala(CN) hydratase/nitrilase activities from to in extracts and from to not In a the of the NIT4 gene ofA. thaliana during was described J. Plant Biol. PubMed Scopus Google Scholar). We therefore compared Ala(CN) hydrolysis in extracts from leaves of plants to and from leaves of plants of enzymatic activities were higher in extracts from senescent Interestingly, the of the two activities from in nonsenescent leaves to in senescent The occurrence of nitrilases in higher plants is known S. PubMed Scopus Google Scholar), but the was to their to convert indole-3-acetonitrile to the plant hormone indole-3-acetic acid (1Bartling D. Seedorf M. Mithöfer A. Weiler E.W. Eur. J. Biochem. 1992; 205: 417-4424Crossref PubMed Scopus (106) Google Scholar, 3Bartel B. Fink G.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6649-6653Crossref PubMed Scopus (168) Google Scholar, S. PubMed Scopus Google Scholar). or cDNA of nitrilases are known from A. thaliana, two species and S. B. K. Fritsch M. J. Physiol. Scopus Google Scholar, M. R. S. T. J. J. Plant Physiol. 1995; Scopus Google Scholar), tobacco M. J. H. Yamaguchi K. PubMed Scopus Google Scholar), and rice accession number AB027054). In expressed tag which The nitrilases of A. thaliana were characterized in most A. thaliana four different nitrilase that are expressed (2Bartling D. Seedorf M. Schmidt R.C. Weiler E.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6021-6025Crossref PubMed Scopus (103) Google Scholar, 3Bartel B. Fink G.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6649-6653Crossref PubMed Scopus (168) Google Scholar, 4Vorwerk, S., Biernacki, S., Hillebrand, H., Janzik, I., Müller, A., Weiler, E. W., and Piotrowski, M. (2000) Planta, in pressGoogle Scholar). nitrilases to Arabidopsis NIT2, and are only known from NIT4 homologs are also found in and that the NIT4 enzymes are not and we therefore to catalyze the same Arabidopsis NIT4 has a substrate specificity for β-cyano-l-alanine which is also converted by the NIT4 homologs of tobacco but is no substrate for Arabidopsis (4Vorwerk, S., Biernacki, S., Hillebrand, H., Janzik, I., Müller, A., Weiler, E. W., and Piotrowski, M. (2000) Planta, in pressGoogle Scholar). In to the report by and Fink (3Bartel B. Fink G.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6649-6653Crossref PubMed Scopus (168) Google Scholar), we no hydrolysis of IAN to IAA by Arabidopsis we a of purified enzyme of the under the is about which may be not to very IAN hydrolysis. The of the with Ala(CN) as The product of hydrolysis of Ala(CN) be aspartic acid we found that NIT4 not only produced Asp but to an higher Asn The of Asn from Ala(CN) be an and Asn may be further to Asp by an activity. NIT4 an and is Asn therefore a intermediate of NIT4-catalyzed Asp from we a in of NIT4 in all from of these with E. this is that it for the of the of Asp showed a in Asp a of Asn which both be Asn were an intermediate of Asp Asn is not an intermediate but one of two products of NIT4-catalyzed Ala(CN) hydrolysis. NIT4 therefore a of from the known of this will be of for the of enzymes that be of for and A for this may be a of the substrate the of the first of the of is not the may be the is the substrate will be further to the Interestingly, an enzyme the same enzymatic characteristics as NIT4 was purified from in H. T. K. Biol. Chem. 47: Scholar), but no of this protein was reported The reported of this enzyme are very similar to NIT4, but for Ala(CN) is about of higher compared with Arabidopsis Ala(CN) is a product of cyanide detoxification of plants. is produced from cyanide and cysteine by cyanoalanine synthase (9Blumenthal S.G. Hendrickson H.R. Conn E.E. J. Biol. Chem. 1968; 243: 5302-5307Abstract Full Text PDF PubMed Google Scholar). indicate that cyanoalanine synthase is an enzyme or identical to cysteine synthase T. T. H. Plant Physiol. 2000; PubMed Scopus Google Scholar, A. Schmidt A. M. K. K. Plant Physiol. 2000; PubMed Scopus Google Scholar). In most species Ala(CN) is converted to although in species it is converted to the dipeptide The catalyzing the of Asn from Ala(CN) (cyanoalanine hydratase Ala(CN) were biochemically from lupine in the of E. Conn in the (10Castric P.A. Farnden K.J.F. Conn E.E. Arch. Biochem. Biophys. 1972; 152: 62-69Crossref PubMed Scopus (85) Google Scholar) and by et al. S.M. Tolosa E.A. Goryachenkova E.V. Biokhimiya. 1982; 47: 1949-1953PubMed Google Scholar), but encoding such enzymes have not been and of corresponding enzymes from plant species are cyanoalanine hydratase and NIT4 the same In using be in Asn but Asp was not (10Castric P.A. Farnden K.J.F. Conn E.E. Arch. Biochem. Biophys. 1972; 152: 62-69Crossref PubMed Scopus (85) Google Scholar, Plant Physiol. PubMed Scopus Google Scholar) a NIT4-catalyzed reaction in which similar of Asp and Asn be We therefore the of plant extracts from A. thaliana, L. B. and lupine to Ala(CN) and Ala(CN) as as Ala(CN) nitrilase in all In extracts of lupine the Ala(CN) was nitrilase was clearly is therefore likely that the Ala(CN) from lupine is a NIT4 is under in in vitro in to the in from the A be that the of Asp is higher than that of Asn in as in Plant Physiol. PubMed Scopus Google Scholar). In this Asp not of cyanide in higher plants is the biosynthesis of the plant hormone ethylene from acid Biochem. Sci. Full Text PDF Scopus Google Scholar). During this reaction acid is produced which to carbon and Interestingly, the Arabidopsis NIT4 was found to be during as shown by E. R. 1995; PubMed Scopus Google Scholar). We higher NIT4 in extracts from senescent leaves of A. thaliana compared with extracts from nonsenescent and the of to nitrilase may indicate that different enzymes are in Ala(CN) metabolism at different but it may also indicate that and nitrilase of NIT4 be by NIT4 or NIT4 expression is to ethylene biosynthesis or cyanide during will be in The in this clearly that NIT4 enzymes thaliana and N. tabacum are Ala(CN) NIT4 are known from different species of quite different position, and it is likely that NIT4 homologs may be present in all higher plants. NIT4 is to part in cyanide in with Ala(CN) We to the gene NIT4 for Ala(CN) Nitrilases from plants belonging to the NIT4 family therefore also be NIT4 of the number of as previously done S. B. K. Fritsch M. J. Physiol. Scopus Google Scholar), tobacco (8Tsunoda H. Yamaguchi K. Plant Physiol. 1995; 109: 339Google Scholar), and We are to Dr. Kazuo Yamaguchi for kindly the TNIT4A and TNIT4B
Piotrowski et al. (Mon,) studied this question.