Proc. Natl. Acad. Sci. USA Vol. 92, pp. 714-718, January 1995 Microbiology

Occurrence of involved in biosynthesis of indole-3-acetic acid from indole-3-acetonitrile in plant-associated bacteria, Agrobacterium and Rhizobium ( hydratase/purification/auxin/amidase) MICHIHIKO KOBAYASHI*, TAKAHISA SUZUKI, TAKAYUKI FUJITA, MASATOSHI MASUDA, AND SAKAYU SHIMIZU Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606, Japan Communicated by Takayoshi Higuchi, Nihon University, Tokyo, Japan, October 12, 1994

ABSTRACT The occurrence of a hitherto unknown path- 1) (16). Yamada and coworkers (17) in our laboratory have way involving the action of two enzymes, a purified and characterized such a from Alcaligenes and an amidase for the biosynthesis ofindole-3-acetic acid was faecalis acting on indole-3-acetonitrile, and we have cloned discovered in phytopathogenic bacteria Agrobacterium tume- (18) the gene. In the other pathway, a nitrile hydratase faciens and in leguminous bacteria Rhizobium. The nitrile catalyzes the hydration of to amides (Eq. 2), which are hydratase acting on indole-3-acetonitrile was purified to then converted to acids and ammonia by amidase (Eq. 3) (15). homogeneity through only two steps from the cell-free extract ofA. tumefaciens. The molecular mass of the purified R-CN + 2H20 -- R-COOH + NH3 [1] estimated by HPLC was about 102 kDa, and the enzyme R-CN + H20 -> R-CONH2 consisted of four subunits identical in molecular mass. The [2] enzyme exhibited a broad absorption spectrum in the visible R-CONH2+ H20 -- R-COOH + NH3 [3] range with absorption maxima at 408 nm and 705 nm, and it contained and . The enzyme stoichiometrically The predicted amino acid sequence of an amidase (19, 20) that catalyzed the hydration of indole-3-acetonitrile into indole-3- presumably is linked with a nitrile hydratase is homologous to acetamide with a specific activity of 13.7 ,umol per min per mg those of indole-3-acetamide from Ag. tumefaciens and a Km of 7.9 ,uM. and Ps. savastanoi (15). A few studies on IAA formation from indole-3-acetonitrile in phytopathogenic microorganisms have Indole-3-acetic acid (IAA) is a well-known phytohormone and been reported (21, 22); these studies concerned the first three biosynthetic routes involving indole-3-pyruvate, pathway described-indoleacetonitrile nitrilase; indoleaceto- tryptamine, or indole-3-acetonitrile have been studied in nitrile hydratase was not considered. higher plants (1, 2). In the indole-3-acetonitrile route, the Ag. tumefaciens, the causative agent of crown gall disease, is nitrile generated from indole-3-acetaldoxime, naturally occur- a member of the family Rhizobiaceae (23) and is closely ring in plants (3), by indoleacetaldoxime (EC related to members of the genus Rhizobium. Rhizobium species 4.2.1.29) (4) or from indole-3-methylglucosinolate by myrosi- are beneficial plant symbionts that induce nitrogen-fixing nase (EC 3.2.3.1) (5), is converted into IAA by nitrilase (EC nodules on the roots of legumes, while Agrobacterium species 3.5.5.1) in Brassicaceae (cabbage group and radish), Gram- are plant pathogens. Agrobacterium and Rhizobium live in ineae (grasses), and Musaceae (banana family) (6, 7). While association with plants; their strains are tentatively named studies on auxin have been made for >100 years (8), none of plant-associated bacteria in this paper. The observations above the genes encoding IAA-forming enzymes in the indole-3- mentioned and the intermediacy of indole-3-acetonitrile in the pyruvate or the tryptamine route have been cloned yet, biosynthesis of IAA in plants prompted the study reported although cDNA cloning of fromArabidopsis thaliana here of plant-associated bacteria, Agrobacterium and Rhizo- (9-11) has been reported. bium. The nitrile hydratase, which hydrates indole-3- Microbial production of plant hormones such as IAA and acetonitrile and forms indole-3-acetamide, was also purified cytokinin seems to be essential for the virulence of bacteria in and characterized from Ag. tumefaciens. their host plants (12). Agrobacterium tumefaciens (Ag. tume- faciens) can infect wound sites on a wide range of dicotyle- donous plants and cause the formation of crown gall tumors by MATERIALS AND METHODS transfer of the transferred DNA (T-DNA) region in the Ti Materials. Indole-3-acetonitrile, indole-3-acetamide, and plasmid from the bacteria into plant cells. The T-DNA genes IAA were obtained from Aldrich. tms-1 and tms-2 encode tryptophan 2-monooxygenase (EC Bacterial Strains. Ag. tumefaciens and Rhizobium strains, 1.13.12.3) and indoleacetamide , respectively (13); which are listed in the relevant table or are described in the IAA is produced from tryptophan by the sequential action of text, were kindly provided from the Ministry of Agriculture, these two enzymes. A similar situation in the genes responsible Forestry and Fisheries (MAFF) collection (Tsukuba, Japan) for IAA synthesis was observed in Pseudomonas savastanoi and from the Institute of Applied Microbiology, The Univer- (Ps. savastanoi), which induced tissue proliferation in olive and sity of Tokyo (IAM) collection. The following Rhizobium oleander plants (14). strains were examined: 12 strains of Rhizobium loti (Rz. loti) In studying nitrile metabolism, we have found that the (MAFF 02-10055, 02-10056, 02-10059, 02-10062, 02-10067, microbial degradation ofnitriles can proceed by two enzymatic 02-10077, 02-10101, 02-10103, 02-10234, 02-10241, 03-03099, pathways (15). In one, a nitrilase catalyzes the direct conver- sion of nitriles into the corresponding acids plus ammonia (Eq. Abbreviations: IAA, indole-3-acetic acid; MAFF, Ministry of Agri- culture, Forestry and Fisheries. The publication costs of this article were defrayed in part by page charge *To whom reprint requests should be addressed at: Department of payment. This article must therefore be hereby marked "advertisement" in Agricultural Chemistry, Kyoto University, Kitashirakawa-oiwake- accordance with 18 U.S.C. §1734 solely to indicate this fact. cho, Sakyo-ku, Kyoto 606, Japan. 714 Downloaded by guest on September 27, 2021 Microbiology: Kobayashi et aL Proc. Natl. Acad. Sci. USA 92 (1995) 715 and 03-03120), 8 strains of Rhizobium leguminosarum (Rz. Osaka) at a flow rate of 1.0 ml/min with the following solvent leguminosarum) (MAFF 02-10041, 02-10049, 02-10051, 02- system: 5 mM KH2PO4-H3PO4, pH 2.9/acetonitrile, 3:1 (vol/ 10228, 02-10230, 02-10231, 03-03021, and 03-03119) and 3 vol). One unit of nitrile hydratase or amidase activity is strains of Rhizobium meliloti (Rz. meliloti) (MAFF 03-03046, defined as the amount of enzyme catalyzing the formation of 03-03097, and 03-03098). These 23 strains of Rhizobium are 1 ,umol of indole-3-acetamide from indole-3-acetonitrile or 1 known to symbiose with the following leguminous plant host: ,umol of IAA from indole-3-acetamide, respectively, per min Leucaena leucocephala, Mimosa pudica, Samanea saman, Al- under the above conditions. The protein was determined bizia procera, Acacia farnesiana, Gliricidia sepium, Cajanus according to Bradford (24). cajan, Lespedeza thunbergii, Samanea saman, Cajanus cajan, Lotus corniculatus var. japonicus, and Lupinus luteus with Rz. loti strains; Phaseolus vulgaris, Pisum sativum, Pisum sativum, RESULTS Trifolium pratense, Canavalia lineata, Vigna marina, Pisum Occurrence of Nitrile Hydratase Activity in Ag. tumefaciens sativum, and Trifolium repens with Rz. leguminosarum strains; Strains. As isovaleronitrile is a good inducer for nitrilase and Medicago sativa, Medicago sativa, andMedicago sativa, with formation in Rhodococcus rhodochrous (Rd. rhodochrous) Jl Rz. meliloti strains, respectively. (25), Rd. rhodochrous K22 (26), Alcaligenes faecalis JM3 (27), Culture Conditions. Each Agrobacterium and Rhizobium and Rhodococcus ATCC 39484 (28), the IAA-forming ni- strain was cultured at 28°C in a 500-ml shaking flask containing trilase activity ofvariousAg. tumefaciens strains grown in 0.1% 90 ml of the basal medium [10 g of glucose/3 g of Polypepton isovaleronitrile/1% glycerol/0.5% Polypepton/0.3% malt ex- (Daigo, Osaka)/1 g of yeast extract (Oriental Yeast, Tokyo)/ tract/0.3% yeast extract was initially examined using indole- 0.5 g of KH2PO4/0.5 g of K2HPO4/0.5 g of MgSO4-7H20/5 mg 3-acetonitrile as a . However, growth of these strains of CoCl2-6H2O/5 mg of FeSO4 7H2O per liter of distilled water was strongly inhibited by isovaleronitrile, and little or no (pH 7.2)] in the presence or in the absence of isovaleronitrile nitrilase activity was detected in the cell-free extracts. or crotonamide in each experiment as indicated. After 48-h We then examined whether IAA synthesis by the combined cultivation, each strain was centrifuged at 12,000 x g for 30 action of nitrile hydratase and amidase could occur in extracts min, washed in 10 mM potassium phosphate (pH 7.5), and of Ag. tumefaciens. Crotonamide (0.2%), a good inducer of suspended in 0.1 M potassium phosphate (pH 7.5). The cell nitrile hydratase in Rd. rhodochrous Ji (29), was added to the suspension was disrupted by sonication for 20 min (19 kHz; basal medium. Metals such as cobalt and iron were also added Insonator Model 201M, Kubota, Tokyo), and centrifuged at to the medium, because cobalt and iron enhance nitrile 12,000 x g for 30 min. The resulting supernatant solutions were hydratase activities in Rd. rhodochrous Jl and Pseudomonas used in the enzyme assay. chlororaphis (Ps. chlororaphis) B23, respectively (15), and their Purification of the Nitrile Hydratase. Ag. tumefaciens IAM purified nitrile hydratases contain cobalt (30) and iron (31), B-261 was grown for 50 h at 28°C in 10 liters of 10 g of L-maleic respectively. acid/0.5 g of K2HPO4/0.5 g of KH2PO4/0.5 g of As shown in Table 1, eight Ag. tumefaciens strains (MAFF MgSO4 7H2O/5 mg of CoCl2 6H2O/5 mg of FeSO4 7H2O/1 g 03-01279, 03-01541, 03-01542, 03-01543, 03-01544, 03-01548, of yeast extract/3 g of malt extract/2 g of e-caprolactam per and 03-01553, and IAM B-261) exhibited both nitrile hy- liter of distilled water (pH 7.2). The harvested cells were dratase and amidase activities, although different media were subjected to the following simple procedures at 0-4°C. required. Surprisingly, cell-free extracts of four strains of Throughout the purification, Hepes-KOH (pH 7.2) containing Agrobacterium (MAFF 03-01279, 03-01541, 03-01544, and 22 mM butyrate (hereafter designated Hepes buffer) was used. 03-01548) grown in the absence of crotonamide exhibited Centrifugation was carried out for 30 min at 12,000 x g. nitrile hydratase activity, whereas those from cells grown in Washed cells (wet mass, 30 g) from 10 liters of culture were suspended in 1.0 liter of 0.1 M Hepes buffer and then disrupted Table 1. Specific activities for nitrile hydratase and amidase in by sonication for 30 min. The cell debris was removed by Ag. tumefaciens centrifugation, and the resulting supernatant solution was used Specific activity, units x as the cell-free extract and dialyzed against 10 mM Hepes 10-3/mg of protein buffer. The dialyzed solution was applied to a DEAE-Sephacel (Pharmacia) column (3.6 X 32 cm) equilibrated with 10 mM Crotonamide Nitrile Hepes buffer. After the column was washed thoroughly with Strain addition hydratase Amidase 10 mM Hepes buffer followed by 10 mM Hepes buffer/0.1 M 03-01279 + 0 27.1 KCI and then by 10 mM Hepes buffer/0.2 M KCl, the enzyme 0.96 0 was eluted with 1.2 liters of 10 mM Hepes buffer/0.3 M KCl. 03-01541 + 0 25.9 Active fractions were pooled, and ammonium sulfate was 0.48 0.17 added to give 80% saturation. After centrifugation of the 03-01542 + 0.89 0 suspension, the precipitate was dissolved in 0.1 M Hepes 0.40 0.23 buffer, followed by dialysis against 10 mM Hepes buffer. The 03-01543 + 0.57 0 resulting supernatant after centrifugation was fractionated 0.87 0.09 with ammonium sulfate (45-50%), followed by dialysis against 03-01544 + 0 15.3 10 mM Hepes buffer, and then preserved in 10 mM Hepes 0.36 0.12 buffer/50% (vol/vol) glycerol at -20°C. 03-01548 + 0 5.78 Enzyme Assay. Nitrile hydratase and nitrilase activities were 0.83 0.29 measured in a reaction mixture (2 ml) containing 100 ,umol of 03-01553 + 0.32 0 potassium phosphate buffer (pH 7.5), 12 ,umol of indole-3- 0.07 0.20 acetonitrile, and an appropriate amount of cell-free extract. B-261 + 410 1.21 Amidase activity was measured in a reaction mixture (2 ml) 0.61 0 containing 100 ,umol of potassium phosphate buffer (pH 7.5), 12 Crotonamide (0.2%) was added to the basal medium. Each strain ,umol of indole-3-acetamide, and an appropriate amount of number, except for the last, stands for the strain accession number of cell-free extract. The amount of indole-3-acetamide or IAA the MAFF collection. The last number (B-261) stands for that of the formed in each reaction mixture was determined by HPLC collection of the Institute and Applied Microbiology (University of with a Shimadzu LC-6A system equipped with an M&S pack Tokyo, Japan). The plant host infected with each strain is Rosa sp. +, C18 column (reversed phase, 4.6 x 150 mm; M&S Instruments, Presence; -, absence. Downloaded by guest on September 27, 2021 716 Microbiology: Kobayashi et al. Proc. Natl. Acad Sci. USA 92 (1995) the presence of crotonamide did not. These unexpected find- medium did not give rise to nitrilase activity in the 23 ings suggest that crotonamide may depress nitrile hydratase Rhizobium strains described above. In general, it can be activity in these strains and that the enzyme may be a concluded that nitrile hydratase and amidase, rather than constitutive enzyme or may be induced by some amide-like nitrilase, are widely distributed in Rhizobium species except in compound in the basal medium. On the whole, there seems to the aforementioned two strains. be various types of nitrile hydratase in each strain of Ag. Purification and Characterization of the Ag. tumefaciens tumefaciens, although the existence of nitrilase cannot be Nitrile Hydratase. Purification of the IAA-synthesizing en- excluded. zyme was performed to clarify whether nitrile hydratase or Occurrence of Nitrile Hydratase Activity in Rhizobium nitrilase is responsible for the IAA synthesis inAg. tumefaciens Strains. The production of IAA from tryptophan via indole- and Rhizobium. Ag. tumefaciens IAM B-261, which exhibited 3-acetamide has been reported in Bradyrhizobium sp. (slow- the highest nitrile hydratase activity for indole-3-acetonitrile growing Rhizobium), but not in Rhizobium sp. (fast-growing of the strains tested, was selected for characterization of the Rhizobium) (32). However, the pathway of IAA synthesis is not enzyme. Initially, we could not purify the enzyme, because it fully understood in Rhizobium. Prompted by the occurrence of was not obtained in good yield and was very labile. Therefore, IAA-synthesizing nitrile hydratase and amidase in Ag. tume- we investigated the culture conditions for the enhancement of faciens described above, we next investigated the distribution nitrile hydratase activity in the strain and found that s-cap- of these two enzymes in Rz. loti, Rz. leguminosarum, and Rz. rolactam showed higher activity than crotonamide, similar to meliloti species, which fix nitrogen symbiotically within the the phenomenon observed in Rhodococcus erythropolis (Rd. root nodules of plants. In 12 collections of Rz. loti examined, erythropolis) (33). The nitrile hydratase formed in the presence nitrile hydratase and amidase activities were detected regard- of s-caprolactam corresponded to 12% of the total soluble less of the presence of crotonamide in the culture medium, protein in the Agrobacterium cell-free extracts, as judged by with the exception of strain MAFF 03-03120 (data not shown), quantitation of the SDS/PAGE track in comparison with the which did not exhibit nitrile hydratase activity in the absence purified enzyme as described below. The optimum culture of crotonamide. The specific activities for nitrile hydratase and medium for the enzyme purification gave abundant growth of amidase observed with Rz. loti MAFF 02-10055 (Table 2) were the strain (unpublished results). Furthermore, the enzyme was similar to those in Rz. loti (MAFF 02-10059, 02-10234, 03- stabilized by the addition of butyric acid, which also stabilized 03099, and 03-03120) and Rz. leguminosarum (MAFF 02- the nitrile hydratase from Ps. chlororaphis B23 (31). 10051). The specific activities for nitrile hydratase and amidase The nitrile was 8.73-fold in Rz. loti MAFF 02-10056 (Table 2) resembled those in Rz. loti hydratase easily purified (final (MAFF 02-10062, 02-10067, 02-10077, 02-10103, and 02- specific activity, 13.7 gmol per min per mg) with 28.4% 10241), Rz. leguminosarum (MAFF 02-10049 and 02-10228) recovery from Ag. tumefaciens IAM B-261, when cultured and Rz. meliloti (MAFF 03-03097 and 03-03098) (data not under the conditions described above. During the purification reported). Three strains of Rz. meliloti also showed nitrile procedure, nitrilase activity was not found in any fractions. hydratase and amidase activities. Two of eight strains of Rz. The purified enzyme showed only one band on SDS/PAGE leguminosarum examined (MAFF 02-10231 and 03-03021) gels (Fig. 1). The molecular mass of the band was estimated to exhibited amidase activity (1.86 x 10-3 to 4.20 x 10-3 be 27 kDa, based on its mobility relative to the mobility of units/mg), but no nitrile hydratase activity either in the reference proteins. The molecular mass of the enzyme was presence or absence of crotonamide in the basal medium. The determined to be '102 kDa by analytical gel-permeation presence of isovaleronitrile instead of crotonamide in the HPLC with TSK G-3000SW column (0.75 X 60 cm, Toyo- Soda, Tokyo), which gave a symmetrical protein peak (data not Table 2. Specific activities for nitrile hydratase and amidase shown). These findings indicated that the nitrile hydratase in Rhizobium consists of four subunits identical in molecular mass and thus resembles the nitrile hydratase from Ps. chlororaphis B23 (31), Specific activity, units although determination of the primary structure is required. x 10-3/mg of protein Qualitative metal analysis in the enzyme with an inductively Crotonamide Nitrile coupled radiofrequency plasma spectrophotometer (Shi- Strain addition hydratase Amidase madzu ICPV-1000, 27120 MHz) revealed that the purified Rz. loti hydratase fromAg. tumefaciens contained both cobalt and iron 02-10055 + 5.32 59.9 ions, in contrast to the cobalt-containing enzyme of Rd. - 6.89 70.9 02-10056 + 1.11 0.80 A - 1.41 0.84 ...... 02-10101 + 116 2.84 ...... :; - 168 5.35

Rz. leguminosarum ...... 02-10041 + 0.27 29.5 - 0 0 7. 02-10230 + 1.12 0 ...... - 0.18 30.3 ...... 03-03119 + 0 43.2 43 - 0.30 23.2 Rz. meliloti FIG. 1. SDS/PAGE of nitrile 03-03046 + 0 1.67 hydratase. Lanes: A, nitrile hy- li 30 dratase (20 jig); B, marker proteins - 0.22 0.96 [phosphorylase b (94 kDa), bovine Crotonamide (0.2%) was added to the basal medium. Each strain serum albumin (67 kDa), ovalbu- number under the species name stands for the strain accession number min (43 kDa), of the MAFF collection. The leguminous plant host symbiosed with ....: (30 kDa), and soybean trypsin in- each strain is described in the text. Specific activities for other . 20 hibitor (20 kDa)]. Electrophoresis Rhizobium strains not listed in this table are shown in the text. +, was from the cathode (top) to the Presence; -, absence. anode. Downloaded by guest on September 27, 2021 Microbiology: Kobayashi et aL Proc. Nati Acad. Sci. USA 92 (1995) 717

1.0 A B DISCUSSION Wright et al. (34) proposed that the nontryptophan route is the primary pathway for the generation of IAA in maize from detailed analysis using a maize tryptophan auxotroph. In experiments using tryptophan auxotrophs of the dicot Arabi- dopsis thaliana, Fink and coworkers (11, 35) also concluded that the major route of IAA biosynthesis does not involve tryptophan; indole-3-acetonitrile, which could arise from in-

0 dole or indole-3-glycerol phosphate, could be a candidate for CD the IAA biosynthetic intermediate. The existence of a biosyn- thesis pathway for IAA from tryptophan via indole-3- acetamide, which occurs inAg. tumefaciens and Ps. savastanoi, has recently been shown in trifoliata orange (Poncirus trifoliata Rafin.) (36). 0 Although auxin biosynthesis has been well studied, the question of whether two enzymes are involved in the conver- sion of indole-3-acetonitrile to IAA has been neglected. In plants other than Arabidopsis (35) and cabbage (37), the presence of indole-3-acetonitrile has not been fully investi- 300 400 500 600 700 800 900 gated, but the biosynthesis of IAA via indole-3-acetonitrile as Wavelength, nm an intermediate cannot be ruled out. Indole-3-acetonitrile and FIG. 2. Absorption spectra of native nitrile hydratase. Concentra- its derivatives were also detected in roots of the Chinese tions of protein were 1.0 mg/ml (trace A) and 6.7 mg/ml (trace B) in cabbage Brassica pekinensis, which enlarge to form "clubs" 10 mM Hepes-KOH (pH 7.2). The same buffer was used as a blank. through infection with a plasmodial fungus Plasmodiophora brassicae (21). Yamada et al. (22) have reported that in virulent rhodochrous Jl (30) and the iron-containing enzyme of Ps. fungal species such as Taphrina wiesneri, Taphrina deformans, chlororaphis B23 (31). The Agrobacterium enzyme did not and Taphrinapruni, which cause hyperplastic diseases in plants contain Be, B, Mg, Al, Si, P, S, Ca, Ti, V, Cr, Mn, Ni, Cu, Zn, such as cherry, peach, and plum, respectively, IAA is synthe- Se, Sr, Zr, Mo, Pd, Ag, Cd, Sn, Sb, Ba, Ta, W, Pt, Au, Hg, Pb, sized not only from tryptophan via indole-3-pyruvate and La, or Ce. The visible and UV absorption spectra of the Ag. indole-3-acetaldehyde as intermediates but also from indole- tumefaciens nitrile hydratase are given in Fig. 2. The UV 3-acetonitrile by the nitrilase (22). On the other hand, the spectrum showed a typical absorption maximum at 280 nm. In existence of nitrile hydratase in these phytopathogenic fungi the visible range, the enzyme exhibited absorption in a broad has never been reported. region with absorption maxima at 408 nm and 705 nm. Since In bacteria that catabolize nitriles by nitrile hydratase, this the Rd. rhodochrous Jl cobalt-containing enzyme exhibits an enzyme, if inducible, is generally induced by amides, not by absorption maximum at 410 nm and the Ps. chlororaphis B23 nitriles such as isovaleronitrile (15). Our findings suggest that iron-containing enzyme exhibits an absorption maximum at a route involving nitrile hydratase and amidase rather than 720 nm in visible region, the absorption maxima at 408 nm and nitrilase occurs commonly in plant-associated bacteria for 705 nm in theAg. tumefaciens enzyme may correspond to some IAA formation from indole-3-acetonitrile. Since nitrile hy- choromophore containing both cobalt and iron. dratase and amidase are closely related in sequential nitrile The stoichiometry of nitrile consumption and amide for- metabolism, and their genes are adjacent in bacteria (15), both mation during the reaction was examined with a reaction enzymes appear to be responsible for the conversion of

Nitrile hydratase Amidase

I-CH2-CN CH2-CONH2 CH2-COOH lo -0- + H20 I + NH3 HH%N.01 H+H20 H H lndole-3-acetonitrile lndole-3-acetamide IAA Scheme I

mixture (2 ml) containing 100 ,umol of potassium phosphate indole-3-acetonitrile to IAA inAg. tumefaciens and Rhizobium buffer (pH 7.5), 6 Atmol of indole-3-acetonitrile, and the (see the following scheme). enzyme in an air-tight tube. After a 20-min incubation, the This two-step pathway is responsible for IAA biosynthesis amounts of residual indole-3-acetonitrile and indole-3- from indole-3-acetonitrile in plant-associated bacteria and acetamide formed were 1.58 ,tmol and 4.36 ,umol, respectively. differs from the tryptophan 2-monooxygenase/indole-3- No IAA was observed. The results show that indole-3- acetamide hydrolase pathway. However, in two strains of Rz. acetamide was formed stoichiometrically with the consump- leguminosarum (MAFF 02-10231 and 03-03021) studied here, tion of indole-3-acetonitrile. When indole-3-acetamide was the amidase activity observed may be coupled with tryptophan added as a substrate, no IAA was detected. The Michaelis 2-monooxygenase, although nitrile hydratase may not have constant for indole-3-acetonitrile with the enzyme was 7.9 ,tM been expressed under our culture conditions. from the Lineweaver-Burk plot. Phenylacetonitrile was re- Both agrobacteria and rhizobia induce plant cells to prolif- markably active as a substrate for the enzyme; the relative erate. In many Rhizobium species, a large plasmid ranging activity was 337% when the synthesis of indole-3-acetamide from 90 to 300 MDa (38) encodes symbiotic functions such as was taken as 100%. The Km value for phenylacetonitrile was 10 nitrogen fixation and nodulation, while inAg. tumefaciens, the ,uM. Ti-plasmid encodes enzymes that direct the synthesis of the Downloaded by guest on September 27, 2021 718 Microbiology: Kobayashi et al. Proc. Natl. Acad ScL USA 92 (1995) plant growth regulators auxin and cytokinin. Although the 13. Klee, H., Montoya, A., Horodyski, F., Lichtenstein, C., Gar- mechanism of bacteroid formation in Rhizobium has not well finkel, D., Fuller, S., Flores, C., Peschon, J., Nester, E. & Gordon, been clarified, Thimann (39) postulated that Rhizobium- M. (1984) Proc. Natl. Acad. Sci. USA 81, 1728-1732. 14. Yamada, T., Palm, C. J., Brooks, B. & Kosuge, T. (1985) Proc. derived IAA might be the factor involved in the root nodu- Natl. Acad. Sci. USA 82, 6522-6526. lation process. Whether or not nitrile hydratase and amidase 15. Kobayashi, M., Nagasawa, T. & Yamada, H. (1992) Trends genes are encoded in the Ti-plasmid of Ag. tumefaciens or in Biotechnol. 10, 402-408. the megaplasmid of Rhizobium should be further investigated. 16. Kobayashi, M. & Shimizu, S. (1994) FEMS Microbiol. Lett. 120, A 3-cyanoalanine hydratase (EC 4.2.1.65) (40), which cat- 217-224. alyzes the hydration of 13-cyanoalanine to form asparagine, is 17. Nagasawa, T., Mauger, J. & Yamada, H. (1990) Eur. J. Biochem. also found in higher plants. This hydratase is responsible for 194, 765-772. 18. Kobayashi, M., Izui, H., Nagasawa, T. & Yamada, H. (1993) Proc. the hydration of 03-cyanoalanine produced by 13-cyanoalanine Natl. Acad. Sci. USA 90, 247-251. synthase (EC 4.4.1.9) (41) from and cyanide, a 19. Kobayashi, M., Komeda, H., Nagasawa, T., Nishiyama, M., coproduct of ethylene biosynthesis (42). This enzyme in lupin Horinouchi, S., Beppu, T., Yamada, H. & Shimizu, S. (1993) Eur. does not utilize indole-3-acetonitrile (40). All nitrile hydrata- J. Biochem. 217, 327-336. ses whose nucleotide sequences have been reported are sig- 20. Hashimoto, Y., Nishiyama, M., Ikehata, O., Horinouchi, S. & nificantly similar to one another and are not homologous to Beppu, T. (1991) Biochim. Biophys. Acta 1088, 225-233. other in the (43); they are also 21. Nomoto, M. & Tamura, S. (1970) Agric. Biol. Chem. 34, 1590- any proteins 1592. clustered into a superfamily (15). However, Cluness et al. (44) 22. Yamada, T., Tsukamoto, H., Shiraishi, T., Nomura, T. & Oku, H. have reported that (EC 4.2.1.66), which (1990) Ann. Phytopathol. Soc. Jpn. 56, 532-540. catalyzes the hydration of cyanide to formamide, showed 23. Jordan, D. C. (1984) in Bergey's Manual of Systematic Bacteriol- significant similarities to the Alcaligenes nitrilase (18). ogy, eds. Krieg, N. R. & Holt, J. G. (Williams & Wilkins, Balti- Since indole-3-acetonitrile and phenylacetonitrile, which more), Vol. 1, pp. 234-254. may be a commonly occurring natural auxin (45), exhibited 24. Bradford, M. (1976) Anal. Biochem. 72, 248-254. smaller Km values with the Ag. tumefaciens nitrile hydratase, 25. Nagasawa, T., Kobayashi, M. & Yamada, H. (1988) Arch. Mi- crobiol. 150, 89-94. these auxin precursors are likely to be physiological substrates 26. Kobayashi, M., Yanaka, N., Nagasawa, T. & Yamada, H. (1991) of the enzyme. Further studies on the enzyme at gene level FEMS Microbiol. Lett. 77, 121-124. should help to elucidate the regulation of IAA biosynthesis. 27. Mauger, J., Nagasawa, T. & Yamada, H. (1990) Arch. Microbiol. 155, 1-6. We dedicate this paper to Emeritus Professor Kenneth V. Thimann 28. Stevenson, D. E., Feng, R., Dumas, F., Groleau, D., Mihoc, A. & (University of California and Harvard University) who is a discoverer Storer, A. C. (1992) Biotechnol. Appl. Biochem. 15, 283-302. of a nitrile-degrading enzyme (nitrilase). We express our sincere 29. Nagasawa, T., Takeuchi, K. & Yamada, H. (1988) Biochem. thanks to Emeritus Professor Hideaki Yamada (Kyoto University) for Biophys. Res. Commun. 155, 1008-1016. his valuable discussion and encouragement. This work was supported 30. Nagasawa, T., Takeuchi, K. & Yamada, H. (1991) Eur. J. Bio- in part by a grant from The Sumitomo Foundation and from The Japan chem. 196, 581-589. Science Society and by a Grant-in-Aid for Scientific Research from the 31. Nagasawa, T., Nanba, H., Ryuno, K., Takeuchi, K. & Yamada, H. Ministry of Education, Science and Culture of Japan. (1987) Eur. J. Biochem. 162, 691-698. 32. Sekine, M., Ichikawa, T., Kuga, N., Kobayashi, M., Sakurai, A. & 1. Schneider, E. A. & Wightman, F. (1978) in Phytohormones and Syono, K. (1988) Plant Cell Physiol. 29, 867-874. Related Compounds: A Comprehensive Treatise, eds. Letham, 33. Duran, R., Nishiyama, M., Horinouchi, S. & Beppu, T. (1993) T. V. Amsterdam), Biosci. Biotechnol. Biochem. 57, 1323-1328. D. S., Goodwin, P. B. & Higgins, J. (Elsevier, 34. Wright, A. D., Sampson, M. B., Neuffer, M. G., Michalczuk, L., Vol. 1, pp. 29-105. Slovin, J. P. & Cohen, J. D. (1991) Science 254, 998-1000. 2. Sembdner, G., Gross, D., Liebisch, H. W. & Schneider, G. (1980) 35. Normanly, J., Cohen, J. D. & Fink, G. R. (1993) Proc. Natl. Acad. in Hormonal Regulation of Development I: Molecular Aspects of Sci. USA 90, 10355-10359. Plant Hormones, ed. MacMillan, J. (Springer, Berlin), Vol. 9, pp. 36. Kawaguchi, M., Fujioka, S., Sakurai, A., Yamaki, Y. T. & Syono, 281-289. K. (1993) Plant Cell Physiol. 34, 121-128. 3. Kindl, H. (1968) Hoppe Seyler's Z. Physiol. Chem. 349, 519-520. 37. Jones, E. R. H., Henbest, H. B., Smith, G. F. & Bentley, J. A. 4. Mahadevan, S. (1963) Arch. Biochem. Biophys. 100, 557-558. (1952) Nature (London) 169, 485-487. 5. Searle, L. M., Chamberlain, K., Rausch, T. & Butcher, D. N. 38. Christensen, A. H. & Schubert, K. R. (1983) J. Bacteriol. 156, (1982) J. Exp. Bot. 33, 935-942. 592-599. 6. Thimann, K. V. & Mahadevan, S. (1964)Arch. Biochem. Biophys. 39. Thimann, K. V. (1936) Proc. Natl. Acad. Sci. USA 22, 511-514. 105, 133-141. 40. Castric, P. A., Farnden, K. J. F. & Conn, E. E. (1972) Arch. 7. Mahadevan, S. & Thimann, K. V. (1964)Arch. Biochem. Biophys. Biochem. Biophys. 152, 62-69. 107, 62-68. 41. Akopyan, T. N., Braunstein, A. E. & Goryachenkova, E. V. 8. Darwin, C. & Darwin, F. (1892) The Power ofMovement in Plants (1975) Proc. Natl. Acad. Sci. USA 72, 1617-1621. (Appleton, New York), pp. 523-545. 42. Peiser, G., Wang, T.-T., Hoffman, N. E., Yang, S. F., Lui, H.-W. 9. Bartling, D., Seedorf, M., Mithofer, A. & Weiler, E. W. (1992) & Walsh, C. T. (1984) Proc. Natl. Acad. Sci. USA 81, 3059-3063. Eur. J. Biochem. 205, 417-424. 43. Ikehata, O., Nishiyama, M., Horinouchi, S. & Beppu, T. (1989) 10. Bartling, D., Seedorf, M., Schmidt, R. C. & Weiler, E. W. (1994) Eur. J. Biochem. 181, 563-570. Proc. Natl. Acad. Sci. USA 91, 6021-6025. 44. Cluness, M. J., Turner, P. D., Clements, E. C., Brown, D. T. & 11. Bartel, B. & Fink, G. R. (1994) Proc. Natl. Acad. Sci. USA 91, O'Reilly, C. (1993) J. Gen. Microbiol. 139, 1807-1815. 6649-6653. 45. Wightman, F. (1977) in Plant Growth Regulation, ed. Pilet, P. E. 12. Morris, R. 0. (1986) Annu. Rev. Plant Physiol. 37, 509-538. (Springer, Berlin), pp. 75-90. Downloaded by guest on September 27, 2021