Division of Applied Microbiology, National Food Research Institute, Kannondai 2-1-12, Tsukuba Ibaraki 305-8642, Japan
Correspondence
Yoshifumi Itoh
yosifumi{at}nfri.affrc.go.jp
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ABSTRACT |
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INTRODUCTION |
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P. aeruginosa PAO1 expresses arginine-inducible ADC, AguA and AguB, which constitute a potential route (the catabolic ADC pathway) for arginine catabolism (Cunin et al., 1986; et al., 1984
; Haas Mercenier et al., 1980
). AguA and AguB are essential for the utilization of agmatine and N-carbamoylputrescine but not arginine (Haas et al., 1984
): the arginine succinyltransferase pathway is the major arginine catabolic pathway in this strain (Jann et al., 1986
) and therefore the role of arginine-inducible ADC in arginine catabolism remains obscure. The aguABR genes have been cloned and characterized (Nakada et al., 2001
). AguR repressor negatively regulates the aguBA operon, and agmatine as well as N-carbamoylputrescine antagonize the repressor function to activate expression of the operon. Since AguAB can be formed at low levels even in the absence of the inducer, they might contribute to biosynthetic conversion of agmatine to putrescine.
To identify the putrescine biosynthetic genes of P. aeruginosa PAO1 and examine the biosynthetic role of AguAB, we constructed knockout mutants of the relevant gene(s). Enzyme assays and growth tests with the mutants confirmed that the predicted speA and speC genes encode biosynthetic ADC and ODC, respectively, and demonstrated that AguAB but not SpeB1 is involved in the biosynthetic ADC pathway like the plant counterparts. We also describe some properties of the P. aeruginosa AguA and AguB enzymes.
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METHODS |
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To maximize the synthesis of AguA and AguB, we cloned the 3·8 kb BglIIKpnI fragment containing the aguAB operon from plasmid pYJ101 (Nakada et al., 2001) into the high-copy vector pNIC6012 (about 15 copies per chromosome) (Nishijyo et al., 2001
) to construct plasmid pYI1005, which was then conjugated into strain PAO4495 (augR : :
Sp/Sm) (Nakada et al., 2001
).
Enzyme assays.
P. aeruginosa strains were cultured in MMP medium containing indicated carbon and nitrogen sources at 20 mM. Cell extracts were prepared by passing cells through a French press (SLM-AMINCO), and AguA and AguB activities measured, as previously described (Nakada et al., 2001). Activities of arginine and ornithine decarboxylases were assayed using L-[U-14C]arginine (11·5 GBq mmol-1; Amersham Biosciences) and L-[1-14C]ornithine (1·92 GBq mmol-1; Amersham Biosciences), respectively, according to Morris & Boeker (1983)
. After incubation at 37 °C for 30 min, the reaction was terminated by trichloroacetic acid and radioactivity of 14CO2 trapped in a filter paper impregnated with 2-aminoethanol and 2-methoxyethanol was measured in a TRI-CARB 3100TR liquid scintillation counter (Packard Bioscience). Protein concentrations were determined using a Protein Assay kit (Bio-Rad) with bovine serum albumin as the standard. One unit of enzyme activity was defined as the amount of the enzyme required to generate 1 µmol product min-1.
Purification of AguA and AguB.
Cells of strain PAO4495 (aguR : : Sp/Sm) harbouring pYI1005 (aguAB+) were grown exponentially (OD660 0·5) in 5 l MMP containing 20 mM glutamate, harvested by centrifugation and suspended in 50 mM potassium phosphate buffer, pH 7·5, containing 1 mM DTT. Cell extracts were prepared by passing cells through a French press, followed by centrifugation at 100 000 g for 1 h. To purify AguA, the enzyme was fractionated from cell extracts using 5075 % saturated ammonium sulphate and then dialysed against the same buffer. The dialysate was eluted from a HiPrep 16/10 DEAE column (Amersham Biosciences) using a linear gradient of KCl (0 to 0·5 M) in 50 mM potassium phosphate buffer, pH 7·5, containing 1 mM DTT, at a flow rate of 2 ml min-1. The eluate was dialysed as described above and then applied onto a column containing agmatine-Sepharose 4B (1 ml bed volume). The affinity-resin was prepared by coupling ECH-Sepharose 4B (Amersham Biosciences) with agmatine in the presence of 0·1 M N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride at room temperature overnight. Fractions were eluted using a linear KCl gradient (0 to 0·5 M) in the potassium phosphate buffer at a flow rate of 0·4 ml min-1. The active fractions were pooled, concentrated using Centriprep 10 (Millipore), and then AguA was finally purified by gel-filtration through a Superose 12 column with 50 mM potassium phosphate buffer, pH 7·5, containing 0·1 M KCl and 1 mM DTT (0·3 ml min-1).
We precipitated AguB from extracts of strain PAO4495/pYI1005 prepared as described above using ammonium sulphate (4070 % saturation). After dialysis the enzyme sample was purified by chromatography using HiPrep 16/10 DEAE as described above. The protein sample was dialysed against 50 mM potassium phosphate buffer containing 0·75 mM KCl and 1 mM DTT. The dialysate was then eluted through a column containing Butyl Toyopearl 650S (Tosoh, 8x75 mm) using a reverse KCl gradient (0·75 to 0·25 M) in the potassium phosphate buffer containing 1 mM DTT, at a flow rate of 0·3 ml min-1. Active fractions were pooled, concentrated and purified by gel filtration using Super se 12 as described above. We determined the purity of the enzyme and the molecular mass of enzyme subunits by SDS-PAGE (Nakada & Itoh, 2002).
Cross-linkage of AguA and AguB.
We incubated AguA (10 µg) in 50 µl 50 mM potassium phosphate buffer, pH 7·6, containing 87·5 µg dithio-bis(propionic acid N-hydroxysuccinimide) ester at room temperature for 15 min (Löster et al., 1995). AguB (10 µg) was cross-linked in a mixture (100 µl) containing 50 mM potassium phosphate, pH 7·6, 100 mM NaCl, 1 mM DTT and 0.01 % glutaraldehyde according to Kostyukova et al. (2000)
. Cross-linked proteins were analysed by SDS-PAGE.
Synthesis of N-carbamoyl compounds.
We synthesized and purified N-carbamoyl compounds using potassium cyanate and the following diamines as previously described (Nakada et al., 2001): N-carbamoylputrescine and N,N'-dicarbamoylputrescine were synthesized from putrescine; N-carbamoyldiaminopropane and N-carbamoylcadaverine were derived from 1,3-diaminopropane and cadaverine, respectively. The molecular mass of the synthetic compounds was confirmed by mass spectrometry (Reflex II 70e: Brucker Daltonics) and purity (
95 %) was estimated by HPLC using a 5-µm C18 3000-Å column (Waters) as previously described (Nakada et al., 2001
). Other substrates were obtained from Sigma.
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RESULTS |
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Identification of AguA and AguB as enzymes in the biosynthetic ADC pathway
To investigate whether speB1 is involved in putrescine biosynthesis, we inactivated this gene by insertion of a FRT-Gm cassette in strain PAO4540 (speC : : FRT). The resultant strain PAO4544 (speC : : FRT speB1 : : FRT-Gm) showed little apparent defect in growth (Fig. 2b), ruling out the participation of speB1 in putrescine biosynthesis. In contrast, when the aguAB genes were inactivated by insertion of the FRT-Gm cassette in the speC background, the resultant mutant PAO4541 (speC : : FRT aguAB : : FRT-Gm) failed to thrive in MMP (Fig. 2b
). As with the speA speC mutant, growth of this mutant was restored to near wild-type levels when 1 mM putrescine was added to the medium (Fig. 2b
). Thus, AguA and AguB appeared to mainly perform the biosynthetic conversion of agmatine to putrescine in P. aeruginosa PAO1.
Purification and subunit structure of AguA and AguB
The aguR gene negatively regulates expression of the aguBA operon (Nakada et al., 2001), and inactivation of the repressor gene results in the formation of AguA and AguB to induced levels (Table 3
). To maximize formation of the enzymes, we cloned the 3·8 kb aguBA region into the multicopy plasmid pNIC6012 (about 15 copies per chromosome) (Nishijyo et al., 2001
) and introduced the resultant plasmid pYI1005 into strain PAO4495 (aguR : :
Sp/Sm), which lacks the AguR repressor. When cultured in MMP containing 20 mM glutamate (non-inducible conditions), this recombinant strain generated about fivefold more AguA and AguB than the levels induced in the wild-type PAO1 strain (Table 3
). Using the procedures described in Methods, AguA and AguB were purified 21- and 13-fold with yields of 7 % and 4 %, respectively. SDS-PAGE indicated that both enzymes had been purified to near homogeneity (Fig. 3
, lanes 1 and 3). The electrophoretic mobility of the purified enzymes compared with the marker proteins on SDS-polyacrylamide gels determined that the molecular masses of the AguA and AguB monomers were 43 and 33 kDa, respectively (Fig. 3
). These are in good agreement with their calculated values (41 190 Da for AguA and 32 672 Da for AguB) (Nakada et al., 2001
). The molecular masses of native AguA (89 kDa) and AguB (230 kDa) determined by gel filtration (not shown) suggested that AguA is a homodimer and AguB is a homohexamer. Cross-linkage experiments confirmed the subunit structures of the proteins. Cross-linked AguA generated an additional band of 85 kDa (dimer) (Fig. 3
, lane 2). On the other hand, cross-linkage of AguB yielded five new protein bands, of 66 kDa (dimer), 100 kDa (trimer), 130 kDa (tetramer), 165 kDa (pentamer) and 200 kDa (hexamer) (Fig. 3
, lane 4).
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DISCUSSION |
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AguA and AguB have been purified either partially or to homogeneity from some plants. The AguA purified homogeneously from maize shoots (Yanagisawa & Suzuki, 1981) is very similar to PAO1 AguA: it is a homodimer of 44 kDa subunits with a Km value of 0·2 mM and sensitivity to p-chloromercuribenzoate. However, others have reported different structures and properties for partially purified rice AguA (a homodimer of 183 kDa, Km value of 15 mM) (Chaudhuri & Ghosh; 1985
) and soybean AguA (a monomer of 70 kDa, Km value of 2·5 mM) (Park & Cho, 1991
). Partially purified maize AguB is a 125 kDa protein with an unknown subunit structure that is sensitive to p-hydroxymercuribenzoate (Yanagisawa & Suzuki, 1982
). No sequence data are available for the plant enzymes. As described below, some plants appear to have enzymes that are homologous to bacterial AguA and AguB.
Hydrolases that can cleave non-peptide CN bonds are distributed over a wide range of protein families and use different reaction mechanisms (Bewley et al., 1999; Dodson & Wlodawer, 1998
; McVey et al., 2001
; Pace & Brenner, 2001
; Perozich et al., 1998
; Shin et al., 2002
). Within a region between residues 60 and 361, AguA is weakly homologous (23 % identity and 38 % similarity) to the corresponding region of the peptidylarginine deiminase of Porphyromonas gingivalis (McGraw et al., 1999
). AguA has no detectable homology with animal proteinarginine deiminases and other known CN hydrolases, including arginine deiminase of P. aeruginosa PAO1 (Baur et al., 1989
). Genome sequencing projects, however, have identified enzymes similar to AguA in various eubacteria and in Arabidopsis (Fig. 4
). These enzymes share high similarity (>50 %) and appear to form a small family of CN hydrolases with a novel structure. The inhibition of AguA by Hg2+ and iodoacetoamide suggests that a cysteine residue is involved in catalysis. A sequence comparison revealed a conserved cysteine in all homologues at a position corresponding to Cys-357 of PAO1 AguA (Fig. 4
). This cysteine residue would form a thioester enzymesubstrate complex with the concomitant release of ammonia, and subsequent nucleophilic attack by water would result in free enzyme and N-carbamoylputrescine, a reaction mechanism analogous to that proposed for amidases (Pace & Brenner, 2001
).
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Importantly, the AguA and AguB homologues occur in pairs in the same bacteria. Most bacteria that have their homologues also appear to possess SpeA arginine decarboxylase but not SpeB agmatinase. Moreover, the homologues are not present in Archaea, nor in animals that have the ODC pathway but not the ADC pathway. This distribution of the AguA and AguB homologues supports the view that the biosynthetic ADC pathway consisting of AguA and AguB occurs in a wide range of eubacteria.
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ACKNOWLEDGEMENTS |
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Received 19 September 2002;
revised 26 November 2002;
accepted 11 December 2002.