Identification of the putrescine biosynthetic genes in Pseudomonas aeruginosa and characterization of agmatine deiminase and N-carbamoylputrescine amidohydrolase of the arginine decarboxylase pathway

Yuji Nakada and Yoshifumi Itoh

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Putrescine can be synthesized either directly from ornithine by ornithine decarboxylase (ODC; the speC product) or indirectly from arginine via arginine decarboxylase (ADC; the speA product). The authors identified the speA and speC genes in Pseudomonas aeruginosa PAO1. The activities of the two decarboxylases were similar and each enzyme alone appeared to direct sufficient formation of the polyamine for normal growth. A mutant defective in both speA and speC was a putrescine auxotroph. In this strain, agmatine deiminase (the aguA product) and N-carbamoylputrescine amidohydrolase (the aguB product), which were initially identified as the catabolic enzymes of agmatine, biosynthetically convert agmatine to putrescine in the ADC pathway: a double mutant of aguAB and speC was a putrescine auxotroph. AguA was purified as a homodimer of 43 kDa subunits and AguB as a homohexamer of 33 kDa subunits. AguA specifically deiminated agmatine with Km and Kcat values of 0·6 mM and 4·2 s-1, respectively. AguB was specific to N-carbamoylputrescine and the Km and Kcat values of the enzyme for the substrate were 0·5 mM and 3·3 s-1, respectively. Whereas AguA has no structural relationship to any known C–N hydrolases, AguB is a protein of the nitrilase family that performs thiol-assisted catalysis. Inhibition by SH reagents and the conserved cysteine residue in AguA and its homologues suggested that this enzyme is also involved in thiol-mediated catalysis.


Abbreviations: ADC, arginine decarboxylase; ODC, ornithine decarboxylase


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Polyamines (putrescine, spermidine and spermine) are a group of ubiquitous polycations that are necessary for optimal cell growth (Glansdorff, 1996). Putrescine and spermidine are major polyamines in bacteria and spermidine is also present in plants and animals (Glansdorff, 1996; Tabor & Tabor, 1972). Putrescine can be formed either directly from ornithine by ornithine decarboxylase (ODC, EC 4.1.1.17; the speC product) or indirectly from arginine by arginine decarboxylase (ADC; EC 4.1.1.19, the speA product). Subsequent transfer of the aminopropyl group of decarboxylated S-adenosylmethionine to putrescine by spermidine synthase (speE) generates spermidine (Fig. 1). Whereas the ODC and ADC pathways operate in animals and in plants, respectively, both pathways operate simultaneously in many bacteria (Cunin et al., 1986; Glansdorff, 1996; Tabor & Tabor, 1972). Two distinct enzymic systems catalyse the biosynthetic conversion of agmatine to putrescine (Cunin et al., 1986). Agmatinase (EC 3.5.3.11, the speB product) catalyses the direct conversion of agmatine to putrescine in bacteria such as Escherichia coli. Plants convert agmatine to putrescine via two enzymes: agmatine deiminase (EC 3.5.3.12; the aguA product) catalyses the conversion of agmatine to N-carbamoylputrescine, which in turn is converted to putrescine by N-carbamoylputrescine amidohydrolase (EC 3.5.1.53; the aguB product) (Fig. 1).



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Fig. 1. Schematic pathways of polyamine biosynthesis. Only ornithine is presented as an intermediate of arginine biosynthesis. The reaction catalysed by ornithine acetyltransferase (N-acetylornithine+glutamate -> N-acetyglutamate+ornithine) in arginine biosynthesis is not indicated. Enzymes encoded by the spe and agu genes are described in the text. SAM, S-adenosylmethionine; dSAM, decarboxylated S-adenosylmethionine.

 
None of the polyamine biosynthetic enzymes has to our knowledge been studied in Pseudomonas aeruginosa PAO1. However, the Pseudomonas Genome Project (http://www.pseudomonas.com) has identified a complete set of the putative speABCDE genes, implying that polyamine biosynthesis by this strain proceeds via both ODC and ADC routes. Two genes (speB1 and speB2) were tentatively annotated as putative speB genes according to their similarities (about 60 %) to the E. coli speB (http://www.pseudomonas.com). Recently we showed that speB2 specifies guanidinobutyrase and renamed the gene as gbuA (Nakada et al., 2001). Thus speB1 remains as a candidate for the agmatinase gene.

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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, plasmids and media.
Bacterial strains and plasmids are listed in Table 1. P. aeruginosa PAO1 and its derivatives were incubated in nutrient-yeast extract broth (NYB) or in minimal medium P (MMP) (Nakada et al., 2001) supplemented with 20 mM glutamate or 20 mM agmatine as carbon and nitrogen sources. E. coli strains were grown in Luria–Bertani (LB) medium (Sambrook et al., 1989). When necessary, either ampicillin (for E. coli) or carbenicillin (for P. aeruginosa) was added to the media at concentrations of 100 µg ml-1 or 125 µg ml-1, respectively.


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Table 1. Bacterial strains and plasmids

 
Strains and plasmid construction.
Plasmid preparation, DNA techniques and conjugation were as described by Sambrook et al. (1989) and Nakada & Itoh (2002). Knockout mutants of P. aeruginosa PAO1 were constructed according to Hoang et al. (1998). The DNA regions of speA and speC were amplified by PCR using PAO1 chromosomal DNA as the template and the following pairs of oligonucleotide primers as described by Nakada & Itoh (2002): 5'-CTCAAGCGCGGTTTCAAGGCGAAGAAGTC-3' (nucleotides [nt] -287 to -259 of speA) and 5'-AAGTAGAAATGAAGGGTTGCGCGGAACTC-3' (complementary to nt 90 to 118 downstream of speA), 5'-AGGTGCAGCAGGCACTGGAGAAATATCCTC-3' (nt -696 to -668 of speC) and 5'-TACACCGTGCCCTTCCTGGTCTGGACTTC-3' (nt 913 to 941 downstream of speC). The amplified DNA fragments were cloned into plasmid pEX18Ap (mob+ sacB+) (Hoang et al., 1998) and the nucleotides were confirmed by sequencing as previously described (Nakada & Itoh, 2002). A 2·3 kb PstI fragment (speB1) and a 4·7 kb KpnI fragment (aguAB) were subcloned from plasmids pGU2 (Nakada et al., 2001) and pYJ101 (Nakada et al., 2001), respectively, into pEX18Ap at the corresponding restriction site. A FRT gentamicin-resistance (Gm) cassette excised from plasmid pPS858 (Hoang et al., 1998) as a SmaI or SalI fragment was then inserted into restriction sites for the target genes on the plasmids: the Eco52I site at nt 1032 of speA, the XhoI site at nt 787 of speC, the PvuII site at nt 799 of speB1, and between the NspV sites of aguA (nt 361) and aguB (nt 271), after blunting incompatible ends when appropriate. The allelic genes were then replaced with the plasmids as described by Hoang et al. (1998). To knock out a second gene from the speC : : FRT-Gm mutant, the Gm-resistance sequence in the chromosome was removed by introducing plasmid pFLP2, which carries the Flp recombinase gene (Hoang et al., 1998). Correct insertions in the constructed mutants (Table 1) were verified by PCR.

To maximize the synthesis of AguA and AguB, we cloned the 3·8 kb BglII–KpnI 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 : : {Omega}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 : : {Omega}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 50–75 % 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 (40–70 % 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.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Construction and characterization of spe mutants
When cultured in MMP with 20 mM each succinate and ammonia as the sources of carbon and nitrogen, respectively, P. aeruginosa PAO1 cells contained 11·6x10-3 units of ADC (mg protein)-1 min-1 and 14·3x10-3 units of ODC (mg protein)-1 min-1. To examine whether these activities are specified by the putative speA (PA4839 of the Pseudomonas Genome Project) or speC (PA4519) gene, we knocked out these genes by insertion of an FRT-Gm cassette or the FRT sequence (Hoang et al., 1998). The speA mutant, PAO4539 (speA : : FRT-Gm), showed no putrescine auxotrophy (Fig. 2a) but had only 0·2x10-3 units of ADC (mg protein)-1 min-1. These results confirmed that speA encodes ADC and indicated that putrescine can be supplied via ODC to fully support growth of strain PAO1 in MMP. The putative ODC encoded by speC shows 48 % similarity with Saccharomyces cerevisiae ODC (Fonzi & Sypherd, 1987) but no significant similarity to E. coli ODC. The speC mutant PAO4540 (speC : : FRT) also grew normally in MMP medium (Fig. 2a). However, the activity of ODC in this mutant was as low as 0·1x10-3 units (mg protein)-1 min-1, confirming that ODC is the product of this gene. Like ODC, the ADC pathway appeared to synthesize enough putrescine for normal growth. When both genes were inactivated, as in PAO4545 (speA : : FRT-Gm speC : : FRT) (Table 2), growth in MMP was severely handicapped (Fig. 2a). The growth of the mutant was restored nearly to the wild-type level when 1 mM putrescine was added to the medium (Fig. 2a).



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Fig. 2. Growth phenotypes of spe and aguAB mutants. Cultures were incubated in MMP containing 20 mM glutamate with the indicated supplements. Plots are means of two independent cultures. (a) {bullet}, Wild-type PAO1 (no supplement); {circ}, PAO4539 (speA : : FRT-Gm) (no supplement); {blacktriangleup}, PAO4540 (speC : : FRT) (no supplement); {triangleup}, PAO4545 (speA : : FRT-Gm speC : : FRT) (no supplement); {square}, PAO4545 (speC : : FRT-Gm speC : : FRT) (1 mM putrescine). (b) {bullet}, Wild-type PAO1 (no supplement); {circ}, PAO4544 (speC : : FRT speB1 : : FRT-Gm) (no supplement); {blacktriangleup}, PAO4541 (speC : : FRT aguAB : : FRT-Gm) (no supplement); {triangleup}, PAO4541 (speC : : FRT aguAB : : FRT-Gm) (1 mM putrescine).

 

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Table 2. Formation of ADC and ODC by the wild-type strain and spe mutants under different growth conditions

Cell extracts were prepared form cells growing exponentially in MMP containing the indicated carbon and nitrogen sources at 20 mM each and were used for enzyme assays. Enzyme activities are presented as 10-3 units (mg protein)-1 min-1. Values are means of two or three measurements. Standard errors (not shown) were below 5 % of the corresponding means. ND, Not determined because of very poor growth (Fig. 2a, b).

 
Regulation of synthesis of ADC and ODC
When cells were cultured in MMP containing 20 mM arginine as carbon and nitrogen source, ADC synthesis was increased twofold, whereas ODC synthesis was diminished to 30–50 % of the level of cells grown in MMP containing succinate and ammonia each at 20 mM (Table 2). Growth in MMP containing 20 mM putrescine as carbon and nitrogen source repressed the formation of both enzymes by 50–70 % (Table 2). Ornithine, an intermediate of arginine biosynthesis (Fig. 1), had little influence on the enzyme synthesis.

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 : : {Omega}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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Table 3. Overproduction of agmatine deiminase and N-carbamoylputrescine amidohydrolase by strain PAO4495 (aguR : : {Omega}Sp/Sm) harbouring an aguAB recombinant plasmid

Cells were grown in MMP containing glutamate (non-inducer) or agmatine (inducer) at 20 mM as carbon and nitrogen sources. The activities of agmatine deiminase (ADI) and N-carbamoylputrescine amidohydrolase (CPAH) were measured in cell extracts. Values, given as 10-2 units (mg protein)-1, are means of two or three measurements under each growth condition. Standard errors were below 5 % of the corresponding means.

 


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Fig. 3. SDS-PAGE of purified AguA and AguB before and after cross-linkage. Purified AguA (2 µg) and AguB (3 µg) were resolved directly or after cross-linking on 10 % polyacrylamide gels with molecular mass markers (carbonic anhydrase, 30 kDa; ovalbumin, 43 kDa; glutamate dehydrogenase, 53 kDa; bovine serum albumin, 67 kDa; transferrin, 76 kDa; {beta}-galactosidase, 116 kDa; {alpha}-2-macroglobulin, 170 kDa; myosin, 212 kDa). Lane 1, AguA without cross-linkage; lane 2, cross-linked AguA; lane 3, AguB without cross-linkage; lane 4, cross-linked AguB.

 
Kinetic and other properties
AguA and AguB were most active at 45 °C and 40 °C, respectively, and their optimal pH value was 8·0 in Tris/HCl buffer. Agmatine was the sole substrate for AguA: activity was undetectable with arginine, creatine, creatinine, guanidinoacetate, 3-guanidinopropionate and 4-guanidinobutytrate. The Km and Kcat values of the enzyme for agmatine were 0·6±0·05 mM and 4·2±0·2 s-1, respectively. AguB preferentially hydrolysed N-carbamoylputrescine [9·9 units (mg protein)-1] and was weakly active towards N-carbamoylcadaverine [0·5 units (mg protein)-1] and N-carbamoyldiaminopropane [0·3 units (mg protein)-1]. Activity was undetectable with N,N'-dicarbamoylputrescine, N-carbamoyl-DL-alanine, N-carbamoylisobutyrate, N-carbamoyl-DL-aminobutyrate, N-carbamoyl-L-glutamate and acetonitrile. The Km and Kcat values of the enzyme for N-carbamoylputrescine were 0·5±0·05 mM and 3·3±0·2 s-1, respectively. None of arginine, ornithine, putrescine, spermidine, spermine, EDTA and all tested divalent cations, except for Hg2+, affected the enzyme activity at 1 mM. Cationic mercury inhibited 90 % and 79 % of AguA and AguB activities, respectively, at 0·1 mM. Iodoacetoamide (1 mM) and p-chloromercuribenzoate (0·1 mM) inhibited AguA by 95 % and 12 %, and AguB by 88 % and 94 %, respectively.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
This study identified the speA and speC putrescine biosynthetic genes and demonstrated that AguA and AguB are involved in putrescine biosynthesis as well as in agmatine catabolism by P. aeruginosa PAO1 (Haas et al., 1984; Nakada et al., 2001). Constitutive levels of AguA and AguB are below 10 % of the induced levels (Table 3) but can complete the biosynthetic reaction (Fig. 2a). The double mutants speA speC and aguAB speC grow very poorly in minimal medium (Fig. 2a, b). Polyamine(s) that support the residual growth of the mutants might be formed from ornithine or lysine by another decarboxylase, e.g. a putative ornithine/arginine/lysine decarboxylase encoded by PA1818 (http://www.psedomonas.com). Expression of speA and speC is partially repressed by putrescine (Table 2) as in E. coli (Glansdorff, 1996). While arginine activates speA expression, it prevents speC expression (Table 2). Arginine exerts feedback inhibition on the first two arginine biosynthetic enzymes, N-acetylglutamate synthase and acetylglutamate kinase (Haas et al., 1972; Haas & Leisinger, 1975). Therefore ornithine pools would be low in cells growing in arginine medium. Inverse regulation of speA and speC expression by arginine could avoid futile synthesis of ODC and could efficiently generate putrescine from exogenous arginine via the ADC pathway.

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 C–N 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 protein–arginine deiminases and other known C–N 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 C–N 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 enzyme–substrate 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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Fig. 4. Putative catalytic cysteine residues conserved at C-terminals of AguA and its orthologues. Sequences were aligned using the CLUSTAL W program at KEGG (http://www.genome.ad.jp). Numbers on the right refer to amino acid positions of each protein. The asterisk above the sequences indicates the conserved catalytic cysteine. Sequence data were obtained from GenBank (http://www.ncbi.nlm.nih.gov): A. thaliana (CAB93718), C. jejuni (CAB73206), C. crescentus (AAK22198), C. tepidum (AAM72735), D. radiodurans (AAF11904), H. pylori (AAD07122), L. sakei (AAL98713), L. lactis (AAK05795), L. monocytogenes (CAC98253), S. pneumoniae (AAK75045), S. coelicolor (CAA19975), X. axonopodis (AAM37155), X. campestris (AAM41478), X. fastidiosa (AAF85241), Y. pestis (CAC89782), Z. mobilis (AAD19715).

 
The nitrilase family includes AguB (Bewley et al., 1999; Bork & Koonin, 1994), as it shares about 45 % similarity with known members of this collection of 13 subfamilies (Bewley et al., 1999). Like N-carbamyl-D-amino acid amidohydrolase of the carbamylase subfamily (Bewley et al., 1999), AguB catalyses amide hydrolysis (R–NH–CO–NH2 -> R–NH2+CO2+NH3), but it does not hydrolyse N-carbamoyl-amino acids and has no close similarity (<50 %) to N-carbamyl-D-amino acid amidohydrolases. Enzymes that share higher similarity to AguA occur in the same bacteria (except for the lactic type), Arabidopsis, rice (accession no. BAB59126) and tomato (CAB45873). In lactic bacteria, putrescine carbamoyltransferase but not AguB catabolically converts N-carbamoylputrescine to putrescine and to carbamoyl phosphate, which is used to synthesize ATP by carbamate kinase (Cunin et al., 1986). Members of the nitrilase family have three conserved regions containing one of the catalytic triads (glutamate, lysine and cysteine) (Bork & Koonin, 1994; Pace & Brenner, 2001). All of these catalytic residues are also conserved in AguB in the corresponding regions. The signatures of the conserved regions in each subfamily are distinct. Regions containing the catalytic cysteine (bold type) in AguB homologues have very similar sequences (R/K-L/I/V-G-V-A/G/L-I/V-C-W-Q-W-Y/F-P-E). This signature is highly conserved and distinct from those of the other subfamilies (Bork & Koonin, 1994). Such structural features and the strict substrate specificity of AguB for N-carbamoylputrescine support the notion that putative AguB constitutes a new subfamily of nitrilases.

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.


   ACKNOWLEDGEMENTS
 
We thank K. Kimura for help with the enzyme purification. We are also grateful to C. D. Lu for plasmid pGU2 and to H. P. Schweizer for plasmids pEX18Ap, pSP858 and pFLP2. This work was supported in part by a grant-in-aid from the Ministry of Education, Culture, Sports, Science and Technology (to Y. I., 14360060). Y. N. is a domestic research fellow supported by the Japan Science and Technology Corporation.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 19 September 2002; revised 26 November 2002; accepted 11 December 2002.