Phenol/cresol degradation by the thermophilic Bacillus thermoglucosidasius A7: cloning and sequence analysis of five genes involved in the pathway

Bacillus thermoglucosidasius A7 degraded phenol at 65 degrees C via the meta cleavage pathway. Five enzymes used in the metabolism of phenol were cloned from B. thermoglucosidasius A7 into pUC18. Nine open reading frames were present on the 8.1kb insert, six of which could be assigned a function in phenol degradation using database homologies and enzyme activities. The phenol hydroxylase is a two-component enzyme encoded by pheA1 and pheA2. The larger component (50kDa) has 49% amino acid identity with the 4-hydroxyphenylacetate hydroxylase of Escherichia coli, while the smaller component (19kDa) is most related (30% amino acid identity) to the styrene monoxygenase component B from Pseudomonas fluorescens. Both components were neccessary for activity. The catechol 2, 3-dioxygenase encoded by pheB has 45% amino acid identity with dmpB of Pseudomonas sp. CF600 and could be assigned to superfamily I, family 2 and a new subfamily of the Eltis and Bolin grouping. The 2-hydroxymuconic acid semialdehyde hydrolase (2HMSH), encoded by pheC, revealed the highest amino acid identity (36%) to the equivalent enzyme from Pseudomonas sp. strain CF600, encoded by dmpD. Based on sequence identity, pheD and pheE were deduced to encode the 2-hydroxypenta-2,4-dienoate hydratase (2HDH), demonstrating 45% amino acid identity to the gene product of cumE from Pseudomonas fluorescens and the acetaldehyde dehydrogenase (acylating) demonstrating 57% amino acid identity to the gene product of bphJ from Pseudomonas LB400.     

Nucleotide sequence and expression of clcD, a plasmid-borne dienelactone hydrolase gene from Pseudomonas sp. strain B13.

The clcD structural gene encodes dienelactone hydrolase (EC 3.1.1.45), an enzyme that catalyzes the conversion of dienelactones to maleylacetate. The gene is part of the clc gene cluster involved in the utilization of chlorocatechol and is carried on a 4.3-kilobase-pair BglII fragment subcloned from the Pseudomonas degradative plasmid pAC27. A 1.9-kilobase-pair PstI-EcoRI segment subcloned from the BglII fragment was shown to carry the clcD gene, which was expressed inducibly under the tac promoter at levels similar to those found in 3-chlorobenzoate-grown Pseudomonas cells carrying the plasmid pAC27. In this study, we present the complete nucleotide sequence of the clcD gene and the amino acid sequence of dienelactone hydrolase deduced from the DNA sequence. The NH2-terminal amino acid sequence encoded by the clcD gene from plasmid pAC27 corresponds to a 33-residue sequence established for dienelactone hydrolase encoded by the Pseudomonas sp. strain B13 plasmid pWR1. A possible relationship between the clcD gene and pcaD, a Pseudomonas putida chromosomal gene encoding enol-lactone hydrolase (EC 3.1.1.24) is suggested by the fact that the gene products contain an apparently conserved pentapeptide neighboring a cysteinyl side chain that presumably lies at or near the active sites; the cysteinyl residue occupies position 60 in the predicted amino acid sequence of dienelactone hydrolase.     

Identification of the functional periplasmic nitrate reductase (nap) gene cluster from the deep-sea denitrifier Pseudomonas sp. strain MT-1

The nap gene cluster encoding periplasmic nitrate reductase was identified from Pseudomonas sp. strain MT-1, a deep-sea denitrifier isolated from the Mariana Trench. The ORFs identified were highly homologous with those of Pseudomonas stutzeri, but the cluster included only four ORFs (napDABC), less than those in other organisms. For other bacteria, some additional small ORFs (such as napE, napF, napG, napH, and napK) are found in the nap gene cluster, although their physiological function is still unclear. The soluble fraction of MT-1 grown under denitrifying condition showed significant nitrate reductase activity. This observation suggests that the periplasmic nitrate reductase encoded by the gene cluster identified in this study is functional. The activity was highest when the organism was grown under denitrifying conditions, suggesting that the enzyme participates in dissimilatory nitrite reduction.     

Cloning and expression of a parathion hydrolase gene from a soil bacterium, Burkholderia sp. JBA3

A bacterium, Burkholderia sp. JBA3, which can mineralize the pesticide parathion, was isolated from an agricultural soil. The strain JBA3 hydrolyzed parathion to p-nitrophenol, which was further utilized as the carbon and energy sources. The parathion hydrolase was encoded by a gene on a plasmid that strain JBA3 harbored, and it was cloned into pUC19 as a 3.7-kbp Sau3AI fragment. The ORF2 (ophB) in the cloned fragment encoded the parathion hydrolase composed of 526 amino acids, which was expressed in E. coli DH10B. The ophB gene showed no significant sequence similarity to most of other reported parathion hydrolase genes.     

Evidence that operons tcb, tfd, and clc encode maleylacetate reductase, the fourth enzyme of the modified ortho pathway.

The maleylacetate reductase from Pseudomonas sp. strain B13 functioning in the modified ortho pathway was purified and digested with trypsin. The polypeptides separated by high-performance liquid chromatography were sequenced. Alignments with the polypeptides predicted from the tfdF and tcbF genes located on plasmids pJP4 of the 2,4-dichlorophenoxyacetate-degrading Alcaligenes eutrophus JMP134 and pP51 of the 1,2,4-trichlorobenzene-degrading Pseudomonas sp. strain P51 as well as polypeptides predicted from the tftE gene located on the chromosome of the 2,4,5-trichlorophenoxyacetate-degrading Burkholderia cepacia AC1100 were obtained. In addition, the deduced protein sequence encoded by the nucleotide sequence downstream of clcD on plasmid pAC27 of the 3-chlorobenzoate-degrading Pseudomonas putida AC866 was tested for homology. Significant sequence similarities with the polypeptides encoded by the tfdF, tcbF, and tftE genes as well as the nucleotide sequence downstream of the clcD gene gave evidence that these genes might encode maleylacetate reductases. A NAD-binding motif in a beta alpha beta-element was detected.     

Cloning and nucleotide sequence analysis of xylE gene responsible for meta-cleavage of 4-chlorocatechol from Pseudomonas sp. S-47

Pseudomonas sp. S-47 expresses catechol 2,3-dioxygenase (C230) catalyzing the conversion of 4-chlorocatechol (4CC) as well as catechol to 5-chloro-2-hydroxymuconic semialdehyde and 2-hydroxymuconic semialdehyde, respectively, through meta-ring cleavage. The xylE gene encoding C230 for meta-cleavage was cloned from strain S-47 and its nucleotide sequence was analyzed. The pRES101 containing the xylE gene exhibited high C230 activity toward catechol and 4CC without altering the substrate specificity from natural strain. The xylE gene was composed of 924 bp and encoded polypeptide of molecular mass 35 kDa containing 307 amino acids. A deduced amino acid sequence of the C230 from strain S-47 exhibited over 80% identity with those of Pseudomonas putida mt-2, Pseudomonas putida G7, and Pseudomonas sp. CF600. However, it shows below 45% identity with those of Pseudomonas cepacia LB400 and Pseudomonas sp. KKS102. The C230 of strain S-47 was conserved in the amino acids (His150, His214, Glu261) for metal binding ligands and those (His199, His242, and Tyr251) for catalytic sites. Therefore, Pseudomonas sp. S-47 can be explained as acting by degrading catechol as well as 4CC by xylE-encoding C230 which was fused by N domain of nahH and C domain of dmpB from other Pseudomonas strains.     

Catechol 2,3-dioxygenase from Pseudomonas sp. strain ND6: gene sequence and enzyme characterization

The catechol 2,3-dioxygenase (C23O) gene in naphthalene catabolic plasmid pND6-1 of Pseudomonas sp. ND6 was cloned and sequenced. The C23O gene was consisted of 924 nucleotides and encoded a polypeptide of molecular weight 36 kDa containing 307 amino acid residues. The C23O of Pseudomonas sp. ND6 exhibited 93% and 89% identities in amino acid sequence with C23Os encoded by naphthalene catabolic plasmid NAH7 from Pseudomonas putida G7 and the chromosome of Pseudomonas stutzeri AN10 respectively. The Pseudomonas sp. ND6 C23O gene was overexpressed in Escherichia coli DH 5alpha using the lac promoter of pUC18, and its gene product was purified by DEAE-Sephacel and Phenyl-Sepharose CL-4B chromatography. The enzymology experiments indicated that the specific activity and thermostability of C23O from Pseudomonas sp. ND6 were better than those of C23O from Pseudomonas putida G7.     

Cloning of a carbofuran hydrolase gene from Achromobacter sp. strain WM111 and its expression in gram-negative bacteria.

A 14-kilobase-pair (kbp) EcoRI DNA fragment that encodes an enzyme capable of rapid hydrolysis of N-methylcarbamate insecticides (carbofuran hydrolase) was cloned from carbofuran-degrading Achromobacter sp. strain WM111. When used to probe Southern blots containing plasmid and total DNAs from WM111, this 14-kbp fragment hybridized strongly to a 14-kbp EcoRI fragment from the greater than 100-kbp plasmid harbored by this strain but weakly to EcoRI-digested total DNA from Achromobacter sp. strain WM111, indicating that the gene for N-methylcarbamate degradation (mcd) is plasmid encoded. Further subcloning localized the mcd gene on a 3-kbp ScaI-ClaI fragment. There was little or no expression of this gene in the alternative gram-negative hosts Pseudomonas putida, Alcaligenes eutrophus, Acinetobacter calcoaceticus, and Achromobacter pestifer. Western blotting (immunoblotting) of the protein products produced by low-level expression in P. putida confirmed that this 3-kbp fragment encodes the two 70+-kilodalton protein products seen in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified carbofuran hydrolase.     

Reductive degradation of pyrazine-2-carboxylate by a newly isolated <Emphasis Type="Italic">Stenotrophomonas</Emphasis> sp. HCU1

A bacterium growing on pyrazine-2-carboxylate broth was isolated, purified and identified as a strain of Stenotrophomonas sp. based on polyphasic taxonomic analyses and designated as strain HCU1. 16S rRNA gene sequence of strain HCU1 showed 98.7% sequence similarity with the type strain of Stenotrophomonas maltophilia belonging to Gammaproteobacteria. Growth of strain HCU1 was demonstrated when pyrazine-2-carboxylate was used as a sole source of nitrogen. Ring reduction of pyrazine-2-carboxylate was shown as increase in absorbance at 268 nm and the reduced product was confirmed as 1,2,5,6-tetrahydropyrazine-2-carboxylate, while a ring opened product, 2-amino-2-hydroxy-3-(methylamino) propanoic acid (with a loss in carbon atom), indicated a reductive degradation of pyrazine-2-carboxylate by strain HCU1.     

Cytochrome c2 is essential for electron transfer to nitrous oxide reductase from physiological substrates in Rhodobacter capsulatus and can act as an electron donor to the reductase in vitro. Correlation with photoinhibition studies.

1. Addition of nitrous oxide to a periplasmic fraction released from Rhodobacter capsulatus strains MT1131, N22DNAR+ or AD2 caused oxidation of c-type cytochrome, as judged by the decrease in absorbance at 550 nm. The periplasmic fraction catalysed reduction of nitrous oxide in the presence of either isoascorbate plus phenazine ethosulphate or reduced methyl viologen. The rates with these two electron donors were similar and were comparable to the activity observed with a quantity of cells equivalent to those from which the periplasm sample had been derived. Activity in the periplasm could not be observed with ascorbate plus 2,3,5,6-tetramethyl-p-phenylenediamine although this reductant was effective with intact cells treated with myxothiazol to block the activity of the cytochrome-bc1 complex. 2. Cells of R. capsulatus MTG4/S4, a mutant from which the gene for cytochrome c2 has been specifically deleted, did not catalyse detectable rates of nitrous-oxide reduction. A nitrous-oxide reductase activity was present, as shown by activity of both cells and a periplasmic fraction with isoascorbate plus phenazine ethosulphate as reductant. The rates in cells and the periplasmic fraction were similar to those observed in the corresponding wild-type strain (MT1131). In contrast to wild-type cells, 2,3,5,6-tetramethyl-p-phenylenediamine and N,N,N',N'-tetramethyl-p-phenylenediamine [Ph(NMe2)2] were ineffective as mediators of electrons from isoascorbate. Visible absorption spectra showed that no detectable cytochromes in either the periplasm or intact cells of the MTG4/S4 mutant were oxidised by nitrous oxide. 3. Purified ferroycytochrome c2 from R. capsulatus was oxidised by nitrous oxide in the presence of periplasm from R. capsulatus MTG4/S4. The rate of oxidation was proportional to the amount of periplasm added, but was considerably lower than the rate of nitrous-oxide reduction observed with the same periplasmic fraction when either ascorbate plus phenazine ethosulphate or reduced methyl viologen were used as substrates. The oxidation of cytochrome c2 was inhibited by acetylene and by low concentrations of NaCl. 4. Oxidation of ferrocytochrome c2 by nitrous oxide was observed when the purified cytochrome was mixed with a preparation of nitrous-oxide reductase. However, oxidation of ferrocytochrome c' by nitrous oxide was not observed in the presence of the reductase. The observations with the mutant MTG4/S4 suggest that cytochrome c2 is the only periplasmic cytochrome involved in nitrous-oxide reduction. 5. Nitrous-oxide-dependent oxidation of a c-type cytochrome was observed in a periplasmic fraction from Paracoccus denitrificans, provided the fraction was first reduced.(ABSTRACT TRUNCATED AT 400 WORDS)     

Periplasmic protein associated with the oligopeptide permeases of Salmonella typhimurium and Escherichia coli.

A periplasmic protein essential for the function of the oligopeptide transport system of Salmonella typhimurium was identified. This protein, encoded by the oppA gene, is one of the most abundant proteins in the periplasm and, with an apparent molecular weight of 52,000, is considerably larger than any other known periplasmic transport component. A similarly abundant periplasmic protein forms part of the oligopeptide transport system of Escherichia coli.     

Cloning, characterization, and sequence analysis of the clcE gene encoding the maleylacetate reductase of Pseudomonas sp. strain B13.

A 3,167-bp PstI fragment of genomic DNA from Pseudomonas sp. strain B13 was cloned and sequenced. The gene clcE consists of 1,059 nucleotides encoding a protein of 352 amino acids with a calculated mass of 37,769 Da which showed maleylacetate reductase activity. The protein had significant sequence similarities with the polypeptides encoded by tcbF of pP51 (59.4% identical positions), tfdF of pJP4 (55.1%), and tftE of Burkholderia cepacia AC1100 (53.1%). The function of TcbF as maleylacetate reductase was established by an enzyme assay.     

Periplasmic location of p-cresol methylhydroxylase in Pseudomonas putida

The cellular location of the flavocytochrome c, p-cresol methylhydroxylase was investigated in two strains of Pseudomonas putida. In both cases the enzymes were shown to be located in the periplasmic fraction by their release during treatment of the bacteria with EDTA and lysozyme in a solution containing a high concentration of sucrose. For strain NCIB 9869 the finding is in accord with the suggestion that the physiological acceptor for the enzyme is azurin as this too was shown to be located mostly in the periplasm.     

Genetic organization of sulphur-controlled aryl desulphonation in Pseudomonas putida S-313

Pseudomonas putida S-313 is able to desulphonate a broad range of aromatic sulphonates to provide sulphur for growth by monooxygenolytic cleavage to yield the corresponding phenol. After miniTn5 transposon mutagenesis of this strain, 11 mutants were isolated that were no longer able to utilize benzenesulphonate as a sulphur source. Three of these mutants were defective in the utilization of all aromatic sulphonates tested, but they grew normally with other sulphur sources. These strains contained independent insertions in the novel 4.2 kb asfRABC gene cluster, encoding a putative reductase (AsfA), a ferredoxin (AsfB), a putative periplasmic binding protein (AsfC), which was localized to the periplasm using alkaline phosphatase fusions, and a divergently oriented fourth gene, asfR, that encoded a LysR-type regulator protein. A further mutant was interrupted in the ssu locus, which includes the gene for a putative desulphonative monooxygenase. Transformation of Pseudomonas aeruginosa with the asfRAB genes was sufficient to allow arylsulphonate utilization by this species, which does not normally use these compounds, suggesting that the AsfAB proteins may constitute an arylsulphonate-specific electron transport system that interacts with a less specific oxygenase. Expression of the asfABC genes in P. putida was induced by benzenesulphonate or toluenesulphonate, and it was repressed in the presence of sulphate in the growth medium. AsfR was a negative regulator of asfABC expression, and toluenesulphonate induced expression of these genes indirectly by reducing the expression of the asfR gene.     

Plasmid dependence of Pseudomonas sp. strain NK87 enzymes that degrade 6-aminohexanoate-cyclic dimer.

A bacterial strain, Pseudomonas sp. strain NK87, that can use 6-aminohexanoate-cyclic dimer as the sole source of carbon and nitrogen was newly isolated from wastewater of a factory which produces nylon-6. Two responsible enzymes, 6-aminohexanoate-cyclic-dimer hydrolase (P-EI) and 6-aminohexanoate-dimer hydrolase (P-EII), were found in the NK87 strain, as is the case with Flavobacterium sp. strain KI72, another 6-aminohexanoate-cyclic-dimer-metabolizing bacterium (H. Okada, S. Negoro, H. Kimura, and S. Nakamura, Nature [London] 306:203-206, 1983). The P-EI enzyme is immunologically identical to the 6-aminohexanoate-cyclic-dimer hydrolase of KI72 (F-EI). However, antiserum against the 6-aminohexanoate-dimer hydrolase purified from KI72 (F-EII) did not react with cell extracts of NK87, indicating that the F-EII and P-EII enzymes are immunologically different. Restriction endonuclease analyses show that the NK87 strain harbors at least six plasmids ranging in size from 20 to 80 kilobase pairs (kbp). The P-EI and P-EII genes were cloned in Escherichia coli. Both the P-EI and F-EI probes strongly hybridized with a 23-kbp plasmid in Southern hybridization analyses. The P-EII probe hybridized specifically with an 80-kbp plasmid, but the F-EII probe hybridized with none of the plasmids harbored in NK87. These results indicate that the P-EI gene and P-EII gene are encoded on the 23-kbp and 80-kbp plasmids, respectively.     

Molecular cloning and partial characterization of the pathway for aniline degradation inPseudomonas sp. strain CIT1

The genes encoding aniline utilization inPseudomonas sp. strain CIT1 have been cloned inEscherichia coli and partially characterized. Molecular cloning of the genes was achieved by construction of a cosmid library, followed by mobilization of the library into mutants ofPs. sp. CIT1 impaired in a number of functions necessary for growth on aniline. A 42-kbSau3A fragment was found to encode the ability to utilize aniline and contained the catechol 2,3-dioxygenase (C230) gene. The regions encoding these activities were subcloned and further characterized. Plasmids containing the aniline oxidase gene encoded a 260-kDa protein complex, which was putatively shown to be composed of 72 kDa and possibly 36 kDa subunits. The fragment required for C230 activity encodes a 35 kDa protein, similar in size to C230 gene products previously characterized.     

Selection of Pseudomonas sp. strain HBP1 Prp for metabolism of 2-propylphenol and elucidation of the degradative pathway.

A mutant of Pseudomonas sp. strain HBP1, originally isolated on 2-hydroxybiphenyl, was selected for the ability to grow on 2-propylphenol as the sole carbon and energy source. In the mutant strain, which was designated as Pseudomonas sp. strain HBP1 Prp, the cellular induction mechanism involved in the synthesis of the NADH-dependent monooxygenase is changed. 2-Propylphenol, which is known to be a substrate of the monooxygenase, does not induce formation of the monooxygenase in the wild type but does have an induction effect in the mutant strain. Furthermore, in contrast to the wild type, mutant strain HBP1 Prp constitutively produces a small amount of monooxygenase and metapyrocatechase. The enzymes from strain HBP1 Prp catalyzing the first three steps in the degradation of 2-propylphenol--the NADH-dependent monooxygenase, the metapyrocatechase, and the meta fission product hydrolase--were partially purified, and their activities were measured. The product of the monooxygenase activity was identified by mass spectrometry as 3-propylcatechol. The metapyrocatechase used this compound as a substrate and produced a yellow meta fission product that was identified by mass spectrometry as 2-hydroxy-6-oxo-nona-2,4- dienoate. Butyrate could be detected as a product of the meta fission product hydrolase in crude cell extract of 2-propylphenol-grown cells, as well as an intermediate in culture broths during growth on 2-propylphenol. All three enzymes expressed highest activities for the metabolites of the degradation of 2-hydroxybiphenyl.     

Mineralization of paraoxon and its use as a sole C and P source by a rationally designed catabolic pathway in Pseudomonas putida

Organophosphate compounds, which are widely used as pesticides and chemical warfare agents, are cholinesterase inhibitors. These synthetic compounds are resistant to natural degradation and threaten the environment. We constructed a strain of Pseudomonas putida that can efficiently degrade a model organophosphate, paraoxon, and use it as a carbon, energy, and phosphorus source. This strain was engineered with the pnp operon from Pseudomonas sp. strain ENV2030, which encodes enzymes that transform p-nitrophenol into beta-ketoadipate, and with a synthetic operon encoding an organophosphate hydrolase (encoded by opd) from Flavobacterium sp. strain ATCC 27551, a phosphodiesterase (encoded by pde) from Delftia acidovorans, and an alkaline phosphatase (encoded by phoA) from Pseudomonas aeruginosa HN854 under control of a constitutive promoter. The engineered strain can efficiently mineralize up to 1 mM (275 mg/liter) paraoxon within 48 h, using paraoxon as the sole carbon and phosphorus source and an inoculum optical density at 600 nm of 0.03. Because the organism can utilize paraoxon as a sole carbon, energy, and phosphorus source and because one of the intermediates in the pathway (p-nitrophenol) is toxic at high concentrations, there is no need for selection pressure to maintain the heterologous pathway.     

南极嗜冷假单胞菌7197 S- 腺苷同型半胱氨酸水解酶 ( SAHH) 基因的克隆与序列分析

从南极普利兹湾深海沉积物中筛选到一株耐冷菌株7197,其16S rDNA序列分析表明该菌株属于假单胞菌属（Pseudomonas）。作者通过设计引物,从该菌的全基因组DNA中克隆到编码S-腺苷-L-高半胱氨酸（SAHH）的完整ORF,全长为1424bp。使用DNAMAN（5,1）软件对全长ORF为1424bp的SAHH基因进行分析,SAHH基因编码一个由474AA残基组成、分子量预计为52523Da的SAHH蛋白质,与Psychrobacter sp．273—4的SAHH有96．84％的相似性;与Acinetobacter sp．ADP1的SAHH有79％的相似性;与Pseudomonas fluorescens Pf-5的SAHH有75％的相似性。     

A novel R-stereoselective amidase from Pseudomonas sp. MCI3434 acting on piperazine-2-tert-butylcarboxamide.

A novel amidase acting on (R,S)-piperazine-2-tert-butylcarboxamide was purified from Pseudomonas sp. MCI3434 and characterized. The enzyme acted R-stereoselectively on (R,S)-piperazine-2-tert-butylcarboxamide to yield (R)-piperazine-2-carboxylic acid, and was tentatively named R-amidase. The N-terminal amino acid sequence of the enzyme showed high sequence identity with that deduced from a gene named PA3598 encoding a hypothetical hydrolase in Pseudomonas aeruginosa PAO1. The gene encoding R-amidase was cloned from the genomic DNA of Pseudomonas sp. MCI3434 and sequenced. Analysis of 1332 bp of the genomic DNA revealed the presence of one open reading frame (ramA) which encodes the R-amidase. This enzyme, RamA, is composed of 274 amino acid residues (molecular mass, 30 128 Da), and the deduced amino acid sequence exhibits homology to a carbon-nitrogen hydrolase protein (PP3846) from Pseudomonas putida strain KT2440 (72.6% identity) and PA3598 protein from P. aeruginosa strain PAO1 (65.6% identity) and may be classified into a new subfamily in the carbon-nitrogen hydrolase family consisting of aliphatic amidase, beta-ureidopropionase, carbamylase, nitrilase, and so on. The amount of R-amidase in the supernatant of the sonicated cell-free extract of an Escherichia coli transformant overexpressing the ramA gene was about 30 000 times higher than that of Pseudomonas sp. MCI3434. The intact cells of the E. coli transformant could be used for the R-stereoselective hydrolysis of racemic piperazine-2-tert-butylcarboxamide. The recombinant enzyme was purified to electrophoretic homogeneity from cell-free extract of the E. coli transformant overexpressing the ramA gene. On gel-filtration chromatography, the enzyme appeared to be a monomer. It had maximal activity at 45 degrees C and pH 8.0, and was completely inactivated in the presence of p-chloromercuribenzoate, N-ethylmaleimide, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Ag+, Cd2+, Hg2+, or Pb2+. RamA had hydrolyzing activity toward the carboxamide compounds, in which amino or imino group is connected to beta- or gamma-carbon, such as beta-alaninamide, (R)-piperazine-2-carboxamide (R)-piperidine-3-carboxamide, D-glutaminamide and (R)-piperazine-2-tert-butylcarboxamide. The enzyme, however, did not act on the other amide substrates for the aliphatic amidase despite its sequence similarity to RamA.