Analysis and sequence of the speB gene encoding agmatine ureohydrolase, a putrescine biosynthetic enzyme in Escherichia coli

Abstract

The speB gene of Escherichia coli encodes the enzyme agmatine ureohydrolase (AUH). AUH catalyzes the hydrolysis of agmatine to urea and putrescine in one of the two polyamine biosynthetic pathways in E. coli. Sequencing of a 2.97-kilobase-pair fragment of the E. coli chromosome containing speB revealed the presence of three intact open reading frames (ORFs), ORF1 and ORF2 on one strand and ORF3 on the opposite strand, as weUl as a truncated ORF, ORF4, which terminated 92 kilobase pairs upstream from ORF3. ORF3 contained the coding sequence of the speB gene, as confirmed by complementation analysis. Two ORF3 transcripts were detected: a shorter transcript that included only ORF3 and a longer transcript that included both ORF3 and ORF4. The short transcript was abundantly expressed when the ORF4 sequences were deleted, but when ORF4 and its upstream sequences were present, the polycistronic message predominated and the amount of the monocistronic message was drastically reduced. The promoter from which the shorter transcript was produced contained a TATACT sequence at position -12, but sequences upstream from the -12 position seemed to be irrelevant for promoter activity. The predicted amino acid sequence of AUH contained three regions of high homology to the arginases of yeasts, rats, and humans.In Escherichia coli putrescine is synthesized either by decarboxylation of ornithine or by decarboxylation of arginine to agmatine followed by hydrolysis of agmatine to putrescine and urea (14). The last two reactions are catalyzed by the enzymes arginine decarboxylase and agmatine ureohydrolase (AUH), respectively. The AUH protein has previously been purified from an E. coli isolate that was transformed with the plasmid pKA5; the enzymatic properties of AUH have been characterized previously (9). The subunit size of AUH, as deduced from its mobility on a sodium dodecyl sulfate-polyacrylamide gel, is 38 kilodaltons. The expression of AUH activity is antagonistically regulated by cyclic AMP and agmatine. In the presence of the cyclic AMP receptor protein, cyclic AMP represses the expression of the speB gene, while agmatine induces it. These two modulators appear to act independently from each other (10); the mechanism of this differential regulation is unknown. The speB gene coding for AUH is located at approximately 63.5 min on the E. coli chromosome. Although a large chromosomal fragment corresponding to this region, including the speB gene, is present in the pKA5 plasmid, the exact location of speB was not previously established. Here we report the nucleotide sequence of the speB structural gene and identify the promoter responsible for transcription of one of two mRNAs encoding AUH. We also report the mapping of the mRNA resulting from transcription that initiated at this promoter. MATERIALS AND METHODSBacterial hosts, media, and growth conditions. E. coli CB806(A1acZ galK phoA8 rpsL thi recA56) (11) was used for all experiments involving the promoter vector pCB267 and its derivatives. E. coli DHSa [F- The bacteria wer...

Identification, cloning, sequencing, and overexpression of the gene encoding proclavaminate amidino hydrolase and characterization of protein function in clavulanic acid biosynthesis

“…Numerous homologies can be seen, notably in the central region of PAH (positions 118 to 122 and 144 to 148) and toward the C terminus (positions 235 to 241). There are 12 invariant residues distributed throughout these proteins and 6 additional residues present in all members except HutG (40), about which only C-terminal information is available. It is highly probable that contained within these conserved amino acids are the catalytic and metal-binding sites.…”

“…Abbreviations for protein sequences: Arg, arginase; Hmfb, ORF in the Hmf locus; hutG, formiminoglutamate hydrolase. Abbreviations (and references) for species: Atu, Agrobacterium tumefaciens plasmid Ti C58 (36); Eco, E. coli (40); Hum, human (42); Kae, K. aerogenes (37); Mfe, Methanothermus fervidus (33); Ncr, N. crassa (21); rat (27) …”

“…However, when the deduced amino acid sequence of PAH was compared with the sequence of AUH (40), 63% similarity and 41% identity were found to exist between the two sequences. AUH catalyzes the hydrolysis of agmatine, the decarboxylation product of arginine, to putrescine (35,40), an important step in polyamine biosynthesis. The amino acid sequence of PAH was also compared with sequences of arginases purified from various species, including Neurospora crassa (21), Xenopus laevis (50), Klebsiella aerogenes (37), Saccharomyces cerevisiae (39), rat liver (27), and human liver (42).…”

“…Therefore, the sequence of 1 to 1.5 kbp of DNA was quickly surveyed upstream and downstream from each gene to test for additional homologies (49). Interestingly, none was evident, although immediately upstream of cs2, a tentative open reading frame (ORF) having greater than 40% identity to agmatine ureohydrolase (AUH) (40) and several arginases (see below) could be identified. The latter carry out the hydrolysis of arginine to ornithine, while the former converts agmatine to putrescine and urea.…”

“…The cla/pah gene encodes a protein of this size. In this study, we have completed the correlation of sequence homology to AUH (40) and arginases to PAH function by overexpression of this gene as a fusion to the maltosebinding protein (MAL) and characterization of its catalytic activities as both a fusion and a cleaved PAH-containing domain. In addition, the role of PAH and its relation to CS are discussed in the wider context of clavulanic acid biosynthesis.…”