Activities and Properties of Putrescine‐Biosynthetic Enzymes in Vibrio parahaemolyticus

Abstract

The biosynthetic pathways for putrescine (Put) in Vibrio p arahaemolyticus were delineated by measuring activities of the enzymes which would be involved in its biosynthesis. Experiments with labeled arginine and ornithine revealed that both of these amino acids were converted into Put by intact cells. The activities of three enzymes, arginine decarboxylase (ADC), ornithine decarboxylase (ODC), and agmatine ureohydrolase (AUH), were detected in cell extracts. ADC and ODC of V. parahaemolyticus were similar in the following properties to the corresponding enzymes of Escherichia coli: 1) both decarboxylases showed a pH optimum at 8.25 and required pyridoxal phosphate and dithiothreitol for full activity; 2) while ODC was considerably activated by GTP, ADC was only slightly; 3) both decarboxylases were inhibited by polyamines ; 4) ADC was inhibited by difluoromethylarginine, a potent inhibitor of bacterial ADC. However, in contrast to the corresponding enzymes of E. coli, the V. parahaemolyticus ADC showed no requirement for Mg2+, and the AUH was active over a wide pH range of 8.5-9.5 with a maximum at pH 9.0. Furthermore, in all 6 strains tested, the activity of ADC was obviously high compared with that of ODC, and AUH was present with a relatively high activity. Cultivation of these strains at a suboptimal NaCl concentration (0.5%) resulted in a pronounced increase in both ADC and AUH activities. These observations suggest that the important pathway for Put biosynthesis in V. parahaemolyticus is the decarboxylation of arginine by ADC and the subsequent hydrolysis of its product, agmatine, by AUH.

Phylogeny of related functions: the case of polyamine biosynthetic enzymes

“…Urea was produced as described by Hirshfield et al (1970) using either agmatine or arginine as substrates (10 mM each). The assay was slightly modified by raising the pH of the Tris\HCl buffer from 7n5 to 9n0 as described by Yamamoto et al (1988). Urea measurement was performed using the Blood urea nitrogen assay kit (Sigma).…”

Characterization of Putrescine Production in Nongrowing Vibrio parahaemolyticus Cells in Response to External Osmolality

“…V. parahaemolyticus AQ 3627 was grown in a synthetic medium (2% NaCl) at 37 C to the late exponential phase (12 hr) as previously described (23). Cells were harvested by centrifugation at 4 C, washed once with cold 2% NaCl solution containing 10 mm MgCl2, and suspended in the same solution to make about 5 mg of cell protein/ml (usually, cells obtained from 1 liter of culture were suspended in 30 ml of the NaCl solution).…”

“…We previously observed a large excretion of Put into the medium even when this bacterium was grown at 0.5% NaCl without added arginine (23). Then, both the intra-and extracellular Put contents were determined at various time periods after suspension of cells in 0.5% NaCl medium with or without added arginine.…”

“…This contrasts with E. coli which derives most of Put from the ODC pathway (7,10). It was also noticeable that cells grown at an optimal NaCl concentration still possessed relatively high specific activities of ADC and AUH despite the low level of Put production (23). This suggested that the activities of ADC and/or AUH might be held in check by certain factor(s) under these growth conditions.…”

“…However, growth at suboptimal NaCl concentrations brought about an enormous increase in Put content with only a minor change in Spd, suggesting that the NaCl concentration of the growth medium may be a major determinant of Put production. Recently, we have found that arginine decaboxylase (ADC) and agmatine ureohydrolase (AUH), but not ornithine decarboxylase (ODC), are the enzymes responsible for biosynthesis of Put in this organism (23). This contrasts with E. coli which derives most of Put from the ODC pathway (7,10).…”