γ-Glutamylputrescine Synthetase in the Putrescine Utilization Pathway of Escherichia coli K-12

Abstract

Glutamate-putrescine ligase (␥-glutamylputrescine synthetase, PuuA, EC 6.3.1.11) catalyzes the ␥-glutamylation of putrescine, the first step in a novel putrescine utilization pathway involving ␥-glutamylated intermediates, the Puu pathway, in Escherichia coli. In this report, the character and physiological importance of PuuA are described. Purified non-tagged PuuA catalyzed the ATP-dependent ␥-glutamylation of putrescine. The K m values for glutamate, ATP, and putrescine are 2.07, 2.35, and 44.6 mM, respectively. There are two putrescine utilization pathways in E. coli: the Puu pathway and the pathway without ␥-glutamylation. Gene deletion experiments of puuA, however, indicated that the Puu pathway was more critical in utilizing putrescine as a sole carbon or nitrogen source. The transcription of puuA was induced by putrescine and in a puuR-deleted strain. The amino acid sequences of PuuA and glutamine synthetase (GS) show high similarity. The molecular weights of the monomers of the two enzymes are quite similar, and PuuA exists as a dodecamer as does GS. Moreover the two amino acid residues of E. coli GS that are important for the metal-catalyzed oxidation of the enzyme molecule involved in protein turnover are conserved in PuuA, and it was experimentally shown that the corresponding amino acid residues in PuuA were involved in the metal-catalyzed oxidation similarly to GS. It is suggested that the intracellular concentration of putrescine is optimized by PuuA transcriptionally and posttranslationally and that excess putrescine is converted to a nutrient source by the Puu pathway.

Metabolic engineering of Escherichia coli for the production of putrescine: A four carbon diamine

“…Also, the putrescine degrading Puu pathway, which is responsible for catabolizing extracellular putrescine (Kurihara et al, 2005) in wild-type E. coli, was deleted; the puuPA genes that encode the dorminant putrescine importer PuuP and glutamate-putrescine ligase (PuuA; the first enzyme of the Puu pathway). However, an alternative putrescine degradation pathway involving YgjG and YdcW was not deleted because its activity is negligible (Kurihara et al, 2008). This WL3110 (DargI DspeE DspeG DpuuPA) strain was named XQ26 and was used as a base strain.…”

Transforming a drug/H + antiporter into a polyamine importer by a single mutation

“…For growth in minimal medium, overnight LB cultures were washed with 1× W-salts (23) and resuspended in minimal medium [which contained 1× W-salts, 2% (wt/vol) glucose, 2.5 μg/mL thiamine, 34 μg/mL chloramphenicol, 200 μM IPTG, and 0.5% putrescine] to a final OD of 0.01, and growth was followed as described above.…”

A Novel Putrescine Importer Required for Type 1 Pili-driven Surface Motility Induced by Extracellular Putrescine in Escherichia coli K-12

“…Analysis of Polyamine Concentration-The concentration of polyamines in the samples was measured as described previously (26).…”

“…The disintegrations per minute of 14 C in the cell were divided by the protein amount of the cell used in the transport assay. The cell protein content was extracted as described previously (26), and the amount of protein was assayed by Lowry's method (27).…”