Abstract
The genome sequence of the hyperthermophilic methanogen Methanococcus jannaschii contains homologs of most genes required for spermidine polyamine biosynthesis. Yet genomes from neither this organism nor any other euryarchaeon have orthologs of the pyridoxal 5'-phosphate-dependent ornithine or arginine decarboxylase genes, required to produce putrescine. Instead, as shown here, these organisms have a new class of arginine decarboxylase (PvlArgDC) formed by the self-cleavage of a proenzyme into a 5-kDa subunit and a 12-kDa subunit that contains a reactive pyruvoyl group. Although this extremely thermostable enzyme has no significant sequence similarity to previously characterized proteins, conserved active site residues are similar to those of the pyruvoyl-dependent histidine decarboxylase enzyme, and its subunits form a similar (alphabeta)(3) complex. Homologs of PvlArgDC are found in several bacterial genomes, including those of Chlamydia spp., which have no agmatine ureohydrolase enzyme to convert agmatine (decarboxylated arginine) into putrescine. In these intracellular pathogens, PvlArgDC may function analogously to pyruvoyl-dependent histidine decarboxylase; the cells are proposed to import arginine and export agmatine, increasing the pH and affecting the host cell's metabolism. Phylogenetic analysis of Pvl- ArgDC proteins suggests that this gene has been recruited from the euryarchaeal polyamine biosynthetic pathway to function as a degradative enzyme in bacteria.
Highlights
Polyamines are ubiquitous organic cations that have been broadly implicated in modulating ion channels, stimulating cell proliferation, and facilitating protein synthesis [1,2,3]
M. jannaschii must have a noncanonical means of producing putrescine that is required for spermidine biosynthesis
S-Adenosylmethionine decarboxylase [11, 12], a homolog of spermidine synthase (MJ0313), a homolog of agmatine ureohydrolase (MJ0309) [17], and a previously uncharacterized open reading frame (MJ0316) are conserved in most euryarchaeal genomes. This group of genes that are required for polyamine biosynthesis suggested that the MJ0316-encoded protein could be an arginine decarboxylase
Summary
Materials—All reagents and synthetic precursors were purchased from Sigma unless otherwise specified. Protein was eluted with a 30-ml linear gradient from 0 to 1 M NaCl in buffer A at a flow rate of 0.5 ml/min. Concentrated protein was applied to a Sephacryl S-200HR size exclusion column (16 mm ϫ 60 cm; Amersham Biosciences) equilibrated in buffer C, which contained 150 mM NaCl and 50 mM Hepes adjusted to pH 7.2 with NaOH. Chromatography was performed in buffer C at a flow rate of 0.5 ml/min, and ArgDC activity eluted maximally at 57 ml. Purified PvlArgDC enzyme (1 g) was incubated with 10 mM amino acid and 50 mM Mes/NaOH (pH 6.0) in a volume of 20 l at 70 °C for 20 min. Fixed time point reactions were initiated with 5 mM L-arginine and 100 nCi L-[1-14C]arginine (55 mCi/mmol), incubated at 70 °C, and terminated after 7 min as described for standard assays. Matrix, neurotensin, gramicidin D, bovine pancreas insulin chain B (oxidized), and horse heart cytochrome c were used for mass calibration
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