Characterization of an Acid-Dependent Arginine Decarboxylase Enzyme from Chlamydophila pneumoniae

Abstract

Genome sequences from members of the Chlamydiales encode diverged homologs of a pyruvoyl-dependent arginine decarboxylase enzyme that nonpathogenic euryarchaea use in polyamine biosynthesis. The Chlamydiales lack subsequent genes required for polyamine biosynthesis and probably obtain polyamines from their host cells. To identify the function of this protein, the CPn1032 homolog from the respiratory pathogen Chlamydophila pneumoniae was heterologously expressed and purified. This protein self-cleaved to form a reactive pyruvoyl group, and the subunits assembled into a thermostable (␣␤) 3 complex. The mature enzyme specifically catalyzed the decarboxylation of L-arginine, with an unusually low pH optimum of 3.4. The CPn1032 gene complemented a mutation in the Escherichia coli adiA gene, which encodes a pyridoxal 5-phosphate-dependent arginine decarboxylase, restoring arginine-dependent acid resistance. Acting together with a putative arginine-agmatine antiporter, the CPn1032 homologs may have evolved convergently to form an arginine-dependent acid resistance system. These genes are the first evidence that obligately intracellular chlamydiae may encounter acidic conditions. Alternatively, this system could reduce the host cell arginine concentration and produce inhibitors of nitric oxide synthase.

From Protease to Decarboxylase

“…Among the different pyruvoyl enzymes, the mechanism of activation of the critical serine, and the amino acids involved in the general base catalysis for ester cleavage are highly divergent (27)(28)(29)(30), and do not provide insights into the likely constituents in the active site of PSDs. Our inspection of the conserved residues and motifs in the PSDs led us to postulate that a canonical D-H-S catalytic triad would likely be involved in the processing of this group of enzymes.…”

“…The autonomous generation of PE at specific subcellular sites appears to play an important role in organelle structure and function (7,8,10). PSDs belong to a small family of enzymes that contain a pyruvoyl prosthetic group, which includes S-adenosylmethionine decarboxylase (27), histidine decarboxylase (28), aspartate decarboxylase (29), and arginine decarboxylase (30) Pyruvoyl enzymes are first synthesized as single polypeptide precursors that undergo autoendoproteolytic cleavage to produce dissimilar ␣-and ␤-subunits. The proteolytic cleavage is initiated by an activated serine residue, which attacks the peptide bond that links the activated serine (e.g.…”

Crenarchaeal Arginine Decarboxylase Evolved from an S-Adenosylmethionine Decarboxylase Enzyme

“…Arginine Decarboxylase Assay-The rate of arginine decarboxylation was determined using a CO 2 capture assay to detect the release of 14 CO 2 from 14 C-labeled arginine (18). Standard reactions (100 l) contained 12 mM citric acid, 26 mM sodium phosphate (pH 6.0), 10 mM L-arginine-HCl, 6.5-300 nCi of L-[1-14 C]arginine (55 mCi mmol…”

“…Protein bands were identified in the gels by silver staining (Pierce) or staining with Coomassie Brilliant Blue R dye. Measurements of native protein mass and Stokes radius were made by analytical size exclusion chromatography (18). Precipitation with trichloroacetic acid was used to concentrate proteins from collected fractions; these were analyzed by SDS-PAGE to determine the subunit composition of oligomers.…”

“…The cells were grown with continuous shaking (250 rpm) at 37°C in LuriaBertani broth with ampicillin (100 g ml Ϫ1 ). Protein expression and cell lysis were performed by standard methods (18). Extracts were heated at 75°C for 20 min and then centrifuged at 18,000 ϫ g for 10 min.…”