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 (alphabeta)(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.

FIG. 1.

The arginine decarboxylase enzyme (ArgDC; EC 4.1.1.19) catalyzes the production of agmatine and CO₂ from l-arginine. The two known types of arginine decarboxylase use either pyridoxal 5′-phosphate or pyruvoyl cofactors. The agmatine urea hydrolase metalloenzyme (AUH; EC 3.5.3.11) catalyzes the hydrolysis of agmatine to produce putrescine, the core diamine of most polyamines. No homolog of AUH is present in chlamydial genomes.

FIG. 2.

Protein sequence alignment of the C. pneumoniae CPn1032 protein (NCBI RefSeq accession no. NP_225226) with homologs from Chlamydia trachomatis (CT373; Swiss-Prot accession no. O84378), Chlorobium tepidum (GenBank accession no. AAM71804), Porphyromonas gingivalis (RefSeq accession no. NP_905146), and Methanocaldococcus jannaschii (Swiss-Prot accession no. Q57764). Conserved amino acid residues are shown in white on a black background. An arrow indicates the site of protein self-cleavage and pyruvoyl group formation. Sequences were aligned using the T-COFFEE program (version 4.96) (32).

FIG. 3.

The C. pneumoniae CPn1032 protein is expressed in E. coli as a mixture of uncleaved π and α and β subunits produced by self-cleavage. Lane M, protein markers with masses shown at the left; lane 1, affinity-purified His₁₀-CPn1032; lane 2, affinity-purified His₁₀-CPn1032-Thr⁵²Ser variant.

FIG. 4.

Purified CPn1032 protein catalyzes l-arginine decarboxylation optimally at pH 3.4 (open boxes). The Thr⁵²Ser variant protein has the same pH optimum for l-arginine decarboxylation (closed circles) but a lower specific activity due to the reduced cleavage shown in Fig. 3. Activity assays were performed as described in Materials and Methods, and data were fit to polynomial functions.

FIG. 5.

Temperature dependence of CPn1032 arginine decarboxylase activity. Purified protein has maximal activity at 47°C but retains significant activity up to 80°C.

FIG. 6.

Expression of CPn1032 complements the adiA mutation in E. coli DEG0121. (A) Mean arginine decarboxylase specific activities of cell extracts from five E. coli strains: DEG0121 (ΔadiA2::kan) (adiA bars), DEG0121 carrying pBAD/HisA (as a vector control), DEG0121 carrying pDG339 (expressing CPn1032), DEG0121 carrying pDG352 (expressing adiA), and MG1655 wild-type cells. Standard deviations are indicated for each strain (n = 3). Decarboxylase activity and total protein assays were performed as described in Materials and Methods. (B) Arginine-dependent acid shock survival rates for the same E. coli strains after 1 h (dark-gray bars) or 2 h (light-gray bars) of incubation at pH 2.5. The mean survival rates and sample standard deviations are shown for each sample (n = 3). Assays were performed with or without 1.5 mM l-arginine (Arg), as indicated. Conditions for cell growth and acid shock are described in the text. Survival rates were less than 0.3% for the adiA mutant and adiA mutant pBAD/HisA strains, as well as in the assays without arginine.