Crenarchaeal arginine decarboxylase evolved from an S-adenosylmethionine decarboxylase enzyme

Abstract

The crenarchaeon Sulfolobus solfataricus uses arginine to produce putrescine for polyamine biosynthesis. However, genome sequences from S. solfataricus and most crenarchaea have no known homologs of the previously characterized pyridoxal 5'-phosphate or pyruvoyl-dependent arginine decarboxylases that catalyze the first step in this pathway. Instead they have two paralogs of the S-adenosylmethionine decarboxylase (AdoMetDC). The gene at locus SSO0585 produces an AdoMetDC enzyme, whereas the gene at locus SSO0536 produces a novel arginine decarboxylase (ArgDC). Both thermostable enzymes self-cleave at conserved serine residues to form amino-terminal beta-domains and carboxyl-terminal alpha-domains with reactive pyruvoyl cofactors. The ArgDC enzyme specifically catalyzed arginine decarboxylation more efficiently than previously studied pyruvoyl enzymes. alpha-Difluoromethylarginine significantly reduced the ArgDC activity of purified enzyme, and treating growing S. solfataricus cells with this inhibitor reduced the cells' ratio of spermidine to norspermine by decreasing the putrescine pool. The crenarchaeal ArgDC had no AdoMetDC activity, whereas its AdoMetDC paralog had no ArgDC activity. A chimeric protein containing the beta-subunit of SSO0536 and the alpha-subunit of SSO0585 had ArgDC activity, implicating residues responsible for substrate specificity in the amino-terminal domain. This crenarchaeal ArgDC is the first example of alternative substrate specificity in the AdoMetDC family. ArgDC activity has evolved through convergent evolution at least five times, demonstrating the utility of this enzyme and the plasticity of amino acid decarboxylases.

FIGURE 1.

Proposed pathway for polyamine biosynthesis in S. solfataricus. AdoMetDC (EC 4.1.1.50) catalyzes dcAdoMet formation. ArgDC (EC 4.1.1.19) produces agmatine and CO₂ from l-arginine. Agmatinase (AUH; EC 3.5.3.11) catalyzes the hydrolysis of agmatine to produce urea and putrescine. Finally, a propylamine transferase enzyme (PAT) transfers one or two aminopropyl groups from dcAdoMet to putrescine, producing spermidine or spermine. The same enzyme transfers aminopropyl groups to 1,3-diaminopropane, producing sym-norspermidine and sym-norspermine. Alternatively, it is possible S. solfataricus uses the N¹-aminopropylagmatine pathway that was described for Thermus thermophilus (41). In that bacterium, PAT transfers a propylamine group to agmatine producing N¹-aminopropylagmatine, which can be hydrolyzed to form spermidine by agmatinase. However, both the S. solfataricus and Pyrococcus furiosus PAT proteins efficiently use putrescine or diaminopropane as substrates (6, 7). The S. solfataricus agmatinase protein is more similar to the Pyrococcus horikoshii agmatinase, which hydrolyzes agmatine, than to the T. thermophilus homolog, which does not hydrolyze agmatine (9). Significant amounts of putrescine were detected in S. solfataricus extracts, supporting the canonical model shown here.

FIGURE 2.

Protein sequence alignment of the S. solfataricus ArgDC protein (SSO0536) with a putative ArgDC from Aeropyrum pernix (APE0079; NP_146956.1), AdoMetDC from S. solfataricus (SSO0585), AdoMetDC from Bacillus subtilis (BsAdoMetDC; NP_390779.1), AdoMetDC from M. jannaschii (MjAdoMetDC; NP_247288.1), and AdoMetDC from T. maritima (TmAdoMetDC; NP_228464.1). The secondary structure of TmAdoMetDC is indicated as β-strands and α-helices below the alignment (from PDB 1VR7) (29). An arrow indicates the site of protein self-cleavage and pyruvoyl group formation at the conserved serine residue of the nascent α-subunit. Asterisks above the alignment indicate positions with amino acid replacements that correlate with substrate specificity. Sequences were aligned using the T-COFFEE program (version 4.96) (23).

FIGURE 3.

Proenzymes (π) of the heterologously expressed His₁₀-SSO0356 and His₁₀-SSO0585 proteins self-cleave to form amino-terminal subunits (β) and carboxyl-terminal subunits (α) with reactive pyruvoyl groups. Affinity-purified proteins were separated by SDS-PAGE and stained with silver. Lane M, polypeptide marker corresponding to the indicated molecular masses; lane 1, 0.9 μgofHis₁₀-SSO0356; lane 2, 1.1 μgof His₁₀-SSO0585; lane 3, 18 μg of His₁₀-β536α585 chimera; lane 4, 9 μg of His₁₀-β585α536 chimera.

FIGURE 4.

The SSO0585 protein has AdoMet decarboxylase activity. Reactions containing M. jannaschii AdoMet synthetase (MAT), ATP, l-[1-¹⁴C]methionine (Met), and buffer salts were preincubated for 30 min at 70 °C before the addition of decarboxylase (DCase), as indicated above the chart. Reactions 1–3 were control reactions omitting AdoMet synthetase and decarboxylase enzymes, ATP, or decarboxylase, respectively. Reaction 4 contained 1 μg of His₁₀-SSO0536 protein, and reaction 5 contained 1 μg of His₁₀-SSO0585 protein. Reaction 6 contained 1 μg of His₁₀-SSO0536 protein but not AdoMet synthetase as a control reaction. Reaction 7 contained 5 μg of His₁₀-β536α585 chimeric protein, and reaction 8 contained 5 μg of His₁₀-β585α536 chimeric protein. Decarboxylase activities are shown with their S.D. values from triplicate experiments.

FIGURE 5.

Arginine decarboxylase activity of the His₁₀-SSO0536 protein. A, the purified enzyme has optimal activity at pH 6, consistent with a biosynthetic function. B, the protein has maximal arginine decarboxylase activity at 80 °C. C, the protein retained 80% arginine decarboxylase activity after preincubation at 90 °C for 10 min.

FIGURE 6.

Phylogeny of the paralogous crenarchaeal AdoMet and arginine decarboxylases, rooted using bacterial and euryarchaeal AdoMetDC sequences. This tree is consistent with an early gene duplication event in the crenarchaeal lineage, leading to the evolution of arginine decarboxylase activity. The phylogeny was inferred from an alignment of protein sequences using the protein maximum likelihood method. The scale bar indicates one amino acid substitution per 10 positions. Bootstrap values are shown near branches supported by a plurality of 100 trees. Another tree inferred by the protein distance and neighbor joining methods produced a similar topology. Enzymes whose functions have been experimentally confirmed are indicated by an asterisk. Boldface labels indicate the predicted functions of the two protein subfamilies. Full names of the organisms and their sequence accession numbers are listed in the supplemental materials.