The First Agmatine/Cadaverine Aminopropyl Transferase: Biochemical and Structural Characterization of an Enzyme Involved in Polyamine Biosynthesis in the Hyperthermophilic Archaeon Pyrococcus furiosus

Abstract

We report here the characterization of the first agmatine/cadaverine aminopropyl transferase (ACAPT), the enzyme responsible for polyamine biosynthesis from an archaeon. The gene PF0127 encoding ACAPT in the hyperthermophile Pyrococcus furiosus was cloned and expressed in Escherichia coli, and the recombinant protein was purified to homogeneity. P. furiosus ACAPT is a homodimer of 65 kDa. The broad substrate specificity of the enzyme toward the amine acceptors is unique, as agmatine, 1,3-diaminopropane, putrescine, cadaverine, and sym-nor-spermidine all serve as substrates. While maximal catalytic activity was observed with cadaverine, agmatine was the preferred substrate on the basis of the k cat /K m value. P. furiosus ACAPT is thermoactive and thermostable with an apparent melting temperature of 108°C that increases to 112°C in the presence of cadaverine. Limited proteolysis indicated that the only proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is not necessary for the integrity of the active site. The crystal structure of the enzyme determined to 1.8-Å resolution confirmed its dimeric nature and provided insight into the proteolytic analyses as well as into mechanisms of thermal stability. Analysis of the polyamine content of P. furiosus showed that spermidine, cadaverine, and sym-nor-spermidine are the major components, with small amounts of sym-nor-spermine and N-(3-aminopropyl)cadaverine (APC). This is the first report in Archaea of an unusual polyamine APC that is proposed to play a role in stress adaptation.Propylamine transferases represent a class of enzymes catalyzing the transfer of a propylamine group from S-adenosyl-(5Ј)-3-methylthiopropylamine (decarboxy-AdoMet) to various amine acceptors (7,39,48,60), according to the following reaction: decarboxy-AdoMet ϩ polyamine substrate 3 5Ј-methylthioadenosine ϩ polyamine product. The reaction involves a nucleophilic attack on the C-3 methylene of the propylamine moiety adjacent to the positively charged sulfur of decarboxy-AdoMet (7,39,48,60).In 1958, Tabor et al. (49) provided the first evidence that decarboxy-AdoMet is the donor of aminopropyl groups in the biosynthesis of spermidine in cell extracts of Escherichia coli. The enzyme catalyzing this reaction was subsequently purified to homogeneity by Bowman et al. (4). The development of rapid and sensitive methods of polyamine analysis, together with the utilization of affinity chromatography, allowed the purification to homogeneity and a complete characterization of spermine and spermidine synthases from bovine brain (36) and rat ventral prostate (41), respectively. Moreover, a propylamine transferase with unusual molecular and kinetic properties has been purified to homogeneity from Sulfolobus solfataricus (S. solfataricus aminopropyl transferase [APT]), an extreme thermophilic archaeon (8). Recently, recombinant forms of the spermidine synthases of the extremely thermophilic bacterium Thermotoga maritima (T. maritima PAPT) (28) and of the human malar...

Identification of a Novel Aminopropyltransferase Involved in the Synthesis of Branched-Chain Polyamines in Hyperthermophiles

“…One group (group A) includes the thermophilic T. kodakarensis BpsA orthologs (PF1111 from P. furiosus, Metig0730 from Methanotorris igneus, and TTHC0171 from T. thermophilus). A second group (group B) consists of the TK0147 orthologs, including E. coli SpeE (43) and P. furiosus PF0127 (48). Group C consists of several other aminopropyltransferases, which act as thermospermine synthases, including PAE1203 from Pyrobaculum aerophilum (49), Hbut0057 and Hbut0383 from Hyperthermus butylicus (49), and At5g19530 from Arabidopsis thaliana (44).…”

Binding and inhibition of human spermidine synthase by decarboxylated S‐adenosylhomocysteine

“…Some aminopropyltransferases such as human spermidine synthase (SpdS) (which acts upon putrescine) and spermine synthase (SpmS) (acting on spermidine) are highly specific for their amine acceptors, [12][13][14] while others, such as those from acute thermophiles, which contain a variety of polyamines not found in mammals, are less discriminating. 12,13,[15][16][17] There are now numerous published structures for aminopropyltransferases including those for SpdS from Thermotoga maritima (TmSpdS), 16 Caenorhabditis elegans, 18 Plasmodium falciparum (PfSpdS), 19 Helicobacter pylori, 20 human (hSpdS), 13 Arabidopsis thaliana (PDB code 2Q41), and Trypanosoma cruzi (PDB code 3BWC), aminopropylagmatine/aminopropylcadaverine synthases from Thermus thermophilus (PDB code 1UIR), Pyrococcus horikoshii (PDB code 2ZSU) and Pyrococcus furiosus, 15 and SpmS from humans (hSpmS). 14 A general mechanism for aminopropyl transfer has been proposed based on kinetic studies of hSpdS, TmSpdS, and hSpmS, their structures with bound substrates and inhibitors, and the results of site-directed mutagenesis of key residues (Fig.…”

Crenarchaeal Arginine Decarboxylase Evolved from an S-Adenosylmethionine Decarboxylase Enzyme

“…In that bacterium, PAT transfers a propylamine group to agmatine producing N 1 -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).…”

“…The complete genome sequences of 13 thermophilic crenarchaea encode orthologs of propylamine transferase (7) and two homologs of the archaeal type AdoMetDC (supplemental Table 1) (8). However, none of the genomes encodes a recognizable ornithine decarboxylase enzyme to produce putrescine.…”