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 degrees C that increases to 112 degrees 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-A 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.

FIG. 1.

Determination of K_m values for 1,3-diaminopropane and putrescine (a), cadaverine (b), and agmatine (c). (d) Chemical structures of the polyamines tested. The enzyme was incubated for 10 min at 70°C in the presence of 100 μM decarboxy-AdoMet and various concentrations of 1,3-diaminopropane (▴), putrescine (•), cadaverine (▪), and agmatine (○), as shown. The amount of the polyamine product was determined by radiochemical or HPLC assay, as described in Materials and Methods. Results are shown as a plot of V_max (V) against the V_max/[substrate] ratio (V/[S]).

FIG. 2.

The effect of temperature on P. furiosus ACAPT activity and stability. (a) The activity observed at 90°C is expressed as 100%. The assay was performed as described in Materials and Methods. Arrhenius plot is reported in the inset. Temperature (T) is measured in kelvins (K). (b) Residual activity after 10-min incubation at temperatures shown in the absence (▪) or in the presence (▴) of 5 mM cadaverine. Apparent T_m values are reported in the insets.

FIG. 3.

Multiple sequence alignment of APTs from different sources. Sequences: TmPAPT, spermidine synthase from Thermotoga maritima; PfACAPT, ACAPT from Pyrococcus furiosus; SsAPT, APT from Sulfolobus solfataricus; EcSpdS, spermidine synthase from E. coli; HsSpdS, spermidine synthase from Homo sapiens; PFSpdS, spermidine synthase from Plasmodium falciparum. Identical and conserved residues in proteins are highlighted in dark and pale gray, respectively. The amino acid residues of T. maritima PAPT involved in the binding with AdoDATO are indicated (▴).

FIG. 4.

Structural features of ACAPT. (a) A stereo view of the ACAPT dimer perpendicular to the dimer axis. The arrow shows the position of the probable proteolytic cleavage site. (b) A cross-eyed stereo view showing the 1.8-Å (Fo-Fc omit) electron density map contoured at 3σ for residues 200 to 212 (helix αF), which were omitted from the REFMAC refinement. (c) Connectivity diagram (residues in parentheses): β1 (5 to 9), β2 (13 to 16), β3 (19 to 27), β4 (32 to 38), β5 (43 to 47), β6 (50 to 54), αA (58 to 73), β7 (79 to 83), αB (87 to 94), β8 (100 to 105), αC (107 to 118), αC’ (123 to 130), β9 (136 to 140), αD (142 to 150), β10 (153 to 159), αE (172 to 182), β11 (183 to 195), αF (199 to 214), β12 (216 to 222), β13 (231 to 238), αG (247 to 253), αH (260 to 269), and αI (271 to 279). (d) A view of the ACAPT binding pocket (cyan, residues Q31, L47, Q52, Y61, H62, G83, G84, D86, E105, I106, D142, D159, S160, T161, D162, P166, L170, Y227, and W231) with the T. maritima PAPT binding pocket (green, residues Q46, L62, M67, Y76, H77, G98, G99, D101, E121, V122, N152, D170, S171, T172, D173, Q178, L182, Y239, and W244) and bound substrate analogue (black) superimposed. Starred residues Tyr227 and Trp231 are lost upon proteolysis.