Purification and characterization of propylamine transferase from Sulfolobus solfataricus, an extreme thermophilic archaebacterium

Abstract

The enzyme propylamine transferase, catalyzing the transfer of the propylamine moiety from S-adenosyl(5')-3-methylthiopropylamine to several amine acceptors, has been purified 643-fold in 20% yield from Sulfolobus solfaturicus, an extreme thermophilic archaebacterium optimally growing at 87 "C. The purified enzyme (specific activity 2.05 units/mg protein), is homogeneous by criteria of gel electrophoresis, gel filtration, isoelectric focusing and ultracentrifugation analysis. The molecular mass of the native enzyme was estimated to be about 110 kDa by gel permeation and ultracentrifugation analysis. The protein migrates on SDS/polyacrylamide gel electrophoresis as a single band of 35 kDa, suggesting that the enzyme is a trimer composed by identical subunits. An optimum pH of 7.5 and an acidic isoelectric point of 5.3 have been calculated. The optimum temperature was 90°C and no loss of activity is observable even after exposure of the purified enzyme to 100°C for 1 h. No reducing agents are required for enzymatic activity.Substrate specificity towards the amine acceptors is rather broad in that 1,3-diaminopropane ( K , = 1675 pM), putrescine (K, = 3850 pM), sym-norspermidine (K, = 954 pM) and spermidine (K, = 1539 pM) are recognized as substrates. Conversely S-adenosyl(5')-3-methylthiopropylamine is the only propylamine donor (K, = 7.9 pM) and the deamination of the sulfonium compound prevents the recognition by the enzyme. The reaction is irreversible and initial-rate kinetic studies indicate that the propylamine transfer is operated through a sequential mechanism. 5'-Methylthioadenosine, a product of the reaction, acts as a powerful competitive inhibitor with a Ki of 3.7 pM. Enzyme-substrate binding sites have been investigated with the aid of several substrate analogs and products. Among the compounds assayed, 5'-methylthiotubercidin, S-adenosyl(5')-3-thiopropylamine and S-adenosyl-3-thio-l,8-diaminooctane are the most active inhibitors.Propylamine transferases represent a group of enzymes catalyzing the transfer of the propylamine moiety from S-adenosyl-(5')-3-methylthiopropylamine (decarboxy-AdoMet) to appropriate amine acceptors [l -41. The reaction involves a nucleophilic attack on the methylene C-3 adjacent to the positively charged sulfur of decarboxy-AdoMet [5]. This class of enzymes is distributed in both prokaryotic and eukaryotic organisms. The recent development of rapid and sensitive methods for polyamine analysis [6 -81 and the utilization of affinity chromatography [9, 101 has made possible their purification and characterization : indeed spermidine Correspondence to V. Zappia, Istituto di Biochimica delle Macromolecole, I Facolta di Medicina e Chirurgia, Universita di Napoli, Via Costantinopoli 16, 1-80138, Napoli, ItalyAbbreviations. Decarboxy-AdoMet, S-adenosyl(S')-3-methylthiopropylamine; sym-norspermidine, 1,7-diamino-4-aza-eptane; symnorspermine, 1,l l-diamino-4,8-diaza-undecane; sym-homospermidine, 1,9-diamin0-5-aza-nonane; caldopentamine, 1,15-diamino- synthase has been purified from ra...

Effect of temperature on the propylamine transferase from Sulfolobus solfataricus, an extreme thermophilic archaebacterium

“…The fifth equation can be obtained assuming that the enzyme possesses its maximum stability at a temperature near its maximum activity. It has been reported (Cacciapuoti et al, 1986) that propylamine transferase shows its activity maximum at about 87'C. Thus, we assume that form B, which is populated above 50 ' C , has its stability maximum at this temperature.…”

“…This is in agreement with and confirms the picture of the enzyme shown in the preceding paper (Facchiano et al, 1992). The structural reorganization at high temperature not only allows a better catalytic efficiency (Cacciapuoti et al, 1986), but also increases the enzyme stability. From Fig.…”

“…Propylamine transferase from Sulfolobus sowataricus was prepared according to Cacciapuoti et al (1986). Protein purity was assessed as described in the preceding paper (Facchiano et al, 1992).…”

“…The protein is a trimer of three identical 35-kDa subunits under physiological conditions, i. e. over 25-90'C (Cacciapuoti et al, 1986). It shows an unusual resistance, both structural and functional, to high temperatures (Cacciapuoti et al, 1986).…”

“…Propylamine transferase is an oligomeric enzyme from the extreme thermophilic archaebacterium Su@lobus solfataricus (Cacciapuoti et al, 1986). The protein is a trimer of three identical 35-kDa subunits under physiological conditions, i. e. over 25-90'C (Cacciapuoti et al, 1986).…”