Putrescine Aminopropyltransferase Is Responsible for Biosynthesis of Spermidine, Spermine, and Multiple Uncommon Polyamines in Osmotic Stress-Tolerant Alfalfa

Abstract

The biosynthesis of polyamines from the diamine putrescine is not fully understood in higher plants. A putrescine aminopropyltransferase (PAPT) enzyme activity was characterized in alfalfa (Medicago sativa L.). This enzyme activity was highly specific for putrescine as the initial substrate and did not recognize another common diamine, 1,3-diaminopropane, or higher-molecular-weight polyamines such as spermidine and spermine as alternative initial substrates. The enzyme activity was inhibited by a general inhibitor of aminopropyltransferases, 5'-methylthioadenosine, and by a specific inhibitor of PAPTs, cyclohexylammonium sulfate. The initial substrate specificity and inhibition characteristics of the enzyme activity suggested that it is a classical example of a PAPT. However, this enzyme activity yielded multiple polyamine products, which is uncharacteristic of PAPTs. l h e major reaction product of PAPT activity i n alfalfa was spermidine. The next most abundant products of the enzyme reaction using putrescine as the initial substrate included the tetramines spermine and thermospermine. These two tetramines were distinguished by thin-layer chromatography to be distinct reaction products exhibiting differential rates of formation. In addition, the uncommon polyamines homocaldopentamine and homocaldohexamine were tentatively identified as minor enzymatic reaction products but only in extracts prepared from osmotic stresstolerant alfalfa cultivars. PAPT activity from alfalfa was highest i n meristematic shoot tip and floral bud tissues and was not detected in older, nonmeristematic tissues. Product inhibition of the enzyme activity was observed after spermidine was added into the i n vitro assay for alfalfa PAPT activity. A biosynthetic pathway is proposed that accounts for the characteristics of this PAPT activity and accommodates a nove1 scheme by which certain uncommon polyamines are produced in plants.The diamine putrescine and the polyamines spermidine and spermine are ubiquitous components of living organisms and are essential primary metabolites for normal growth and development (Flores et al., 1989). These three compounds are often referred to as common polyamines, and polyamines with a restricted natural occurrence may

A Polyamine Metabolon Involving Aminopropyl Transferase Complexes in Arabidopsis

“…It has been suggested that plant SPDS may occur in the form of a multienzyme complex (Bagga et al, 1997). Thus, characterization of SPDS in crude alfalfa enzyme extracts showed that although alfalfa SPDS was highly specific for putrescine as the initial substrate, extended enzymatic reaction produced, besides spermidine, spermine and several uncommon polyamines (Bagga et al, 1997).…”

“…It also prevents the loss or dilution of substrate by diffusion and protects chemically labile intermediates (Ovadi and Srere, 2000). An early report on the purification of SPDS from alfalfa suggested that this enzyme might produce different polyamines, including spermine and other uncommon compounds, in addition to spermidine (Bagga et al, 1997). The fact that the different products were produced at different rates indicated that these reactions cannot be catalyzed by a single SPDS enzyme; most likely, an enzyme complex is required.…”

Polyamines in Eukaryotes, Bacteria, and Archaea

“…2) as a co-product of the oxidation of Spd (47). However, the alfalfa aminopropyltransferase did not recognize Dap as a substrate, although it was able to produce Spd, Spm, and Tspm from Put (48). Production of Nspd in eukaryotes therefore was a mystery until very recently, when it was shown that a plant polyamine oxidase produces Nspd as a catabolic product from Tspm (49).…”

Heterologous Expression and Biochemical Characterization of a Polyamine Oxidase from Arabidopsis Involved in Polyamine Back Conversion

“…Furthermore, uncommon polyamines, such as norspermidine (Nor-Spd), homospermidine, norspermine (Nor-Spm), homospermine, thermospermine, caldopentamine, caldohexamine, homocaldopentamine, and homocaldohexamine, are abundant in the extreme thermophilic bacterium Thermus thermophilus and have also been detected in bacteria, algae, fungi, animals, and higher plants (Cohen, 1998). In plants, a putative role for these molecules in growth, differentiation, and stress tolerance has been hypothesized (RodriguezGaray et al, 1989;Bagga et al, 1997;Koc et al, 1998).…”