A barley polyamine oxidase isoform with distinct structural features and subcellular localization

Two cDNAs encoding polyamine oxidase (PAO) isoforms (BPAO1 and BPAO2) and t he corresponding gene copies were isolated from barley cultivar Aura. Gene organization is not conserved between these two nonallelic coding sequences . Both precursor proteins include a cleavable N-terminal leader of 25 amino acids. N-terminal sequencing of PAO purified from barley seedlings reveals a unique amino-acid sequence corresponding to the BPAO2 N-terminus as pred icted from the corresponding cDNA. BPAO2 has been purified, characterized a nd compared to maize PAO (MPAO), the best characterized member of this enzy me class. The two proteins show different pH optima for catalytic activity, K-m and V-max values with spermidine and spermine as substrates. Molecular modelling of BPAO2 reveals the same global fold as in MPAO. However, subst itution of the active site residue Phe403 by a tyrosine, provides a rationa le for the different catalytic properties of the two enzymes. In barley lea ves PAO-specific activity is higher in isolated mesophyll protoplasts than in the extracellular fluids, whereas in maize the reverse is true. The C-te rminus of BPAO2 shows homology with the endoplasmic reticulum retention sig nal that might be responsible for the subcellular localization observed. We conclude that BPAO2 is a symplastic PAO in barley mesophyll cells. Product ion of BPAO2 mRNA and the corresponding protein is induced by light, and ha s a different pattern of accumulation in leaves and coleoptiles.