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

Author:
Cervelli, Manuela, Cona, Alessandra, Angelini, Riccardo, Polticelli, Fabio, Federico, Rodolfo, Mariottini, Paolo
Source:
European journal of biochemistry 2001 v.268 no.13 pp. 3816-3830
ISSN:
0014-2956
Subject:
active sites, amino acid sequences, barley, catalytic activity, coleoptiles, complementary DNA, corn, cultivars, endoplasmic reticulum, enzymes, extracellular fluids, genes, leaves, mesophyll, messenger RNA, models, pH, proteins, protoplasts, seedlings, spermidine, spermine, tyrosine
Abstract:
Two cDNAs encoding polyamine oxidase (PAO) isoforms (BPAO1 and BPAO2) and the 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 predicted from the corresponding cDNA. BPAO2 has been purified, characterized and compared to maize PAO (MPAO), the best characterized member of this enzyme class. The two proteins show different pH optima for catalytic activity, Km and Vmax values with spermidine and spermine as substrates. Molecular modelling of BPAO2 reveals the same global fold as in MPAO. However, substitution of the active site residue Phe403 by a tyrosine, provides a rationale for the different catalytic properties of the two enzymes. In barley leaves PAO‐specific activity is higher in isolated mesophyll protoplasts than in the extracellular fluids, whereas in maize the reverse is true. The C‐terminus of BPAO2 shows homology with the endoplasmic reticulum retention signal that might be responsible for the subcellular localization observed. We conclude that BPAO2 is a symplastic PAO in barley mesophyll cells. Production of BPAO2 mRNA and the corresponding protein is induced by light, and has a different pattern of accumulation in leaves and coleoptiles.
Agid:
177848