Identification, biochemical characterization, and evolution of the Rhizopus oryzae 99-880 polygalacturonase gene family.

A search of the recently sequenced Rhizopus oryzae strain 99-880 genome database uncovered 18 putative polygalacturonase genes with two genes being identical and only one with similarity to a previously reported R. oryzae polygalacturonase gene. The 17 different genes share 50% to greater than 90% identity at the nucleotide level as well as the deduced protein sequence level. The cDNA of the different genes was isolated directly or recombinantly and used to express the encoded proteins in Pichia pastoris. Recombinant protein expression demonstrated that 15 of the 17 genes encode active enzymes with twelve genes encoding for endo-polygalacturonase enzymes and three genes encoding for exo-polygalacturonase enzymes. Phylogenetic analysis indicates that the genes form a distinct monophyletic group among fungal polygalacturonase enzymes. Finally, our results also suggest that the ancestral form of polygalacturonase in fungi is endolytic and exolytic function evolved later, at least two independent times.