Cloning, expression, and substrate specificity of a fungal chymotrypsin - Evidence for lateral gene transfer from an actinomycete bacterium

Unlike trypsins, chymotrypsins have not until now been found in fungi. Expr essed sequence tag analysis of the deuteromycete Metarhizium anisopliae ide ntified two trypsins (family S1) and a novel chymotrypsin (CHY1), CHY1 rese mbles actinomycete (bacterial) chymotrypsins (family S2) rather than other eukaryote enzymes (family S1) in being synthesized as a precursor species ( 374 amino acids, pI/MW: 5.07/88,279) containing a large N-terminal fragment (186 amino acids). Chy1 was expressed in Pichia pastoris yielding an enzym e with a chymotrypsin specificity for branched aliphatic and aromatic C-ter minal amino acids, This is predictable as key catalytic residues determinin g the specificity of Streptomyces griseus chymotrypsins are conserved with CHY1, Mature (secreted) CHY1 (pI/MW: 8.29/18,499) shows closest overall ami no acid identity to S. griseus protease C (55%) and clustered with other se creted bacterial S2 chymotrypsins that diverged widely from animal and endo cellular bacterial enzymes in phylogenetic trees of the chymotrypsin superf amily. Conversely, actinomycete chymotrypsins are much more closely related to fungal proteases than to other eubacterial sequences. Complete genomes of yeast, gram eubacteria, archaebacteria, and mitochondria do not contain paralogous genes. Expressed sequence tag data bases from other fungi also l ack chymotrypsin homologs, In light of this patchy distribution, we conclud e that chy1 probably arose by lateral gene transfer from an actinomycete ba cterium.