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. Expressed sequence tag analysis of the deuteromycete Metarhizium anisopliae identified two trypsins (family S1) and a novel chymotrypsin (CHY1). CHY1 resembles actinomycete (bacterial) chymotrypsins (family S2) rather than other eukaryote enzymes (family S1) in being synthesized as a precursor species (374 amino acids, pI/MW: 5.07/38,279) containing a large N-terminal fragment (186 amino acids). Chy1 was expressed in Pichia pastoris yielding an enzyme with a chymotrypsin specificity for branched aliphatic and aromatic C-terminal amino acids. This is predictable as key catalytic residues determining the specificity of Streptomyces griseus chymotrypsins are conserved with CHY1. Mature (secreted) CHY1 (pI/MW: 8.29/18,499) shows closest overall amino acid identity to S. griseus protease C (55%) and clustered with other secreted bacterial S2 chymotrypsins that diverged widely from animal and endocellular bacterial enzymes in phylogenetic trees of the chymotrypsin superfamily. 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 lack chymotrypsin homologs. In light of this patchy distribution, we conclude that chy1 probably arose by lateral gene transfer from an actinomycete bacterium.
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Department of Entomology, University of Maryland, College Park, Maryland 20742, USA.
St Leger
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RJ
D000192:Actinomycetales / Q000201:enzymology / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D002918:Chymotrypsin / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D020224:Expressed Sequence Tags; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics*; D005658:Fungi / Q000201:enzymology*; D018014:Gene Transfer Techniques; D008969:Molecular Sequence Data; D010843:Pichia; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D013379:Substrate Specificity
D005656:Fungal Proteins; D011994:Recombinant Proteins; D002918:Chymotrypsin