Abstract
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.
Highlights
Unlike trypsins, chymotrypsins have not until now been found in fungi
Chy1 was expressed in Pichia pastoris yielding an enzyme with a chymotrypsin specificity for branched aliphatic and aromatic C-terminal amino acids
Cloning and DNA Sequencing of Chy1 and Two Trypsins Try1 and Try2—Total RNA from M. anisopliae cultures grown in the presence of 1% cockroach cuticle for 24 h was isolated with Tri-reagent (Sigma) from finely homogenized fungal mycelia as described previously [10, 11] and used to construct a cDNA library in the unidirectional ZAP vector (Stratagene)
Summary
Expressed sequence tag data bases from other fungi lack chymotrypsin homologs In light of this patchy distribution, we conclude that chy probably arose by lateral gene transfer from an actinomycete bacterium. Proteases of the chymotrypsin superfamily, which includes the pancreatic chymotrypsins, trypsins, and elastases (chymotrypsin family S1) and the bacterial lytic endopeptidases (chymotrypsin family S2) possess an active site serine that attacks the scissile peptide bond [1] Such stereotypical chymotrypsin superfamily proteases as chymotrypsin itself and protease A (Streptomyces griseus) are synthesized as preproenzymes and are secreted to act extracellularly, endocellular forms are known. Finding that the fungal sequence falls between the two classes of organisms, Rypniewski et al [5, 6] suggested that no interspecies gene transfer is necessary to explain the homology between trypsins and posited a continuos evolutionary divergence from a common ancestor of prokaryotes and eukaryotes. Fungal Chymotrypsin fer from bacteria that actively secrete proteases may have played a major role in the evolution of secreted (digestive) proteases of some fungi and perhaps higher eukaryotes
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