The sequence and X-ray structure of the trypsin from <i>Fusarium oxysporum</i>

Rypniewski, W.; Hastrup, Sven; Betzel, C.; Dauter, Mirosława; Dauter, Z.; Papendorf, Gerlind; Branner, Sven; Wilson, K.S.

doi:10.1093/protein/6.4.341

Cited by 47 publications

(29 citation statements)

References 3 publications

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“…The closure abrogates Na ϩ binding, but isolates D189 from the apparently detrimental effects of a three-way channel shaped as in the Y225I mutant of thrombin. This correlation is reinforced by the crystal structure of the trypsin from Fusarium oxysporum (30), carrying Ser at position 225. In this structure, the carbonyl O atom of residue 224 is oriented as in the proteases with P225 (6) and the channel is shaped as in the Y225P mutant of thrombin.…”

Section: Resultsmentioning

confidence: 92%

Unexpected crucial role of residue 225 in serine proteases

Guinto

Caccia

Rose

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

122

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Residue 225 in serine proteases of the chymotrypsin family is Pro or Tyr in more than 95% of nearly 300 available sequences. Proteases with Y225 (like some blood coagulation and complement factors) are almost exclusively found in vertebrates, whereas proteases with P225 (like degradative enzymes) are present from bacteria to human. Saturation mutagenesis of Y225 in thrombin shows that residue 225 affects ligand recognition up to 60,000-fold. With the exception of Tyr and Phe, all residues are associated with comparable or greatly reduced catalytic activity relative to Pro. The crystal structures of three mutants that differ widely in catalytic activity (Y225F, Y225P, and Y225I) show that although residue 225 makes no contact with substrate, it drastically inf luences the shape of the water channel around the primary specificity site. The activity profiles obtained for thrombin also suggest that the conversion of Pro to Tyr or Phe documented in the vertebrates occurred through Ser and was driven by a significant gain (up to 50-fold) in catalytic activity. In fact, Ser and Phe are documented in 4% of serine proteases, which together with Pro and Tyr account for almost the entire distribution of residues at position 225. The unexpected crucial role of residue 225 in serine proteases explains the evolutionary selection of residues at this position and shows that the structural determinants of protease activity and specificity are more complex than currently believed. These findings have broad implications in the rational design of enzymes with enhanced catalytic properties.

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Section: Resultsmentioning

confidence: 92%

Unexpected crucial role of residue 225 in serine proteases

Guinto

Caccia

Rose

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

122

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“…Crystals were grown as described elsewhere (15), with the pH lowered to 5.0 instead of 6.0 using 100 mM sodium citrate buffer. A combination of streak seeding and macroseeding was applied to initiate nucleation.…”

Section: Methodsmentioning

confidence: 99%

Trypsin Revisited

Schmidt

Jelsch

Østergaard

et al. 2003

Journal of Biological Chemistry

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A series of crystal structures of trypsin, containing either an autoproteolytic cleaved peptide fragment or a covalently bound inhibitor, were determined at atomic and ultra-high resolution and subjected to ab initio quantum chemical calculations and multipole refinement. Quantum chemical calculations reproduced the observed active site crystal structure with severe deviations from standard stereochemistry and indicated the protonation state of the catalytic residues. Multipole refinement directly revealed the charge distribution in the active site and proved the validity of the ab initio calculations. The combined results confirmed the catalytic function of the active site residues and the two water molecules acting as the nucleophile and the proton donor. The crystal structures represent snapshots from the reaction pathway, close to a tetrahedral intermediate. The de-acylation of trypsin then occurs in true S N 2 fashion.

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“…However, examination of all available data bases, including those of the most gene-rich mitochondrial DNA known (62 protein-encoding genes in Reclinomonas americana) (33), the Rickettsia genome, the Rhodobacter (free living ␣-proteobacteria) genome, the Bacillus subtilis (grampositive bacteria) genome, and various archaeobacterium genomes revealed no non-actinomycete eubacterial or archaeobacterium genes that could be paralogous to the metallocarboxypeptidase (7), chymotrypsins, and trypsins found in actinomycetes and eukaryotes. The argument of Rypniewski et al (5,6) that animal, fungal, and Streptomyces trypsins are derived from gene divergence predating the prokaryote/eukaryote split does not explain the patchy and very narrow distribution of these enzymes among fungi and bacteria. Plant and insect pathogenic members of the Ascomycotina and actinomycete bacteria are the only known microbial sources of S1 trypsins.…”

Section: Discussionmentioning

confidence: 99%

“…Obtaining sequence data from a trypsin from a lower eukaryote, the fungus Fusarium oxysporum, made it possible to reassess the relationship between the microbial and mammalian trypsins. 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. However, recently we cloned a class of metallocarboxypeptidase from Metarhizium anisopliae, not found previously in fungi, that also fell between Streptomyces and animal sequences and had only distant homologs in other bacteria (7).…”

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confidence: 99%

Cloning, Expression, and Substrate Specificity of a Fungal Chymotrypsin

Screen

Leger

2000

Journal of Biological Chemistry

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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.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, although endocellular forms are also known. They play a role in cancer and other diseases, and they have a diverse array of important functions, including digestion and degradative processes, blood coagulation, fibrinolysis, cellular and humoral immunity, embryonic development, and fertilization (2), that underscore the need to better understand how these enzymes have become adapted to diverse activities. Most chymotrypsin-like enzymes and their associated functions are confined to animals, which suggests that the responsible enzymes arose in the ancestor of that lineage. However there are a few prokaryotic systems and lower eukaryotes in which to study the evolution and divergence of the chymotrypsin superfamily.

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The sequence and X-ray structure of the trypsin from Fusarium oxysporum

Cited by 47 publications

References 3 publications

Unexpected crucial role of residue 225 in serine proteases

Unexpected crucial role of residue 225 in serine proteases

Trypsin Revisited

Cloning, Expression, and Substrate Specificity of a Fungal Chymotrypsin

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