Structure of inhibited trypsin from Fusarium oxysporum at 1.55 Å

Abstract

The structure of trypsin from the fungus Fusarium oxysporum has been refined at 1.55 A resolution by restrained least-squares minimization to an R-factor of 14.4%. The data were recorded from a single-crystal on the X31 beamline at EMBL, Hamburg, using a locally developed image-plate scanner. The final model consists of 1557 protein atoms, 400 water molecules, one molecule of isopropanol and one monoisopropyl phosphoryl inhibitor group covalently bound to the catalytic Ser195. Comparison of the structure with bovine trypsin reveals significant differences in the active site and suggests a possible explanation for the difference in substrate specificity between the two enzymes. In F. oxysporum trypsin the specificity pocket is larger than in bovine trypsin. This explains the preference of F. oxysporum trypsin for the bulkier arginine over lysine and the reverse preference in bovine trypsin. The binding cavity on the C-terminal side of the substrate is more restricted in F. oxysporum trypsin than in mammalian and Streptomyces griseus trypsins, which explains the relative inactivity of F. oxysporum trypsin towards peptide-pNA substrate analogues as an unfavourable steric interaction between the side of the binding cavity and the para-nitroanilino group of peptide-pNA. The observed restriction of the binding cavity does not lead to a reduced catalytic activity compared to other trypsins.

Inhibition of coagulation factors by recombinant barley serpin BSZx

“…corroborated by reports on monoclonal antibodies recognizing neoantigenic epitopes on C1 inhibitor and PAI-1 generated both by cleavage of the reactive center loop and by complex formation [22,23]. Mammalian and Fusarium trypsins are very similar in their secondary structural elements and overall conformation but significant differences are observed in the active site region [24]. Although Fusarium trypsin preferentially binds Arg in the specificity pocket and is inhibited by the very different Kunitz inhibitors of pancreas and soy bean [24], no inhibition by BSZx was observed.…”

“…Mammalian and Fusarium trypsins are very similar in their secondary structural elements and overall conformation but significant differences are observed in the active site region [24]. Although Fusarium trypsin preferentially binds Arg in the specificity pocket and is inhibited by the very different Kunitz inhibitors of pancreas and soy bean [24], no inhibition by BSZx was observed. Recently cq-proteinase inhibitor and cq-antichymotrypsin were shown to inhibit and form SDS stable complexes with subtilisins [25].…”

Structural and Dynamic Basis of Serine Proteases from Nematophagous Fungi for Cuticle Degradation

“…As the consequence of weaker interaction of His69 with Ser224, Ser224 exhibits a higher degree of conformational freedom compared to Asp39 and His69, as indicated by B- factors of 5.5, 6.9 and 15.0 Å 2 for Asp39, His69 and Ser224, respectively. This is consistent with the unfavorable geometry of the Ser O  observed in some crystal structures of serine proteases (Dauter et al, 1991;Rypniewski et al, 1995). Functionally, the high flexibility of the serine hydroxyl allows it to adopt different orientations to suit the needs for proton transfer, nucleophilic attack and subsequent release of the cleaved peptide product.…”

Fusarium oxysporumtrypsin at atomic resolution at 100 and 283 K: a study of ligand binding

“…The accepted reaction mechanism proceeds via a tetrahedral transition-state intermediate. This is hard to observe owing to its transient nature (West et al, 1990), but a number of model compounds have been found to give rise to tetrahedral covalently bound adducts at the serine (Chen et al, 1995;Mac Sweeney et al, 2000;Rypniewski, Dambmann et al, 1995). Several authors have also reported enzyme±product complexes (Harel et al, 1991), acyl-enzyme intermediates (Strynadka et al, 1992) or even a trapped tetrahedral reaction intermediate (Yennawar et al, 1994).…”

“…The trypsin from F. oxysporum has been investigated previously, inhibited by diisopropyl¯uorophosphate (DFP), which forms an irreversible covalently bound adduct at the catalytic serine (Rypniewski, Dambmann et al, 1995). However, the enzyme is also stable for extensive periods of time in the presence of boric acid, which acts as a mild reversible inhibitor.…”