Crystal structures of Aflatoxin-oxidase from Armillariella tabescens reveal a dual activity enzyme.

Aflatoxin-oxidase (AFO), a newly discovered oxidase isolated from Armillariella tabescens, was reported to perform aflatoxin B1 (AFB1) detoxification through breaking the bisfuran ring of AFB1. However, based on sequence alignment, we found that AFO shares high sequence identities with dipeptidyl peptidase III (DPP III) family members. To understand the functions of AFO, we determined its crystal structures in the absence and presence of zinc, copper ion, and employed HPLC to test if AFO could cleave the substrates of DPP III. Our structures reveal that AFO contains the classic DPP III activity center and the HPLC results further confirm that AFO possesses the dipeptidyl peptidase activity. Therefore, AFO should belong to DPP III family. Interestingly, unlike reported classic DPP III structure that has a large domain movement upon substrate binding, the AFO structures all adopt the closed conformation, independent of substrate binding. This conformation characteristic of AFO may be related to its enzyme activities. Taken together, our results demonstrate that AFO is a dual activity enzyme with both aflatoxin-oxidase and dipeptidyl peptidase activities and its unique conformation feature expands our understanding on the mode of reaction for this enzyme family.

Hybrid molecular structure of the giant protease tripeptidyl peptidase II.

Tripeptidyl peptidase II (TPP II) is the largest known eukaryotic protease (6 MDa). It is believed to act downstream of the 26S proteasome, cleaving tripeptides from the N termini of longer peptides, and it is implicated in numerous cellular processes. Here we report the structure of Drosophila TPP II determined by a hybrid approach. We solved the structure of the dimer by X-ray crystallography and docked it into the three-dimensional map of the holocomplex, which we obtained by single-particle cryo-electron microscopy. The resulting structure reveals the compartmentalization of the active sites inside a system of chambers and suggests the existence of a molecular ruler determining the size of the cleavage products. Furthermore, the structure suggests a model for activation of TPP II involving the relocation of a flexible loop and a repositioning of the active-site serine, coupling it to holocomplex assembly and active-site sequestration.

Localization of the lysosomal protease dipeptidyl peptidase II in the young normal rat lens: a correlative light and electron microscopic analysis.

This investigation was follow-up to an earlier biochemical and light microscopic histochemical study, in which the lysosomal protease dipeptidyl peptidase II (DPP II) was demonstrated in rodent lenses. In the present study, a method was employed that allowed a more precise histochemical localization of the enzyme, one that was suitable for ultrastructural as well as light microscopic analysis. Successful demonstration of the enzyme using either of two synthetic substrates, and the significant reduction of the enzyme reaction by phenylmethylsulphonyl fluoride (PMSF), a serine protease inhibitor, pointed to the sensitivity of the method. A flat-embedding technique allowed the correlative light and electron microscopic analysis of specific areas of the specimen. Examination of the epithelium and outer cortical regions of the lens revealed the compartmentalization of DPP II activity within lysosomal dense bodies that were concentrated primarily in the equatorial and sutural regions, and also an association of the reaction product with larger bodies that were confined to the sutural regions. The latter structures appeared to represent fiber cell fragments that were enwrapped with narrow extensions of the surrounding fiber cells. The location of enzyme activity within the sutural bodies and also within the intercellular spaces of the modified fiber cell extensions surrounding these bodies suggested that lysosomal proteases may play a role in the segregation and degradation of specific regions of normal lens fiber cells.