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Abstract
NlpC/P60 superfamily papain-like enzymes play important roles in all kingdoms of life. Two members of this superfamily, LRAT-like and YaeF/YiiX-like families, were predicted to contain a catalytic domain that is circularly permuted such that the catalytic cysteine is located near the C-terminus, instead of at the N-terminus. These permuted enzymes are widespread in virus, pathogenic bacteria, and eukaryotes. We determined the crystal structure of a member of the YaeF/YiiX-like family from Bacillus cereus in complex with lysine. The structure, which adopts a ligand-induced, “closed” conformation, confirms the circular permutation of catalytic residues. A comparative analysis of other related protein structures within the NlpC/P60 superfamily is presented. Permutated NlpC/P60 enzymes contain a similar conserved core and arrangement of catalytic residues, including a Cys/His-containing triad and an additional conserved tyrosine. More surprisingly, permuted enzymes have a hydrophobic S1 binding pocket that is distinct from previously characterized enzymes in the family, indicative of novel substrate specificity. Further analysis of a structural homolog, YiiX (PDB 2if6) identified a fatty acid in the conserved hydrophobic pocket, thus providing additional insights into possible function of these novel enzymes.
Highlights
NlpC/P60 superfamily proteins [1] are ubiquitous papain-like cysteine peptidases or other functionally related enzymes
The selenomethionine derivative of BcPPNE was expressed in E. coli with an N-terminal tobacco etch virus (TEV) cleavable His-tag and purified by metal affinity chromatography
The crystal structure indicates that the cleavage site is not readily accessible to TEV since it is located at the start of a helix
Summary
NlpC/P60 superfamily proteins [1] are ubiquitous papain-like cysteine peptidases or other functionally related enzymes. Characterized members of this superfamily have diverse enzymatic functions, such as peptidases, amidases, transglutaminases and acetyltransferases. P60-like and AcmB/LytN-like enzymes are hydrolases with specificity for amide linkages in cell-wall components, such as those in D-c-glutamylmeso-diaminopimelate and N-acetylmuramate-L-alanine. These two families are canonical papain-like NlpC/P60 enzymes (CPNEs) with a catalytic core similar to that of papain, which has been confirmed by structural studies [2,3,4,5]. The latter two families were predicted to contain a conserved catalytic triad (Cys, His and a polar third residue) in a circularly permuted catalytic domain where the relative positions of the cysteine and histidine/ polar residue are swapped in the primary sequence [1], which we will refer to as permuted papain-like NlpC/P60 enzymes (PPNEs)
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