Unusual extra space at the active site and high activity for acetylated hydroxyproline of prolyl aminopeptidase from Serratia marcescens

Abstract

The prolyl aminopeptidase complexes of Ala-TBODA [2-alanyl-5-tert-butyl-(1, 3, 4)-oxadiazole] and Sar-TBODA [2-sarcosyl-5-tert-butyl-(1, 3, 4)-oxadiazole] were analyzed by X-ray crystallography at 2.4 angstroms resolution. Frames of alanine and sarcosine residues were well superimposed on each other in the pyrrolidine ring of proline residue, suggesting that Ala and Sar are recognized as parts of this ring of proline residue by the presence of a hydrophobic proline pocket at the active site. Interestingly, there was an unusual extra space at the bottom of the hydrophobic pocket where proline residue is fixed in the prolyl aminopeptidase. Moreover, 4-acetyloxyproline-betaNA (4-acetyloxyproline beta-naphthylamide) was a better substrate than Pro-betaNA. Computer docking simulation well supports the idea that the 4-acetyloxyl group of the substrate fitted into that space. Alanine scanning mutagenesis of Phe139, Tyr149, Tyr150, Phe236, and Cys271, consisting of the hydrophobic pocket, revealed that all of these five residues are involved significantly in the formation of the hydrophobic proline pocket for the substrate. Tyr149 and Cys271 may be important for the extra space and may orient the acetyl derivative of hydroxyproline to a preferable position for hydrolysis. These findings imply that the efficient degradation of collagen fragment may be achieved through an acetylation process by the bacteria.

FIG. 1.

The ribbon and stick model of enzyme-inhibitor complexes. |F_o| − |F_c| omit maps of the enzyme-Ala-TBODA complex (A) and the enzyme-Sar-TBODA complex (B) were calculated without the structure factor of inhibitor. Each inhibitor is shown in red. The electron density map is shown at the 2σ contour level. (C) Stereo drawing of superimposed enzyme-Pro-TBODA (orange), Ala-TBODA (green), and Sar-TBODA (blue) complexes. Panels A and B were produced by the POVscript⁺ and Raster3D programs, and panel C was drawn by the MOLSCRIPT and Raster3D programs.

FIG. 2.

Stereograms of the substrate binding pocket at the active site. (A) The hydrophobic pocket was drawn as emerald surface with stick models of Pro-TBODA (orange), Ala-TBODA (green), and Sar-TBODA (sky blue). (B) Computer-designed enzyme-AcHyp-TBODA complex model. It was suggested that the 4-acetyloxy group was fitted into the unusual extra space. The figures were drawn using the POVscript⁺ and Raster3D programs.

FIG. 3.

Stereogram of the enzyme-AcHyp-TBODA complex model, which was drawn using the MOLSCRIPT and Raster3D programs, viewed from the bottom of the hydrophobic pocket. 4-Acetyloxyproline is shown in pink, and the residues around the active site are shown as stick models.