Time-resolved X-ray Crystal Structure Analysis of Protein

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The crystal structure of a zinc endoprotease from Streptomyces caespitosus (ScNP) determined at 1 Å resolution has been analyzed to investigate geometrical properties of a catalytically essential zinc ion. The zinc ion is tetrahedrally coordinated by three side-chains (His83, His87 and Asp93) and a water molecule. The distances between the zinc ion and the coordinating atoms are 2.01 Å, 2.01 Å and 1.95 Å for His83Nε, His87Nε and Asp93Oδ, respectively. These distances agree very well with those normally found in crystal structures of small zinc-containing compounds deposited in the Cambridge Structural Database. On the other hand, the distance between the zinc ion and the coordinating water molecule (1.93 Å) is slightly shorter than the typical value (2.01 Å) found in the Database. In addition, Glu84Oε makes a strong hydrogen bond to this water molecule with the distance of 2.54 Å. Thus, the water molecule is in a highly polarized state. Two hydrogen bonds (His83Nδ-Leu102O, His87Nδ-Leu91O) and van der Waals interactions between the side-chain of Met103 and the two imidazole rings of His83 and His87 are also observed. These interactions are probably important for His83 and His87 to construct the tetrahedral coordination arrangement to the zinc ion. This crystal structure of ScNP is the highest resolution and accuracy among crystal structures of zinc endoproteases ever determined, and is not only important for zinc coordination chemistry and manifestation in biological systems but useful for the design of organo-metallic catalyst including zinc as well. The enzymatic reaction mechanism of the N-acetylmuramidase produced by Streptomyces globisporus was also clarified. The glycosidic linkage between NAM and NAG is cut by Asp98, which as an acid catalyst hands proton to the oxygen atom linking NAM and NAG. The reaction intermediate, oxyocarbenium ion, is stabilized by the negative charge of Asp198.