Protein secretion controlled by a synthetic gene in Escherichia coli

Abstract

We report here the X-ray crystal structure of native subtilisin Carlsberg, solved at 2.5 Å resolution by molecular replacement and refined by restrained least squares to a crystal-lographic residual of 0.206. we compare this structure to the crystal structure of subtilisin BPN'. We find that, despite 82 amino acid substitutions and one deletion in subtilisin Carlsberg relative to subtilisin BPN', the structures of these enzymes are remarkably similar. We calculate an r.m.s. difference between equivalent a-carbon positions in subtilisin Carlsberg and subtilisin BPN' of only 0.55 Å. This confirms previous reports of extensive structural bomology between these two subtilisins based on X-ray crystal structures of the complex of eglin-c with subtilisin Carlsberg [McPhalen,C.A., Schnebli.H.P. and James,M.N.G. (1985) FEBS Lett., 188, 55; Bode,W., Papamokos,E. and Musil,D. (1987) Eur. J. Biochem., 166, 673-692]. In addition, we find that the native active sites of subtilisins Carlsberg and BPN' are virtually identical. While conservative substitutions at residues 217 and 156 may have subtle effects on the environments of substrate-binding sites SI' and SI respectively, we find no obvious structural correlate for reports that subtilisins Carlsberg and BPN' differ in their recognition of model substrates. In particular, we find no evidence that the hydrophobic binding pocket SI in subtilisin Carlsberg is ‘deeper’, ‘narrower’ or 'less polar' than the corresponding binding site hi subtilisin BPN' [Karasaki and Ohno (1978) J. Biochem., Tokyo, 84, 531–538].