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PEDS Advance Access published online on May 21, 2008
Protein Engineering Design and Selection, doi:10.1093/protein/gzn026
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Alteration of substrate specificity of rat neurolysin from matrix metalloproteinase-2/9-type to -3-type specificity by comprehensive mutation

Tetsuya Kadonosono, Michiko Kato-Murai and Mitsuyoshi Ueda1

Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo, Kyoto 606-8502, Japan

1 To whom correspondence should be addressed. E-mail: miueda{at}kais.kyoto-u.ac.jp

The substrate specificity of rat brain neurolysin was rapidly modified by semirational mutagenesis coupled with a yeast molecular display system. Neurolysin mainly recognizes substrates with sequential six residues close to the scissile bond in polypeptides, cleaving a peptide bond in the center position of the six residues. To alter the recognition of the P2' amino acid of substrates by neurolysin, six residues of neurolysin, Asp467, Arg470, Glu510, Tyr606, Tyr610 and Tyr611, which might be involved in the formation of the neurolysin S2' subsite, were individually and comprehensively substituted. The protein libraries of mutant neurolysins comprising 120 species were displayed on the yeast cell surface and screening was carried out using two fluorescence-quenching peptides, the matrix metalloproteinase-2/9- (MMPs-2/9-) and MMP-3-specific substrates, which consisted of similar amino acids, except for alanine (for MMPs-2/9) or glutamic acid (for MMP-3) at the P2' amino acid position. Among mutant neurolysins, the Y610L mutant neurolysin exhibited a marked change in substrate specificity. Steady-state kinetic analysis of the purified Y610L mutant neurolysin revealed that the binding efficiency toward the MMP-3-specific substrate was about 3-fold higher than that toward the MMP-2/9-specific substrate. These results indicate that Tyr610 of neurolysin is the important residue to recognize the P2' amino acid of substrates.

Keywords: matrix metalloproteinase/molecular display/neurolysin/peptidase/substrate specificity

Received November 23, 2007; revised April 11, 2008; accepted April 11, 2008.


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