Protein Science
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lauble, H.
Right arrow Articles by Effenberger, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lauble, H.
Right arrow Articles by Effenberger, F.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?
Protein Science (2002), 11:65-71.
Copyright © 2002 The Protein Society

Structure determinants of substrate specificity of hydroxynitrile lyase from Manihot esculenta

Hanspeter Lauble1, Burkhard Miehlich1, Siegfried Förster1, Christoph Kobler1, Harald Wajant2 and Franz Effenberger1

1 Institut für Organische Chemie der Universität Stuttgart, D-70569 Stuttgart, Germany
2 Institut für Zellbiologie und Immunologie der Universität Stuttgart, D-70569 Stuttgart, Germany

Reprint requests to: Hanspeter Lauble, Institut für Organische Chemie, Universität Stuttgart, Pfaffenwaldring 55, D-70569 Stuttgart, Germany; e-mail: PeterLauble{at}t-online.de; fax: 49-711-685-4269.

Tryptophan 128 of hydroxynitrile lyase of Manihot esculenta (MeHNL) covers a significant part of a hydrophobic channel that gives access to the active site of the enzyme. This residue was therefore substituted in the mutant MeHNL-W128A by alanine to study its importance for the substrate specificity of the enzyme. Wild-type MeHNL and MeHNL-W128A showed comparable activity on the natural substrate acetone cyanohydrin (53 and 40 U/mg, respectively). However, the specific activities of MeHNL-W128A for the unnatural substrates mandelonitrile and 4-hydroxymandelonitrile are increased 9-fold and ~450-fold, respectively, compared with the wild-type MeHNL. The crystal structure of the MeHNL-W128A substrate-free form at 2.1 Å resolution indicates that the W128A substitution has significantly enlarged the active-site channel entrance, and thereby explains the observed changes in substrate specificity for bulky substrates. Surprisingly, the MeHNL-W128A–4-hydroxybenzaldehyde complex structure at 2.1 Å resolution shows the presence of two hydroxybenzaldehyde molecules in a sandwich type arrangement in the active site with an additional hydrogen bridge to the reacting center.

Keywords: Substrate specificity; active-site tunnel mutant; crystal structure


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
K. Gruber, G. Gartler, B. Krammer, H. Schwab, and C. Kratky
Reaction Mechanism of Hydroxynitrile Lyases of the {alpha}/{beta}-Hydrolase Superfamily: THE THREE-DIMENSIONAL STRUCTURE OF THE TRANSIENT ENZYME-SUBSTRATE COMPLEX CERTIFIES THE CRUCIAL ROLE OF LYS236
J. Biol. Chem., May 7, 2004; 279(19): 20501 - 20510.
[Abstract] [Full Text] [PDF]


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Copyright © 2002 by The Protein Society.