HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wiegert, T. Articles by Sprenger, G. A. Search for Related Content PubMed PubMed Citation Articles by Wiegert, T. Articles by Sprenger, G. A. Social Bookmarking                      What's this?

Volume 272, Number 20, Issue of May 16, 1997 pp. 13126-13133
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

The Substitution of a Single Amino Acid Residue (Ser-116  Asp) Alters NADP-containing Glucose-Fructose Oxidoreductase of Zymomonas mobilis into a Glucose Dehydrogenase with Dual Coenzyme Specificity

(Received for publication, October 28, 1996, and in revised form, March 10, 1997)

From the Institut für Biotechnologie 1 der Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany

Glucose-fructose oxidoreductase (GFOR, EC 1.1.1.99.-) from the Gram-negative bacterium Zymomonas mobilis contains the tightly bound cofactor NADP. Based on the revision of the gfo DNA sequence, the derived GFOR sequence was aligned with enzymes catalyzing reactions with similar substrates. A novel consensus motif (AGKHVXCEKP) for a class of dehydrogenases was detected. From secondary structure analysis the serine-116 residue of GFOR was predicted as part of a Rossmann-type dinucleotide binding fold. An engineered mutant protein (S116D) was purified and shown to have lost tight cofactor binding based on (a) altered tryptophan fluorescence; (b) lack of NADP liberation through perchloric acid treatment of the protein; and (c) lack of GFOR enzyme activity. The S116D mutant showed glucose dehydrogenase activity (3.6 ± 0.1 units/mg of protein) with both NADP and NAD as coenzymes (K_m for NADP, 153 ± 9 µM; for NAD, 375 ± 32 µM). The single site mutation therefore altered GFOR, which in the wild-type situation contains NADP as nondissociable redox cofactor reacting in a ping-pong type mechanism, to a dehydrogenase with dissociable NAD(P) as cosubstrate and a sequential reaction type. After prolonged preincubation of the S116D mutant protein with excess NADP (but not NAD), GFOR activity could be restored to 70 units/mg, one-third of wild-type activity, whereas glucose dehydrogenase activity decreased sharply. A second site mutant (S116D/K121A/K123Q/I124K) showed no GFOR activity even after preincubation with NADP, but it retained glucose dehydrogenase activity (4.2 ± 0.2 units/mg of protein).

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

This article has been cited by other articles:

				S. Watanabe, T. Kodak, and K. Makino Cloning, Expression, and Characterization of Bacterial L-Arabinose 1-Dehydrogenase Involved in an Alternative Pathway of L-Arabinose Metabolism J. Biol. Chem., February 3, 2006; 281(5): 2612 - 2623. [Abstract] [Full Text] [PDF]

				A. Kuhn, S. Yu, and F. Giffhorn Catabolism of 1,5-Anhydro-D-Fructose in Sinorhizobium morelense S-30.7.5: Discovery, Characterization, and Overexpression of a New 1,5-Anhydro-D-Fructose Reductase and Its Application in Sugar Analysis and Rare Sugar Synthesis Appl. Envir. Microbiol., February 1, 2006; 72(2): 1248 - 1257. [Abstract] [Full Text] [PDF]

				S. Watanabe, T. Kodaki, and K. Makino Complete Reversal of Coenzyme Specificity of Xylitol Dehydrogenase and Increase of Thermostability by the Introduction of Structural Zinc J. Biol. Chem., March 18, 2005; 280(11): 10340 - 10349. [Abstract] [Full Text] [PDF]

				U. Johnsen and P. Schonheit Novel Xylose Dehydrogenase in the Halophilic Archaeon Haloarcula marismortui J. Bacteriol., September 15, 2004; 186(18): 6198 - 6207. [Abstract] [Full Text] [PDF]

				T. Hauck, F. Bruhlmann, and W. Schwab Formation of 4-Hydroxy-2,5-Dimethyl-3[2H]-Furanone by Zygosaccharomyces rouxii: Identification of an Intermediate Appl. Envir. Microbiol., July 1, 2003; 69(7): 3911 - 3918. [Abstract] [Full Text] [PDF]

				H. J. Hektor, H. Kloosterman, and L. Dijkhuizen Identification of a Magnesium-dependent NAD(P)(H)-binding Domain in the Nicotinoprotein Methanol Dehydrogenase from Bacillus methanolicus J. Biol. Chem., November 27, 2002; 277(49): 46966 - 46973. [Abstract] [Full Text] [PDF]

				N. Blaudeck, G. A. Sprenger, R. Freudl, and T. Wiegert Specificity of Signal Peptide Recognition in Tat-Dependent Bacterial Protein Translocation J. Bacteriol., January 15, 2001; 183(2): 604 - 610. [Abstract] [Full Text]