title: The structure of substrate-free 1,5-anhydro-D-fructose reductase from Sinorhizobium meliloti 1021 reveals an open enzyme conformation.
abstract: 1,5-Anhydro-D-fructose (1,5-AF) is an interesting building block for enantioselective and stereoselective organic synthesis. Enzymes acting on this compound are potential targets for structure-based protein/enzyme design to extend the repertoire of catalytic modifications of this and related building blocks. Recombinant 1,5-anhydro-D-fructose reductase (AFR) from Sinorhizobium meliloti 1021 was produced in Escherichia coli, purified using a fused 6×His affinity tag and crystallized in complex with the cofactor NADP(H) using the hanging-drop technique. Its structure was determined to 1.93 Å resolution using molecular replacement. The structure displays an empty substrate-binding site and can be interpreted as an open conformation reflecting the enzyme state shortly after the release of product, presumably with bound oxidized cofactor NADP⁺. Docking simulations indicated that amino-acid residues Lys94, His151, Trp162, Arg163, Asp176 and His180 are involved in substrate binding, catalysis or product release. The side chain of Lys94 seems to have the ability to function as a molecular switch. The crystal structure helps to characterize the interface relevant for dimer formation as observed in solution. The crystal structure is compared with the structure of the homologue from S. morelense, which was solved in a closed conformation and for which dimer formation in solution could not be verified but seems to be likely based on the presented studies of S. meliloti AFR.
authors.affiliation: Institute of Zoology - Structural Biology, Christian-Albrechts University Kiel, Am Botanischen Garten 1-9, 24118 Kiel, Germany.
authors.forename: Mario
authors.initials: M
authors.lastname: Schu
authors.forename: Annette
authors.initials: A
authors.lastname: Faust
authors.forename: Beata
authors.initials: B
authors.lastname: Stosik
authors.forename: Gert Wieland
authors.initials: GW
authors.lastname: Kohring
authors.forename: Friedrich
authors.initials: F
authors.lastname: Giffhorn
authors.forename: Axel J
authors.initials: AJ
authors.lastname: Scheidig
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites / Q000502:physiology; D018360:Crystallography, X-Ray; D005632:Fructose / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011487:Protein Conformation; D016962:Sinorhizobium meliloti / Q000201:enzymology / Q000235:genetics*; D013379:Substrate Specificity / Q000502:physiology
chemical_list: D001426:Bacterial Proteins; D005632:Fructose; C048927:1,5-anhydrofructose
keywords: biotechnology; carbohydrate metabolism; redox enzymes
references.citation: Acta Crystallogr D Biol Crystallogr. 2011 Apr;67(Pt 4):355-67
references.pmid: 21460454
references.citation: Nature. 1974 Jul 19;250(463):194-9
references.pmid: 4368490
references.citation: Acta Crystallogr D Biol Crystallogr. 1999 Jan;55(Pt 1):191-205
references.pmid: 10089410
references.citation: J Comput Chem. 2009 Dec;30(16):2785-91
references.pmid: 19399780
references.citation: Acta Crystallogr D Biol Crystallogr. 1999 Mar;55(Pt 3):583-601
references.pmid: 10089455
references.citation: Acta Crystallogr D Biol Crystallogr. 2010 Feb;66(Pt 2):133-44
references.pmid: 20124693
references.citation: J Biochem. 1999 Jan;125(1):166-72
references.pmid: 9880813
references.citation: Acta Crystallogr D Biol Crystallogr. 2010 Apr;66(Pt 4):486-501
references.pmid: 20383002
references.citation: Acta Crystallogr D Biol Crystallogr. 2000 Apr;56(Pt 4):442-50
references.pmid: 10739917
references.citation: J Bacteriol. 1986 Sep;167(3):863-9
references.pmid: 3745122
references.citation: Curr Opin Struct Biol. 2009 Jun;19(3):341-8
references.pmid: 19481444
references.citation: Nucleic Acids Res. 2009 Jan;37(Database issue):D355-9
references.pmid: 18996896
references.citation: Acta Crystallogr D Biol Crystallogr. 2010 Jan;66(Pt 1):22-5
references.pmid: 20057045
references.citation: Biochim Biophys Acta. 2003 Oct 13;1623(2-3):82-7
references.pmid: 14572905
references.citation: Carbohydr Res. 2010 Jan 26;345(2):181-90
references.pmid: 20004890
references.citation: Structure. 1996 Dec 15;4(12):1413-28
references.pmid: 8994968
references.citation: Carbohydr Res. 2002 May 13;337(10):873-90
references.pmid: 12007470
references.citation: Biochim Biophys Acta. 2004 May 3;1672(2):120-9
references.pmid: 15110094
references.citation: Biochemistry. 2001 Nov 20;40(46):13857-67
references.pmid: 11705375
references.citation: Planta Med. 2010 Oct;76(15):1635-41
references.pmid: 20645241
references.citation: J Biol Chem. 1997 May 16;272(20):13126-33
references.pmid: 9148926
references.citation: Eur J Biochem. 1996 Dec 15;242(3):832-8
references.pmid: 9022716
references.citation: Biosci Biotechnol Biochem. 2000 Nov;64(11):2462-5
references.pmid: 11193417
references.citation: Nucleic Acids Res. 2003 Jul 1;31(13):3381-5
references.pmid: 12824332
references.citation: J Mol Biol. 2007 Sep 21;372(3):774-97
references.pmid: 17681537
references.citation: Appl Environ Microbiol. 2006 Feb;72(2):1248-57
references.pmid: 16461673
references.citation: Dis Markers. 2005;21(3):127-32
references.pmid: 16276006
references.citation: Nature. 1992 Jan 30;355(6359):472-5
references.pmid: 18481394
references.citation: Eur J Pharmacol. 2000 May 26;397(1):219-25
references.pmid: 10844116
references.citation: Nat Struct Biol. 1997 Apr;4(4):269-75
references.pmid: 9095194
references.citation: Biochim Biophys Acta. 2006 Sep;1760(9):1314-22
references.pmid: 16822618
references.citation: Acta Crystallogr D Biol Crystallogr. 2010 Feb;66(Pt 2):125-32
references.pmid: 20124692
references.citation: Biochim Biophys Acta. 1993 Mar 21;1156(3):313-20
references.pmid: 8461323
references.citation: Nucleic Acids Res. 2003 Jul 1;31(13):3320-3
references.pmid: 12824317
references.citation: Glycobiology. 2000 Dec;10(12):1283-9
references.pmid: 11159920
references.citation: Biochemistry. 2006 Aug 22;45(33):10030-42
references.pmid: 16906761
references.citation: IUBMB Life. 2008 Dec;60(12):798-809
references.pmid: 18785261
references.citation: Planta. 1993;191(1):137-42
references.pmid: 7763826
references.citation: J Biochem. 1998 Jan;123(1):189-93
references.pmid: 9504428
references.citation: Biochemistry. 2003 Nov 11;42(44):13081-90
references.pmid: 14596624