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2glx

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{{STRUCTURE_2glx| PDB=2glx | SCENE= }}
{{STRUCTURE_2glx| PDB=2glx | SCENE= }}
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'''Crystal Structure Analysis of bacterial 1,5-AF Reductase'''
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===Crystal Structure Analysis of bacterial 1,5-AF Reductase===
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==Overview==
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Recombinant 1,5-anhydro-d-fructose reductase (AFR) from Sinorhizobium morelense S-30.7.5 was crystallized in complex with the cofactor NADP(H) and its structure determined to 2.2 A resolution using selenomethionine SAD (refined R(work) and R(free) factors of 18.9 and 25.0%, respectively). As predicted from the sequence and shown by the structure, AFR can be assigned to the GFO/IDH/MocA protein family. AFR consists of two domains. The N-terminal domain displays a Rossmann fold and contains the cofactor binding site. The intact crystals contain the oxidized cofactor NADP(+), whose attachment to the cofactor binding site is similar to that of NADP(+) in glucose-fructose oxidoreductase (GFOR) from Zymomonas mobilis. Due to variations in length and sequence within loop regions L3 and L5, respectively, the adenine moiety of NADP(+) adopts a different orientation in AFR caused by residue Arg38 forming hydrogen bonds with the 2'-phosphate moiety of NADP(+) and cation-pi stacking interactions with the adenine ring. Amino acid replacements in AFR (S10G, A13G, and S33D) showed that Ala13 is crucial for the discrimination between NADPH and NADH and yielded the A13G variant with dual cosubstrate specificity. The C-terminal domain contains the putative substrate binding site that was occupied by an acetate ion. As determined by analogy to GFOR and by site-directed mutagenesis of K94G, D176A, and H180A, residues Lys94, Asp176, and His180 are most likely involved in substrate binding and catalysis, as substitution of any of these residues resulted in a significant decrease in k(cat) for 1,5-AF. In this context, His180 might serve as a general acid-base catalyst by polarizing the carbonyl function of 1,5-AF to enable the transfer of the hydride from NADPH to the substrate. Here we present the first structure of an AFR enzyme catalyzing the stereoselective reduction of 1,5-AF to 1,5-anhydro-d-mannitol, the final step of a modified anhydrofructose pathway in S. morelense S-30.7.5. We also emphasize the importance of the A13G variant in biocatalysis for the synthesis of 1,5-AM and related derivatives.
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(as it appears on PubMed at http://www.pubmed.gov), where 16906761 is the PubMed ID number.
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{{ABSTRACT_PUBMED_16906761}}
==About this Structure==
==About this Structure==
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==Reference==
==Reference==
Crystal structure of NADP(H)-dependent 1,5-anhydro-D-fructose reductase from Sinorhizobium morelense at 2.2 A resolution: construction of a NADH-accepting mutant and its application in rare sugar synthesis., Dambe TR, Kuhn AM, Brossette T, Giffhorn F, Scheidig AJ, Biochemistry. 2006 Aug 22;45(33):10030-42. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/16906761 16906761]
Crystal structure of NADP(H)-dependent 1,5-anhydro-D-fructose reductase from Sinorhizobium morelense at 2.2 A resolution: construction of a NADH-accepting mutant and its application in rare sugar synthesis., Dambe TR, Kuhn AM, Brossette T, Giffhorn F, Scheidig AJ, Biochemistry. 2006 Aug 22;45(33):10030-42. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/16906761 16906761]
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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., Kuhn A, Yu S, Giffhorn F, Appl Environ Microbiol. 2006 Feb;72(2):1248-57. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/16461673 16461673]
[[Category: 1,5-anhydro-D-fructose reductase]]
[[Category: 1,5-anhydro-D-fructose reductase]]
[[Category: Ensifer adhaerens]]
[[Category: Ensifer adhaerens]]
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[[Category: Rossmann-fold]]
[[Category: Rossmann-fold]]
[[Category: Sugar metabolism]]
[[Category: Sugar metabolism]]
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''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sun May 4 05:15:11 2008''
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''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sun Jul 27 21:01:25 2008''

Revision as of 18:01, 27 July 2008

Image:2glx.png

Template:STRUCTURE 2glx

Crystal Structure Analysis of bacterial 1,5-AF Reductase

Template:ABSTRACT PUBMED 16906761

About this Structure

2GLX is a Single protein structure of sequence from Ensifer adhaerens. Full crystallographic information is available from OCA.

Reference

Crystal structure of NADP(H)-dependent 1,5-anhydro-D-fructose reductase from Sinorhizobium morelense at 2.2 A resolution: construction of a NADH-accepting mutant and its application in rare sugar synthesis., Dambe TR, Kuhn AM, Brossette T, Giffhorn F, Scheidig AJ, Biochemistry. 2006 Aug 22;45(33):10030-42. PMID:16906761

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., Kuhn A, Yu S, Giffhorn F, Appl Environ Microbiol. 2006 Feb;72(2):1248-57. PMID:16461673

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