X-ray crystallographic structures of D-xylose isomerase-substrate complexes position the substrate and provide evidence for metal movement during catalysis.
The X-ray crystallographic structures of the metal-activated enzyme xylose isomerase from Streptomyces olivochromogenes with the substrates D-glucose, 3-O-methyl-D-glucose and in the absence of substrate were determined to 1.96-, 2.19-, and 1.81-A resolution and refined to R-factors of 16.6%, 15.9%, and 16.1%, respectively. Xylose isomerase catalyzes the interconversion between glucose and fructose (xylose and xylulose under physiological conditions) by utilizing two metal cofactors to promote a hydride shift; the metals are bridged by a glutamate residue. This puts xylose isomerase in the small but rapidly growing family of enzymes with a bridged bimetallic active site, in which both metals are involved in the chemical transformation. The substrate 3-O-methylglucose was chosen in order to position the glucose molecule in the observed electron density unambiguously. Of the two essential magnesium ions per active site, Mg-2 was observed to occupy two alternate positions, separated by 1.8 A, in the substrate-soaked structures. The deduced movement was not observed in the structure without substrate present and is attributed to a step following substrate binding but prior to isomerization. The substrates glucose and 3-O-methylglucose are observed in their linear extended forms and make identical interactions with the enzyme by forming ligands to Mg-1 through O2 and O4 and by forming hydrogen bonds with His53 through O5 and Lys182 through O1. Mg-2 has a water ligand that is interpreted in the crystal structure in the absence of substrate as a hydroxide ion and in the presence of substrate as a water molecule. This hydroxide ion may act as a base to deprotonate the glucose O2 and subsequently protonate the product fructose O1 concomitant with hydride transfer. Calculations of the solvent-accessible surface of possible dimers, with and without the alpha-helical C-terminal domain, suggest that the tetramer is the active form of this xylose isomerase.
Lavie
A
A
Department of Biochemistry, Brandeis University, Massachusetts 02254-9110.
Allen
K N
KN
Petsko
G A
GA
Ringe
D
D
D019325:3-O-Methylglucose; D019747:Aldose-Ketose Isomerases; D000595:Amino Acid Sequence; D001483:Base Sequence; D002238:Carbohydrate Epimerases / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D018360:Crystallography, X-Ray; D005632:Fructose / Q000378:metabolism; D005947:Glucose / Q000737:chemistry* / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry* / Q000378:metabolism; D008757:Methylglucosides / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D012997:Solvents; D013302:Streptomyces / Q000201:enzymology; D013379:Substrate Specificity
D008757:Methylglucosides; D012997:Solvents; D019325:3-O-Methylglucose; D005632:Fructose; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase; D008274:Magnesium; D005947:Glucose