Protein engineering of xylose (glucose) isomerase from Actinoplanes missouriensis. 3. Changing metal specificity and the pH profile by site-directed mutagenesis.
Aldose-ketose isomerization by xylose isomerase requires bivalent cations such as Mg2+, Mn2+, or Co2+. The active site of the enzyme from Actinoplanes missouriensis contains two metal ions that are involved in substrate binding and in catalyzing a hydride shift between the C1 and C2 substrate atoms. Glu 186 is a conserved residue located near the active site but not in contact with the substrate and not with a metal ligand. The E186D and E186Q mutant enzymes were prepared. Both are active, and their metal specificity is different from that of the wild type. The E186Q enzyme is most active with Mn2+ and has a drastically shifted pH optimum. The X-ray analysis of E186Q was performed in the presence of xylose and either Mn2+ or Mg2+. The Mn2+ structure is essentially identical to that of the wild type. In the presence of Mg2+, the carboxylate group of residue Asp 255, which is part of metal site 2 and a metal ligand, turns toward Gln 186 and hydrogen bonds to its side-chain amide. Mg2+ is not bound at metal site 2, explaining the low activity of the mutant with this cation. Movements of Asp 255 also occur in the wild-type enzyme. We propose that they play a role in the O1 to O2 proton relay accompanying the hydride shift.
van Tilbeurgh
H
H
Plant Genetic Systems N.V., Gent, Belgium.
Jenkins
J
J
Chiadmi
M
M
Janin
J
J
Wodak
S J
SJ
Mrabet
N T
NT
Lambeir
A M
AM
D000192:Actinomycetales / Q000201:enzymology*; D019747:Aldose-Ketose Isomerases; D001665:Binding Sites; D002238:Carbohydrate Epimerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D003035:Cobalt / Q000737:chemistry; D003461:Crystallography; D005632:Fructose / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry*; D008345:Manganese / Q000737:chemistry*; D008667:Metalloproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D014867:Water / Q000737:chemistry; D014961:X-Ray Diffraction; D014994:Xylose / Q000378:metabolism
D008667:Metalloproteins; D014867:Water; D005632:Fructose; D003035:Cobalt; D008345:Manganese; D014994:Xylose; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase; D008274:Magnesium; D005947:Glucose