Protein engineering of xylose (glucose) isomerase from Actinoplanes missouriensis. 2. Site-directed mutagenesis of the xylose binding site.
Site-directed mutagenesis in the active site of xylose isomerase derived from Actinoplanes missouriensis is used to investigate the structural and functional role of specific residues. The mutagenesis work together with the crystallographic studies presented in detail in two accompanying papers adds significantly to the understanding of the catalytic mechanism of this enzyme. Changes caused by introduced mutations emphasize the correlation between substrate specificity and cation preference. Mutations in both His 220 and His 54 mainly affect the catalytic rate constant, with catalysis being severely reduced but not abolished, suggesting that both histidines are important, but not essential, for catalysis. Our results thus challenge the hypothesis that His 54 acts as an obligatory catalytic base for ring opening; this residue appears instead to be implicated in governing the anomeric specificity. With none of the active site histidines acting as a catalytic base, the role of the cations in catalyzing proton transfer is confirmed. In addition, Lys 183 appears to play a crucial part in the isomerization step, by assisting the proton shuttle. Other residues also are important but to a lesser extent. The conserved Lys 294 is indirectly involved in binding the activating cations. Among the active site aromatic residues, the tryptophans (16 and 137) play a role in maintaining the general architecture of the substrate binding site while the role of Phe 26 seems to be purely structural.
Lambeir
A M
AM
Plant Genetic Systems N.V., Gent, Belgium.
Lauwereys
M
M
Stanssens
P
P
Mrabet
N T
NT
Snauwaert
J
J
van Tilbeurgh
H
H
Matthyssens
G
G
Lasters
I
I
De Maeyer
M
M
Wodak
S J
SJ
D000192:Actinomycetales / Q000201:enzymology*; D019747:Aldose-Ketose Isomerases; D001665:Binding Sites; D002238:Carbohydrate Epimerases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002384:Catalysis; D005818:Genetic Engineering; D006639:Histidine / Q000737:chemistry; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000737:chemistry; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014364:Tryptophan / Q000737:chemistry; D014994:Xylose / Q000378:metabolism
D010649:Phenylalanine; D006639:Histidine; D014364:Tryptophan; D014994:Xylose; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase; D008239:Lysine