Glucose isomerase: insights into protein engineering for increased thermostability.
Thermostable glucose isomerases are desirable for production of 55% fructose syrups at >90 degrees C. Current commercial enzymes operate only at 60 degrees C to produce 45% fructose syrups. Protein engineering to construct more stable enzymes has so far been relatively unsuccessful, so this review focuses on elucidation of the thermal inactivation pathway as a future guide. The primary and tertiary structures of 11 Class 1 and 20 Class 2 enzymes are compared. Within each class the structures are almost identical and sequence differences are few. Structural differences between Class 1 and Class 2 are less than previously surmised. The thermostabilities of Class 1 enzymes are essentially identical, in contrast to previous reports, but in Class 2 they vary widely. In each class, thermal inactivation proceeds via the tetrameric apoenzyme, so metal ion affinity dominates thermostability. In Class 1 enzymes, subunit dissociation is not involved, but there is an irreversible conformational change in the apoenzyme leading to a more thermostable inactive tetramer. This may be linked to reversible conformational changes in the apoenzyme at alkaline pH arising from electrostatic repulsions in the active site, which break a buried Arg-30-Asp-299 salt bridge and bring Arg-30 to the surface. There is a different salt bridge in Class 2 enzymes, which might explain their varying thermostability. Previous protein engineering results are reviewed in light of these insights.
Hartley
B S
BS
Department of Biochemistry, Imperial College, SW7 2AZ, London, UK. b.hartley@bc.ic.ac.uk
Hanlon
N
N
Jackson
R J
RJ
Rangarajan
M
M
D019747:Aldose-Ketose Isomerases / Q000737:chemistry* / Q000145:classification / Q000235:genetics; D000595:Amino Acid Sequence; D001051:Apoenzymes / Q000737:chemistry; D001105:Archaea; D001173:Arthrobacter; D001665:Binding Sites; D002384:Catalysis; D002413:Cations, Divalent; D004220:Disulfides / Q000737:chemistry; D004795:Enzyme Stability; D006358:Hot Temperature; D008670:Metals / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering; D013379:Substrate Specificity; D020860:Subtilisin; D013820:Thermolysin
D001051:Apoenzymes; D002413:Cations, Divalent; D004220:Disulfides; D008670:Metals; D020860:Subtilisin; D013820:Thermolysin; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase