Isomaltulose synthase (PalI) of Klebsiella sp. LX3. Crystal structure and implication of mechanism.
Isomaltulose synthase from Klebsiella sp. LX3 (PalI, EC 5.4.99.11) catalyzes the isomerization of sucrose to produce isomaltulose (alpha-D-glucosylpyranosyl-1,6-D-fructofuranose) and trehalulose (alpha-D-glucosylpyranosyl-1,1-d-fructofuranose). The PalI structure, solved at 2.2-A resolution with an R-factor of 19.4% and Rfree of 24.2%, consists of three domains: an N-terminal catalytic (beta/alpha)8 domain, a subdomain between N beta 3 and N alpha 3, and a C-terminal domain having seven beta-strands. The active site architecture of PalI is identical to that of other glycoside hydrolase family 13 members, suggesting a similar mechanism in substrate binding and hydrolysis. However, a unique RLDRD motif in the proximity of the active site has been identified and shown biochemically to be responsible for sucrose isomerization. A two-step reaction mechanism for hydrolysis and isomerization, which occurs in the same pocket is proposed based on both the structural and biochemical data. Selected C-terminal truncations have been shown to reduce and even abolish the enzyme activity, consistent with the predicted role of the C-terminal residues in the maintenance of enzyme conformation and active site topology.
Zhang
Daohai
D
Department of Pathology, National University of Singapore, 5 Lower Kent Ridge Road, Singapore 119074.
Li
Nan
N
Lok
Shee-Mei
SM
Zhang
Lian-Hui
LH
Swaminathan
Kunchithapadam
K
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D018360:Crystallography, X-Ray; D005822:Genetic Vectors; D019751:Intramolecular Transferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007709:Klebsiella / Q000201:enzymology*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D019751:Intramolecular Transferases; C042002:isomaltulose synthase