Crystal structure of levansucrase from the Gram-negative bacterium Gluconacetohacter diazotrophicus

TY - JOUR

T1 - Crystal structure of levansucrase from the Gram-negative bacterium Gluconacetohacter diazotrophicus

AU - Martinez-Fleites, C

AU - Ortiz-Lombardia, M

AU - Pons, T

AU - Tarbouriech, N

AU - Taylor, E J

AU - Arrieta, J G

AU - Hernandez, L

AU - Davies, G J

PY - 2005/8/15

Y1 - 2005/8/15

N2 - The endophytic Gram-negative bacterium Gluconacetobacter diazotrophicus SRT4 secretes a constitutively expressed levansucrase (LsdA, EC 2.4. 1.10), which converts sucrose into fructooligosaccharides and levan. The enzyme is included in GH (glycoside hydrolase) family 68 of the sequence-based classification of glycosidases. The three-dimensional structure of LsdA has been determined by X-ray crystallography at a resolution of 2.5 angstrom (1 angstrom= 0. 1 nm). The structure was solved by molecular replacement using the homologous Bacillus subtilis (Bs) levansucrase (Protein Data Bank accession code IOYG) as a search model. LsdA displays a five-bladed beta-propeller architecture, where the catalytic residues that are responsible for sucrose hydrolysis are perfectly superimposable with the equivalent residues of the Bs homologue. The comparison of both structures, the mutagenesis data and the analysis of GH68 family multiple sequences alignment show a strong conservation of the sucrose hydrolytic machinery among levansucrases and also a structural equivalence of the Bs levansucrase Ca2+-binding site to the LsdA Cys(339)-Cys(395) disulphide bridge, suggesting similar fold-stabilizing roles. Despite the strong conservation of the sucrose-recognition site observed in LsdA, Bs levansucrase and GH32 family Thermotoga maritima invertase, structural differences appear around residues involved in the transfructosylation reaction.

AB - The endophytic Gram-negative bacterium Gluconacetobacter diazotrophicus SRT4 secretes a constitutively expressed levansucrase (LsdA, EC 2.4. 1.10), which converts sucrose into fructooligosaccharides and levan. The enzyme is included in GH (glycoside hydrolase) family 68 of the sequence-based classification of glycosidases. The three-dimensional structure of LsdA has been determined by X-ray crystallography at a resolution of 2.5 angstrom (1 angstrom= 0. 1 nm). The structure was solved by molecular replacement using the homologous Bacillus subtilis (Bs) levansucrase (Protein Data Bank accession code IOYG) as a search model. LsdA displays a five-bladed beta-propeller architecture, where the catalytic residues that are responsible for sucrose hydrolysis are perfectly superimposable with the equivalent residues of the Bs homologue. The comparison of both structures, the mutagenesis data and the analysis of GH68 family multiple sequences alignment show a strong conservation of the sucrose hydrolytic machinery among levansucrases and also a structural equivalence of the Bs levansucrase Ca2+-binding site to the LsdA Cys(339)-Cys(395) disulphide bridge, suggesting similar fold-stabilizing roles. Despite the strong conservation of the sucrose-recognition site observed in LsdA, Bs levansucrase and GH32 family Thermotoga maritima invertase, structural differences appear around residues involved in the transfructosylation reaction.

KW - five-bladed beta-propeller

KW - fructosyltransferase

KW - Gluconacetobacter diazotrophicus

KW - glycoside hydrolase

KW - glycoside hydrolase 68 family (GH68 family)

KW - levansucrase

KW - BACILLUS-SUBTILIS LEVANSUCRASE

KW - GLUCONACETOBACTER-DIAZOTROPHICUS

KW - MOLECULAR CHARACTERIZATION

KW - ENZYMATIC-PROPERTIES

KW - PROTEIN

KW - SRT4

KW - PURIFICATION

KW - REFINEMENT

KW - RESIDUES

KW - GENE

U2 - 10.1042/BJ20050324

DO - 10.1042/BJ20050324

M3 - Article

SN - 0264-6021

VL - 390

SP - 19

EP - 27

JO - Biochemical journal

JF - Biochemical journal

ER -