title: Aglycone specificity of Thermotoga neapolitana β-glucosidase 1A modified by mutagenesis, leading to increased catalytic efficiency in quercetin-3-glucoside hydrolysis.
abstract: BACKGROUND
  The thermostable β-glucosidase (TnBgl1A) from Thermotoga neapolitana is a promising biocatalyst for hydrolysis of glucosylated flavonoids and can be coupled to extraction methods using pressurized hot water. Hydrolysis has however been shown to be dependent on the position of the glucosylation on the flavonoid, and e.g. quercetin-3-glucoside (Q3) was hydrolysed slowly. A set of mutants of TnBgl1A were thus created to analyse the influence on the kinetic parameters using the model substrate para-nitrophenyl-β-D-glucopyranoside (pNPGlc), and screened for hydrolysis of Q3.
  
  
  RESULTS
  Structural analysis pinpointed an area in the active site pocket with non-conserved residues between specificity groups in glycoside hydrolase family 1 (GH1). Three residues in this area located on β-strand 5 (F219, N221, and G222) close to sugar binding sub-site +2 were selected for mutagenesis and amplified in a protocol that introduced a few spontaneous mutations. Eight mutants (four triple: F219L/P165L/M278I, N221S/P165L/M278I, G222Q/P165L/M278I, G222Q/V203M/K214R, two double: F219L/K214R, N221S/P342L and two single: G222M and N221S) were produced in E. coli, and purified to apparent homogeneity. Thermostability, measured as Tm by differential scanning calorimetry (101.9°C for wt), was kept in the mutated variants and significant decrease (ΔT of 5-10°C) was only observed for the triple mutants. The exchanged residue(s) in the respective mutant resulted in variations in KM and turnover. The KM-value was only changed in variants mutated at position 221 (N221S) and was in all cases monitored as a 2-3 × increase for pNPGlc, while the KM decreased a corresponding extent for Q3.Turnover was only significantly changed using pNPGlc, and was decreased 2-3 × in variants mutated at position 222, while the single, double and triple mutated variants carrying a mutation at position 221 (N221S) increased turnover up to 3.5 × compared to the wild type. Modelling showed that the mutation at position 221, may alter the position of N291 resulting in increased hydrogen bonding of Q3 (at a position corresponding to the +1 subsite) which may explain the decrease in KM for this substrate.
  
  
  CONCLUSIONS
  These results show that residues at the +2 subsite are interesting targets for mutagenesis and mutations at these positions can directly or indirectly affect both KM and turnover. An affinity change, leading to a decreased KM, can be explained by an altered position of N291, while the changes in turnover are more difficult to explain and may be the result of smaller conformational changes in the active site.
authors.affiliation: Biotechnology, Dept of Chemistry, Lund University, P,O, Box 124, SE-221 00 Lund, Sweden.
authors.forename: Samiullah
authors.initials: S
authors.lastname: Khan
authors.forename: Tania
authors.initials: T
authors.lastname: Pozzo
authors.forename: Márton
authors.initials: M
authors.lastname: Megyeri
authors.forename: Sofia
authors.initials: S
authors.lastname: Lindahl
authors.forename: Anders
authors.initials: A
authors.lastname: Sundin
authors.forename: Charlotta
authors.initials: C
authors.lastname: Turner
authors.forename: Eva Nordberg
authors.initials: EN
authors.lastname: Karlsson
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D055162:Biocatalysis; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011794:Quercetin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D046949:Thermotoga neapolitana / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001617:beta-Glucosidase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; C016527:isoquercitrin; D011794:Quercetin; D001617:beta-Glucosidase