Rational engineering of mannosyl binding in the distal glycone subsites of Cellulomonas fimi endo-β-1,4-mannanase: mannosyl binding promoted at subsite -2 and demoted at subsite -3

TY - JOUR

T1 - Rational engineering of mannosyl binding in the distal glycone subsites of Cellulomonas fimi endo-β-1,4-mannanase

T2 - mannosyl binding promoted at subsite -2 and demoted at subsite -3

AU - Hekmat, Omid

AU - Lo Leggio, Leila

AU - Rosengren, Anna

AU - Kamarauskaite, Jurate

AU - Kolenova, Katarina

AU - Stålbrand, Henrik

PY - 2010/6/15

Y1 - 2010/6/15

N2 - To date, rational redesign of glycosidase active-site clefts has been mainly limited to the removal of essential functionalities rather than their introduction. The glycoside hydrolase family 26 endo-β-1,4-mannanase from the soil bacterium Cellulomonas fimi depolymerizes various abundant plant mannans. On the basis of differences in the structures and hydrolytic action patterns of this wild-type (but recombinantly expressed) enzyme and a homologous mannanase from Cellvibrio japonicus, two nonconserved amino acid residues at two distal glycone-binding subsites of the C. fimi enzyme were substituted, Ala323Arg at subsite -2 and Phe325Ala at subsite -3, to achieve inverted mannosyl affinities in the respective subsites, mimicking the Ce. japonicus enzyme that has an Arg providing mannosyl interactions at subsite -2. The X-ray crystal structure of the C. fimi doubly substituted mannanase was determined to 2.35 Å resolution and shows that the introduced Arg323 is in a position suitable for hydrogen bonding to mannosyl at subsite -2. We report steady-state enzyme kinetics and hydrolysis-product analyses using anion-exchange chromatography and a novel rapid mass spectrometric profiling method of 18O-labeled products obtained using H218O as a solvent. The results obtained with oligosacharide substrates show that although the catalytic efficiency (kcat/Km) is wild-type-like for the engineered enzyme, it has an altered hydrolytic action pattern that stems from promotion of substrate binding at subsite -2 (due to the introduced Arg323) and demotion of it at subsite -3 (to which removal of Phe325 contributed). However, kcat/Km decreased ∼1 order of magnitude with polymeric substrates, possibly caused by spatial repositioning of the substrate at subsite -3 and beyond for the engineered enzyme.

AB - To date, rational redesign of glycosidase active-site clefts has been mainly limited to the removal of essential functionalities rather than their introduction. The glycoside hydrolase family 26 endo-β-1,4-mannanase from the soil bacterium Cellulomonas fimi depolymerizes various abundant plant mannans. On the basis of differences in the structures and hydrolytic action patterns of this wild-type (but recombinantly expressed) enzyme and a homologous mannanase from Cellvibrio japonicus, two nonconserved amino acid residues at two distal glycone-binding subsites of the C. fimi enzyme were substituted, Ala323Arg at subsite -2 and Phe325Ala at subsite -3, to achieve inverted mannosyl affinities in the respective subsites, mimicking the Ce. japonicus enzyme that has an Arg providing mannosyl interactions at subsite -2. The X-ray crystal structure of the C. fimi doubly substituted mannanase was determined to 2.35 Å resolution and shows that the introduced Arg323 is in a position suitable for hydrogen bonding to mannosyl at subsite -2. We report steady-state enzyme kinetics and hydrolysis-product analyses using anion-exchange chromatography and a novel rapid mass spectrometric profiling method of 18O-labeled products obtained using H218O as a solvent. The results obtained with oligosacharide substrates show that although the catalytic efficiency (kcat/Km) is wild-type-like for the engineered enzyme, it has an altered hydrolytic action pattern that stems from promotion of substrate binding at subsite -2 (due to the introduced Arg323) and demotion of it at subsite -3 (to which removal of Phe325 contributed). However, kcat/Km decreased ∼1 order of magnitude with polymeric substrates, possibly caused by spatial repositioning of the substrate at subsite -3 and beyond for the engineered enzyme.

KW - Amino Acid Substitution

KW - Binding Sites

KW - Carbohydrate Sequence

KW - Cellulomonas

KW - Conserved Sequence

KW - Crystallography, X-Ray

KW - Hydrolysis

KW - Mannose

KW - Mannosidases

KW - Mutagenesis, Site-Directed

KW - Protein Binding

KW - Protein Engineering

KW - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

KW - Substrate Specificity

U2 - 10.1021/bi100097f

DO - 10.1021/bi100097f

M3 - Journal article

C2 - 20426480

SN - 0006-2960

VL - 49

SP - 4884

EP - 4896

JO - Biochemistry

JF - Biochemistry

IS - 23

ER -