Dataset Information

NMR and Fluorescence Spectroscopies Reveal the Preorganized Binding Site in Family 14 Carbohydrate-Binding Module from Human Chitotriosidase.

ABSTRACT: Carbohydrate-binding modules (CBM) play important roles in targeting and increasing the concentration of carbohydrate active enzymes on their substrates. Using NMR to get the solution structure of CBM14, we can gain insight into secondary structure elements and intramolecular interactions with our assigned nuclear overhauser effect peaks. This reveals that two conserved aromatic residues (Phe437 and Phe456) make up the hydrophobic core of the CBM. These residues are also responsible for connecting the two ?-sheets together, by being part of ?2 and ?4, respectively, and together with disulfide bridges, they create CBM14's characteristic "hevein-like" fold. Most CBMs rely on aromatic residues for substrate binding; however, CBM14 contains just a single tryptophan (Trp465) that together with Asn466 enables substrate binding. Interestingly, an alanine mutation of a single residue (Leu454) located behind Trp465 renders the CBM incapable of binding. Fluorescence spectroscopy performed on this mutant reveals a significant blue shift, as well as a minor blue shift for its neighbor Val455. The reduction in steric hindrance causes the tryptophan to be buried into the hydrophobic core of the structure and therefore suggests a preorganized binding site for this CBM. Our results show that both Trp465 and Asn466 are affected when CBM14 interacts with both (GlcNAc)3 and ?-chitin, that the binding interactions are weak, and that CBM14 displays a slightly higher affinity toward ?-chitin.

SUBMITTER: Madland E

PROVIDER: S-EPMC6933781 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

NMR and Fluorescence Spectroscopies Reveal the Preorganized Binding Site in Family 14 Carbohydrate-Binding Module from Human Chitotriosidase.

Madland Eva E Crasson Oscar O Vandevenne Maryléne M Sørlie Morten M Aachmann Finn L FL

ACS omega 20191209 26

Carbohydrate-binding modules (CBM) play important roles in targeting and increasing the concentration of carbohydrate active enzymes on their substrates. Using NMR to get the solution structure of CBM14, we can gain insight into secondary structure elements and intramolecular interactions with our assigned nuclear overhauser effect peaks. This reveals that two conserved aromatic residues (Phe437 and Phe456) make up the hydrophobic core of the CBM. These residues are also responsible for connecti ...[more]

PMID: 31891077

Similar Datasets

Project description:BackgroundHighly efficient enzymatic saccharification of pretreated lignocellulose is a key step in achieving lignocellulosic biorefinery. Cellobiohydrolase I (Cel7A) secreted by Trichoderma reesei is an industrially used cellulase that possesses carbohydrate-binding module 1 (TrCBM1) at the C-terminal domain. The nonproductive binding of TrCBM1 to lignin significantly decreases the enzymatic saccharification efficiency and increases the cost of biomass conversion because of the additionally required enzymes. Understanding the interaction mechanism between lignin and TrCBM1 is essential for realizing a cost-effective biofuel production; however, the binding sites in lignin have not been clearly elucidated.ResultsThree types of 13C-labeled β-O-4 lignin oligomer models were synthesized and characterized. The 2D 1H-13C heteronuclear single-quantum correlation (HSQC) spectra of the 13C-labeled lignin models confirmed that the three types of the 13C labels were correctly incorporated in the (1) aromatic rings and β positions, (2) α positions, and (3) methoxy groups, respectively. The TrCBM1-binding sites in lignin were analyzed by observing NMR chemical shift perturbations (CSPs) using the synthetic 13C-labeled β-O-4 lignin oligomer models. Obvious CSPs were observed in signals from the aromatic regions in oligomers bound to TrCBM1, whereas perturbations in the signals from aliphatic regions and methoxy groups were insignificant. These findings indicated that hydrophobic interactions and π-π stacking were dominating factors in nonproductive binding. The synthetic lignin models have two configurations whose terminal units were differently aligned and donated C(I) and C(II). The C(I) ring showed remarkable perturbation compared with the C(II), which indicated that the binding of TrCBM1 was markedly affected by the configuration of the lignin models. The long-chain lignin models (degree of polymerization (DP) 4.16-4.70) clearly bound to TrCBM1. The interactions of TrCBM1 with the short-chain lignin models (DP 2.64-3.12) were insignificant, indicating that a DP greater than 4 was necessary for TrCBM1 binding.ConclusionThe CSP analysis using 13C-labeled β-O-4 lignin oligomer models enabled the identification of the TrCBM1 binding sites in lignins at the atomic level. This specific interaction analysis will provide insights for new molecular designs of cellulase having a controlled affinity to cellulose and lignin for a cost-effective biorefinery process.

Dataset Information

NMR and Fluorescence Spectroscopies Reveal the Preorganized Binding Site in Family 14 Carbohydrate-Binding Module from Human Chitotriosidase.

Publications

NMR and Fluorescence Spectroscopies Reveal the Preorganized Binding Site in Family 14 Carbohydrate-Binding Module from Human Chitotriosidase.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets