Crystal structure of ChbG from Klebsiella pneumoniae reveals the molecular basis of diacetylchitobiose deacetylation

Abstract

The chitobiose (chb) operon is involved in the synthesis of chitooligosaccharide and is comprised of a BCARFG gene cluster. ChbG encodes a chitooligosaccharide deacetylase (CDA) which catalyzes the removal of one acetyl group from N,N'-diacetylchitobiose. It is considered a novel type of CDA due to its lack of sequence homology. Although there are various structural studies of CDAs linked to the kinetic properties of the enzyme, the structural information of ChbG is unavailable. In this study, the crystal structure of ChbG from Klebsiella pneumoniae is provided. The molecular basis of deacetylation of diacetylchitobiose by ChbG is determined based on structural analysis, mutagenesis, biophysical analysis, and in silico docking of the substrate, diacetylchitobiose. This study contributes towards a deeper understanding of chitin and chitosan biology, as well as provides a platform to engineer CDA biocatalysts.

Fig. 1. Crystal structure of ChbG from Klebsiella pneumoniae (kpChbG).

a Catalytic reaction of ChbG. b Size-exclusion chromatography (SEC) of Ni²⁺-affinity purified ChbG. Loaded fractions are indicated by the horizontal black bar. Inset: SDS-PAGE assessment of purity. M: size marker and B: before SEC loading. c ESI-MS analysis of deacetylation of N,N’-diacetylchitobiose ((GlcNAc)₂) to N-acetyl-β-glucosaminyl-glucosamine (GlcN-GlcNAc) by kpChbG. d Cartoon representation of two kpChbG molecules (molecule A and B) presented in an asymmetric unit. e Superposition of the structures in one asymmetric unit. f Rainbow-colored cartoon representation of monomeric kpChbG. The polypeptide chain from the N-terminus to the C-terminus is colored blue to red. Helices and sheets are labeled α and β, respectively. ACT represents acetate and Ion is the metal ion. g Putty representation showing B-factor distribution in the order of B-factor values using rainbow colors (red to violet).

Fig. 2. Dimeric structure of kpChbG.

a Multi-angle light scattering (MALS) profile of the SEC eluted kpChbG peak. The red line indicates the experimental molecular mass analyzed by MALS. b Crystallographic packing symmetry analyzed by PyMOL. Two kpChbG molecules (A and B) found in the asymmetric unit are colored in blue and orange ribbons, while the other symmetric molecules are colored gray. c Table summarizing the interaction details of each type of interface analyzed by the PISA server. ACT and CSS indicate acetate and the complex formation significance score, respectively. Cartoon representation of the putative dimeric structure of the A/B complex (d) and the A/B’ complex (e). The red-dashed lines of the A/B complex and the A/B’ complex represent the magnified region of the interfaces shown in f and g, respectively. Red-dashed lines and black-dashed lines indicate salt bridges and hydrogen bonds, respectively. Residues that are involved in dimer formation are labeled. h SEC profiles of wild type kpChbG and mutant kpChbG at putative interface binding sites. i ESI-MS analysis of deacetylation of N,N’-diacetylchitobiose to N-acetyl-β-glucosaminyl-glucosamine by the dimer disruption mutant, E23K/D224K.

Fig. 3. Identification of the Zn2+ (Ion) and acetate (ACT) ion in the putative active site of kpChbG.

a The electron density map around the metal ion binding site of kpChbG. The 2Fo-Fc map contoured at the 1σ level is shown. Residues coordinated with the metal ion are labeled. b Electrostatic surface representation of kpChbG. The scale bar ranges from −7.3 kT/e (red) to 7.3 kT/e (blue). The black dashed box shows the magnified region of the putative active site. c Cartoon representation of the putative active site of kpChbG. Residues involved in coordination of the metal ion and acetate ion are colored red and blue, respectively. Critical residues involved in kpChbG catalysis (D10 and H206) are colored green. d Table showing the metal ion concentration in kpChbG (µg/kg) analyzed by ICP-MS. n.d.: not determined.

Fig. 4. Comparison of kpChbG with its structural homologs: hypothetical proteins EF3048 and TTHB029.

a Sequence alignment using Clustal Omega. Completely conserved and partially conserved residues are colored red and blue, respectively. The positions of the α3/β3 connecting loop and the α9 helix are indicated by black squares. Conserved residues that are involved in the formation of the putative active site are indicated by asterisks (*). Structural superposition of kpChbG (light blue) with EF3048 (yellow) (b) and TTHB029 (magenta) (c). Black-dashed boxes indicate the magnified region of the putative active site. d Cartoon representation showing the specific region containing the α3/β3 connecting loop and the α9 helix around the putative active site which might be critical for substrate specificity. kpChbG (light blue color), EF3048 (yellow color), and TTHB029 (magenta color) were superposed for structural comparison. e Overall surface feature of the three homologous proteins. The black-dashed circle indicates the ChbG active site containing the α3/β3 connecting loop and the α9 helix.

Fig. 5. Comparison of kpChbG with other CE4 enzyme family members.

a Sequence alignment based on structural alignment using PROMALS3D. The five residues forming the active site, which are critical for activity of CE4 enzymes, are indicated by asterisks (*). Four of the five residues forming the active site structurally aligned with each other are shown in red, whereas one residue, H125 on kpChbG, which does not structurally aligned, is shown in blue. The putative corresponding histidine residue with H125 of kpChbG, identified by locating the similar position in the active site of the CE4 enzyme, is linked by a dashed black line. Six loops (Loop 1~6) involved in the substrate specificity control as characterized in CE4 enzymes are indicated by colored lines under the corresponding residues. Residues used to form α7 helix in kpChbG are highlighted using orange color. b Structural superposition of kpChbG (light blue) with spPgdA (gray) and vcCDA (cyan). c Magnified region of the active site that is marked by a black-dashed box in b. Conserved residues involved in the formation of the active site are labeled. d Structural comparison of six loops in kpChbG with those of other CE4 enzymes. α7 helix, which is only present in kpChbG, is indicated by an orange-colored circle.

Fig. 6. Sequence comparison of prokaryote and eukaryote ChbG homologs.

a Sequence alignment using Clustal Omega Completely conserved and partially conserved residues are shown in red and blue, respectively. The position of the α3/β3 connecting loop is indicated by a black square. Conserved residues that are involved in the formation of the putative active site and the A/B’ dimer are indicated by asterisks (*) and hashes (#), respectively. Kp: Klebsiella pneumoniae, Ec: Escherichia coli, St: Salmonella typhimurium, Yp: Yersinia pestis, Hs: Homo sapiens, Mm: Mus musculus, Dr: Danio rerio. b Cartoon representation of kpChbG colored according to the degree of amino acid sequence conservation generated by the ConSurf server. c Surface representation of kpChbG. d Three close-up views of panel (b) showing the amino acids of the putative active site, the first dimer PPI, and the second dimer PPI.

Fig. 7. Proposed model of diacetylchitobiose deacetylation mechanism by kpChbG.

a Cartoon representation of diacetylchitobiose substrate docking into the putative active site of kpChbG. Magnified view of docking site in the surface figure is provided. b Close-up of the kpChbG active site after diacetylchitobiose docking. c The electron density map around the active site of kpChbG. The 2Fo-Fc density map contoured at the 1σ level is shown. d Structural superposition of kpChbG/diacetylchitobiose complex model (blue color) with vcCDA/disaccharide DP2 complex (Cyan color). The location of two substrates are indicated by black arrows. Five core residues at the active site were labeled. e Surface representation of diacetylchitobiose docking in the putative active site of kpChbG. The locations of α7 helix and α9 helix are indicated on the surface. Red-colored surface indicates Zn²⁺ ion binding region. f The final dimeric model of kpChbG with bound substrate in the active site. g Proposed Zn²⁺-assisted enzymatic mechanism of kpChbG.