Structure, activity, and stability of metagenome-derived glycoside hydrolase family 9 endoglucanase with an N-terminal Ig-like domain

Abstract

A metagenome-derived glycoside hydrolase family 9 enzyme with an N-terminal immunoglobulin-like (Ig-like) domain, leaf-branch compost (LC)-CelG, was characterized and its crystal structure was determined. LC-CelG did not hydrolyze p-nitrophenyl cellobioside but hydrolyzed CM-cellulose, indicating that it is endoglucanase. LC-CelG exhibited the highest activity at 70°C and >80% of the maximal activity at a broad pH range of 5-9. Its denaturation temperature was 81.4°C, indicating that LC-CelG is a thermostable enzyme. The structure of LC-CelG resembles those of CelD from Clostridium thermocellum (CtCelD), Cel9A from Alicyclobacillus acidocaldarius (AaCel9A), and cellobiohydrolase CbhA from C. thermocellum (CtCbhA), which show relatively low (29-31%) amino acid sequence identities to LC-CelG. Three acidic active site residues are conserved as Asp194, Asp197, and Glu558 in LC-CelG. Ten of the thirteen residues that form the substrate binding pocket of AaCel9A are conserved in LC-CelG. Removal of the Ig-like domain reduced the activity and stability of LC-CelG by 100-fold and 6.3°C, respectively. Removal of the Gln40- and Asp99-mediated interactions between the Ig-like and catalytic domains destabilized LC-CelG by 5.0°C without significantly affecting its activity. These results suggest that the Ig-like domain contributes to the stabilization of LC-CelG mainly due to the Gln40- and Asp99-mediated interactions. Because the LC-CelG derivative lacking the Ig-like domain accumulated in Escherichia coli cells mostly in an insoluble form and this derivative accumulated in a soluble form exhibited very weak activity, the Ig-like domain may be required to make the conformation of the active site functional and prevent aggregation of the catalytic domain.

Keywords: GH family 9; crystal structure; endoglucanase; immunoglobulin-like (Ig-like) domain; metagenome; thermal stability.

Figure 1

Alignment of amino acid sequences of LC-CelG, CtCelD, AaCel9A, and CtCbhA. The accession numbers are KF626654 for LC-CelG, CAA28255 for CtCelD, ACV59481 for AaCel9A, and ABN51651 for CtCbhA. The amino acid sequence of LC-CelG without a putative signal peptide (Residues 20–577) and the corresponding regions of other three proteins are shown. The amino acid residues, which are conserved in all four proteins, are denoted with white letters and highlighted in black. The amino acid residues, which are conserved in two or three different proteins, are highlighted in gray. The ranges of the secondary structures of LC-CelG (βa–βh strands for the Ig-like domain, and β1–β6 strands and α1–α13 helices for the cellulase domain) are shown above its sequence based on its crystal structure. The residues that form the catalytic site (Asp194, Asp197, and Glu558), substrate binding site, Zn site (Cys150, Asp166, His167, and His193), Ca1 site (Asp225, Glu232, Asn235, Val237, Asp239, and Asp242), and Ca2 site (Asp351, Asp353, Asp356, Asp357, and Val395) are indicated by “*,” “+,” “z,” “1,” and “2,” respectively, above the LC-CelG sequence. The residues that form the Ca3 site of CtCelD and AaCel9A are indicated by “3” below the sequences. The position, at which N-terminal Ig-like domain of LC-CelG is truncated to construct His-ΔIg-CelG, is shown by solid arrow head above the LC-CelG sequence. Likewise, Gln40 and Asp99 of LC-CelG, which are mutated in this study, are indicated by open arrow heads.

Figure 2

Optimum pH and temperature for activity of His-LC-CelG. The pH (A) and temperature (B) dependencies of the enzymatic activity of His-LC-CelG are shown. The activity was determined at 60°C at the pH indicated (A) or at pH 7.0 and the temperatures indicated (B) using 1% (w/v) CM-cellulose as a substrate, as described in Materials and Methods. The buffers used to analyze the pH dependence of the activity were 100 mM sodium citrate (pH 4.0–6.5), 100 mM sodium phosphate (pH 6.0–8.0), and 100 mM Glycine-NaOH (pH 8.0–10.0). The experiment was performed at least twice, and errors from the average values are indicated by vertical lines.

Figure 3

Thermal denaturation curves of His-LC-CelG and its derivatives. The thermal denaturation curves of His-LC-CelG (thin solid line), His-ΔIg-CelG (thick solid line), His-Q40A-CelG (thin dashed line), His-D99A-CelG (thick dotted line), and His-Q40A/D99A-CelG (thick dashed line) are shown. These curves were obtained in the presence of 5 mM CaCl₂ at pH 7.0 by monitoring the change in CD values at 222 nm as described in Materials and Methods.

Figure 4

Crystal structure of LC-CelG. (A) A stereo view of the overall structure of LC-CelG. The structure of LC-CelG is superimposed on that of CtCelD (PDB code 1CLC). For the LC-CelG structure, the Ig-like and catalytic domains are colored cyan and green, respectively. One zinc and two calcium ions (Ca1 and Ca2) are shown as orange and yellow spheres, with the numbers of the calcium ions indicated. Three active site residues (Asp194, Asp197, and Glu558) are indicated by green stick models, in which the oxygen and nitrogen atoms are colored red and blue, respectively. The entire CtCelD structure, including one zinc and three calcium ions (Ca1–Ca3), is colored gray. Three active site residues (Asp198, Asp201, and Glu555) are indicated by gray stick models. They are labeled in parentheses. (B) The structure around the substrate binding pocket. The structure around the substrate binding pocket of LC-CelG is superimposed on those of AaCel9A (PDB code 3H3K) and CtCbhA (PDB code 1RQ5) in complex with cellotetraose. The residues forming the substrate binding pocket of LC-CelG are indicated by green stick models, the corresponding residues and cellotetraose in the AaCel9A structure are indicated by gray stick models, and the corresponding residues and cellotetraose in the CtCbhA structure are indicated by yellow stick models. In these stick models, which are labeled with the same colors, the oxygen and nitrogen atoms are colored red and blue, respectively. The six subsites are labeled from −4 to +2. The water molecule is shown as red sphere. Dashed lines represent hydrogen bonds. (C–E) Electron density around the binding sites of zinc ion (C), and calcium ions Ca1 (D) and Ca2 (E). The zinc and calcium ions are shown as orange and yellow spheres, respectively. The water molecule is shown as red sphere. The residues co-ordinated with these metal ions are indicated as shown in (B). In (C) and (E), the 2F_o–F_c maps contoured at the 2.0σ and 4.0σ levels are shown in magenta and blue, respectively. In (D), the 2F_o–F_c map contoured at the 1.5σ level is shown in orange. In these figures, the F_o–F_c omit maps contoured at the 5.0σ level are also shown in red. Numbers represent the coordinate bond lengths (Å). (F) The structure around Gln40 and Asp99. Gln40 and Asp99 located in the Ig-like domain are shown as cyan stick models. The residues that are located in the catalytic domain and form hydrogen bonds with Gln40 (Ala494, Ser503, and As504) or salt bridge with Asp99 (Arg545) are shown by green stick models. In these stick models, the oxygen and nitrogen atoms are colored red and blue, respectively.

Figure 5

CD spectra of His-LC-CelG and its derivatives. The far-UV CD spectra of His-LC-CelG (thin solid line), His-ΔIg-CelG (thick solid line), His-Q40A-CelG (thin dashed line), His-D99A-CelG (thick dotted line), and His-Q40A/D99A-CelG (thick dashed line) were measured at pH 7.0 and 25°C, as described in Materials and Methods.