βγ-Crystallination Endows a Novel Bacterial Glycoside Hydrolase 64 with Ca2+-Dependent Activity Modulation

Abstract

The prokaryotic βγ-crystallins are a large group of uncharacterized domains with Ca²⁺-binding motifs. We have observed that a vast number of these domains are found appended to other domains, in particular, the carbohydrate-active enzyme (CAZy) domains. To elucidate the functional significance of these prospective Ca²⁺ sensors in bacteria and this widespread domain association, we have studied one typical example from Clostridium beijerinckii, a bacterium known for its ability to produce acetone, butanol, and ethanol through fermentation of several carbohydrates. This novel glycoside hydrolase of family 64 (GH64), which we named glucanallin, is composed of a βγ-crystallin domain, a GH64 domain, and a carbohydrate-binding module 56 (CBM56). The substrates of GH64, β-1,3-glucans, are the targets for industrial biofuel production due to their plenitude. We have examined the Ca²⁺-binding properties of this protein, assayed its enzymatic activity, and analyzed the structural features of the β-1,3-glucanase domain through its high-resolution crystal structure. The reaction products resulting from the enzyme reaction of glucanallin reinforce the mixed nature of GH64 enzymes, in contrast to the prevailing notion of them being an exotype. Upon disabling Ca²⁺ binding and comparing different domain combinations, we demonstrate that the βγ-crystallin domain in glucanallin acts as a Ca²⁺ sensor and enhances the glycolytic activity of glucanallin through Ca²⁺ binding. We also compare the structural peculiarities of this new member of the GH64 family to two previously studied members.IMPORTANCE We have biochemically and structurally characterized a novel glucanase from the less studied GH64 family in a bacterium significant for fermentation of carbohydrates into biofuels. This enzyme displays a peculiar property of being distally modulated by Ca²⁺ via assistance from a neighboring βγ-crystallin domain, likely through changes in the domain interface. In addition, this enzyme is found to be optimized for functioning in an acidic environment, which is in line with the possibility of its involvement in biofuel production. Multiple occurrences of a similar domain architecture suggest that such a "βγ-crystallination"-mediated Ca²⁺ sensitivity may be widespread among bacterial proteins.

Keywords: Ca2+ binding; calcium binding; calcium-induced activity; crystal structure; glucanase; glycoside hydrolase; βγ-crystallins.

FIG 1

Modular organization of different domains of glucanallin protein from Clostridium beijerinckii. The protein comprises three distinct modules. This protein is a representative of the modular architecture observed in at least six other proteins: WP_088448808.1 (from Roseateles terrae), WP_073463826.1 (from Rhizobacter sp. strain OV335), WP_082011168.1 (from Methylibium sp. strain YR605), KQV60387.1 (from Pelomonas sp. strain Root1217), KQW44766.1 (from Pelomonas sp. strain Root405), and WP_088481862.1 (from Pelomonas puraquae). These example sequences have been aligned to depict the shared regions.

FIG 2

(A) Trp fluorescence emission spectra of glucanallin with increasing Ca²⁺ concentrations. Spectra are recorded by excitation at 295 nm. (B) Near-UV CD spectra of glucanallin upon Ca²⁺ titrations. (C) ITC thermogram of Ca²⁺ binding to glucanallin.

FIG 3

TLC pattern of hydrolytic products of laminarin (laminaribiose, L2; laminaritriose, L3; laminaritetraose, L4; and laminaripetaose, L5) as reaction products by glucanallin and CbLPHase.

FIG 4

DNS reagent-based enzyme activity assessment. (A) The activities of glucanallin and CbLPHase are compared in the absence or presence of Ca²⁺ at pH 5.0. Although glucanallin exhibits a strong enhancement in activity due to Ca²⁺, the change in CbLPHase is minor. (B) The activity shown by the glucanallin two-domain protein is similar to glucanallin, while disabling the Ca²⁺-binding motif in the glucanallin-CBD construct eliminates its Ca²⁺ responsiveness. (C) The activity of the glucanallin two-domain protein was compared at pH 5.0 and 7.5. The sensitivity to Ca²⁺ is greater at pH 5.0. (D) The activities of all the four proteins were compared without or with Ca²⁺. In all of the panels, “apo” refers to the condition in which Ca²⁺ was chelated away using EGTA, whereas “holo”’ means the presence of Ca²⁺. The values of standard deviations were used to plot the error bars in all of the graphs.

FIG 5

Schematic representation of four protein constructs used in this work (B to E), along with the parental molecule (A). (A) Natural protein encoded by the gene corresponding to C. beijerinckii glucanase. (B) Glucanallin was prepared and studied. It lacks the N-terminal signal sequence presented in the naturally translated protein. (C) Glucanallin two-domain protein consists of the βγ-crystallin and LPHase domains. (D) Glucanallin-CBD differs from glucanallin only in that the βγ-crystallin Ca²⁺-binding motifs in the former have been disabled. (E) CbLPHase, a single-domain protein.

FIG 6

Equilibrium unfolding studies of glucanallin and CbLPHase at pH 7.5 and 5.0. To evaluate the chemical stability, Trp fluorescence emmision maxima were plotted against increasing concentrations of GdmCl for glucanallin at pH 7.5 (A), CbLPHase at pH 7.5 (B), glucanallin at pH 5.0 (D), and CbLPHase at pH 5.0 (E). During the unfolding of CbLPHase at pH 7.5, precipitation of partially unfolded species led to the absence of data points between 1 and 3 M in panel B. The far UV-CD spectra at selected temperatures were also plotted to compare the secondary structural changes for glucanallin at pH 7.5 (C) and glucanallin at pH 5.0 (F).

FIG 7

(A) Cartoon representation of the crystal structure of CbLPHase solved at 1.86-Å resolution. Secondary structures were rendered using the DSSP plug-in in PyMOL. (B) Electrostatic surface representation of the crystal structure showing the wide groove of the ligand-binding cleft. A triple-helical polymer curdlan is modeled to highlight that the groove can accommodate complex helical polymers. The coordinates for the triple- helical curdlan were downloaded from PolySacDb (63, 64). (C) Stereo representation of the superposition of SmLPHase in ligand-bound (blue, PDB ID 3GD9) and unbound (yellow, PDB ID 3GD0) forms with CbLPHase (purple) highlighting the catalytic residues.

FIG 8

(A) Structural comparison of CbLPHase with different TLPs. TLP from banana is shown in gray (PDB ID 1Z3Q), that from cherry is in cyan (PDB ID 2AHN), and that from apple is in teal (PDB ID 3ZS3). (B) Structure of CbLPHase showing four distinct regions (in blue, pink, red, and yellow). The topology diagram of CbLPHase shows the corresponding regions with highlighted colors. (C) Structural overlap of SmLPHase (yellow, PDB ID 3GD0) and CbLPHase (green, 5H4E) to highlight the differences between two structures. (D) Structural comparison between CbLPHase (PDB ID 5H4E) and PbBgl64A (PBD ID 5H9Y). Region III is different in CbLPHase, whereas region IV is missing in PbBgl64A. PbBgl64A was crystallized with laminarihexaose (23).