Characterization of a new rhamnogalacturonan acetyl esterase from Bacillus halodurans C-125 with a new putative carbohydrate binding domain

Abstract

BH1115 is a gene from Bacillus halodurans strain C-125 that hypothetically encodes a rhamnogalacturonan acetyl esterase (RGAE) of the CE-12 family. As confirmation, this gene was cloned, and the product was expressed in Escherichia coli strain Rosetta (DE3) cells and purified. The enzyme obtained was monomeric, with a molecular mass of 45 kDa, and exhibited alkaliphilic properties. A study of the inhibition of the activity by some modulators confirmed that the catalytic triad for the esterase activity was Ser-His-Asp. This enzyme also presents broad substrate specificity and is active toward 7-aminocephalosporanic acid, cephalosporin C, p-nitrophenyl acetate, beta-naphthyl acetate, glucose pentaacetate, and acetylated xylan. Moreover, RGAE from B. halodurans achieves a synergistic effect with xylanase A toward acetylated xylan. As a member of the SGNH family, it does not adopt the common alpha/beta hydrolase fold. The homology between the folds of RGAE from Aspergillus aculeatus and the hypothetical YxiM precursor from Bacillus subtilis, which both belong to the SGNH family, illustrates the divergence of such proteins from a common ancestor. Furthermore, the enzyme possesses a putative substrate binding region at the N terminus of the protein which has never been described to date for any RGAE.

FIG. 1.

SDS-PAGE of the BH1115 gene product (BhRGAE) after 14 h of induction with 1 mM IPTG. Each lane contained 5 mg protein. Lane M, molecular mass markers (maltose-binding protein β-galactosidase, 175 kDa; maltose-binding protein paramyosin, 83 kDa; glutamic dehydrogenase, 62 kDa; aldolase, 47.5 kDa; triosephosphate isomerase, 32.5 kDa; β-lactoglobulin A, 25 kDa; lysozyme, 16.5 kDa; aprotinin, 6.5 kDa); lane 1, crude extract; lane 2, Resource Q column pooled fraction; lane 3, His-trap FF column pooled fraction.

FIG. 2.

Multiple alignment of some members of the SGNH family. ESPript (9) outputs were obtained from the BhRGAE, AaRGAE (Protein Data Bank accession no. 1deo), and hypothetical YxiM precursor (Protein Data Bank accession no. 2o14) sequences from the SWISSPROT databank (10) and aligned with CLUSTAL-W (32). Block I has the characteristic GDS sequence motif. Block II has a Gly as the only conserved residue in the members of this family. GXND is the consensus sequence in block III. Finally, block V has the DXXHP conserved sequence that is placed in a variable loop, whereas the other blocks are found at the C-terminal ends of the central β-sheet (20). Sequences are grouped according to similarity. Rectangles below the sequences show accessible residues in black, intermediate ones in gray, and buried ones in white. Residues strictly conserved among groups are shown in white font on a dark background. Residues conserved within a group but showing significant differences between groups are in gray font. The symbols above the blocks of sequences represent the secondary structure. Triangles represent the locations of the active sites.

FIG. 3.

Ribbon diagram of the monomeric structure of recombinant BhRGAE. (A) Overview of the whole modeled structure. (B) Representation of the catalytic domain. (C) Representation of the putative substrate binding domain. The amino acids of the active site in panels A and B are represented in the same shade. Dark-gray parts of the three representations indicate the presence of β-turns. These figures were rendered using SWISS-MODEL and Swiss-PdbViewer (10).

FIG. 4.

Topology diagrams. (A) Diagram of recombinant BhRGAE. (B) Representation of RGAE (Protein Data Bank accession no. 1deo). (C) Representation of hypothetical YxiM precursor (Protein Data Bank accession no. 2o14). Helices are represented by circles, and β-strands are represented by triangles. White circles represent 3₁₀ or other small helices in the structures. White triangles represent those strands not contained in the central β-sheet. The diagrams were made using the TOPS program (19).