Structural Dissection of the Active Site of <i>Thermotoga maritima</i> β‑Galactosidase Identifies Key Residues for Transglycosylating Activity

Glycoside hydrolases, specifically β-galactosidases, can be used to synthesize galacto-oligosaccharides (GOS) due to the transglycosylating (secondary) activity of these enzymes. Site-directed mutagenesis of a thermoresistant β-galactosidase from <i>Thermotoga maritima</i> has been carried out to study the structural basis of transgalactosylation and to obtain enzymatic variants with better performance for GOS biosynthesis. Rational design of mutations was based on homologous sequence analysis and structural modeling. Analysis of mutant enzymes indicated that residue W959, or an alternative aromatic residue at this position, is critical for the synthesis of β-3′-galactosyl-lactose, the major GOS obtained with the wild-type enzyme. Mutants W959A and W959C, but not W959F, showed an 80% reduced synthesis of this GOS. Other substitutions, N574S, N574A, and F571L, increased the synthesis of β-3′-galactosyl-lactose about 40%. Double mutants F571L/N574S and F571L/N574A showed an increase of about 2-fold

Thermal stability of the mutants obtained by rational design.

<p>(A) Relative initial GOX activity in the culture medium of the yeast transformants. (B) Residual GOX activity after incubation at 60°C for 45 (dark grey bars) or 80 (light grey bars) minutes. Error bars represent standard deviation of triplicates. Significant differences (p < 0.05 or p < 0.01) with the wild-type enzyme are indicated by one or two asterisks, respectively.</p

Structural detail in the vicinity of residues critical for GOX stability.

<p>Relevant interactions are depicted with dashed lines. (A) T554 in wild-type enzyme (left panel) and M554 in T554M mutant (right panel). Θ = 60°; d = 5 Å. (B) Q345 in wild-type enzyme. (C) R90 and E509 in the double mutant Q90R/Y509E. The subunit of origin is indicated in parenthesis. N-acetyl-glucosamine modification is colored in orange.</p

Thermal stability of T554M mutant obtained by random mutagenesis.

<p>(A) Relative initial GOX activity in the culture medium of the yeast transformants. (B) Residual GOX activity after incubation at 60°C for 45 (dark grey bars) or 80 (light grey bars) minutes. Error bars represent standard deviation of triplicates. Significant differences (p < 0.05 or p < 0.01) with the wild-type enzyme are indicated by one or two asterisks, respectively.</p

Analysis of glycosylation pattern and specific activity of selected GOX mutants.

<p>(A) Proteins released to the culture medium were analyzed without (C) or with (E) EndoH treatment. GOX was identified as a differential band compared to a yeast control transformed with the same plasmid lacking the GOX gene. Migration of the deglycosylated GOX (dGOX) in the E lanes is indicated by an arrow and that of the glycosylated GOX (gGOX) in the C lanes is shown by a bracket. (B) Relative intrinsic activity of GOX mutants. Error bars represent standard deviation of analytical triplicates. Significant differences (p < 0.01) with the wild-type enzyme are indicated by asterisks.</p

Mutations designed to introduce new salt bridges in <i>A</i>. <i>niger</i> GOX.

<p>(A) Q469K/L500D; (B) Q142R/L569E; (C) Q90R/Y509E; (D) H172K/H220D; (E) H447K; (F) Q345K. In A-D, sequence alignments with homologous enzymes from thermo-tolerant organisms are shown. Sequence codes are as follows: An_GOX: GOX from <i>A</i>. <i>niger</i> (Uniprot code P13006); Af_GOX: GOX from <i>A</i>. <i>fumigatus</i> (Uniprot code BOXU64); Hrt_GMC: glucose-methanol-choline oxidoreductase from <i>Halorubrum tebenquichense</i> (Genbank code WP_006628503.1); Htt_GMC: glucose-methanol-choline oxidoreductase from <i>Haloterrigena thermotolerans</i> (Genbank code WP_006648055.1); Tc_GMC: Glucose-methanol-choline oxidoreductase from <i>Thermomonospora curvata</i> (Uniprot code D1A2Y2); Tb_GMC: Glucose-methanol-choline oxidoreductase from <i>Thermobispora bispora</i> (Uniprot code D6Y5M6). Residues involved in the predicted salt bridges in An_GOX-homologous enzymes are highlighted in blue (cationic partner) and red (anionic partner). Panels on the right show details of An_GOX structure (PDB code 1CF3) and homology-based models of Af_GOX (green) and Htt_GOX (orange). An_GOX residues to be mutated and those involved in putative salt bridges in the homologues are shown. Panels E and F display the position of two single mutations. The residue to be mutated and the putative partner to form a salt bridge are shown. The two subunits of An_GOX structure are depicted in grey and blue.</p

Thermal stability of the enzymes with combined mutations.

<p>(A) Relative initial GOX activity in the culture medium of the yeast transformants. (B) Residual GOX activity after incubation at 60°C for 25 (dark grey bars) or 45 (light grey bars) minutes. Error bars represent standard deviation of triplicates. Significant differences (p < 0.05 or p < 0.01) with the wild-type enzyme are indicated by one or two asterisks, respectively.</p

Analysis of Domain Architecture and Phylogenetics of Family 2 Glycoside Hydrolases (GH2)

<div><p>In this work we report a detailed analysis of the topology and phylogenetics of family 2 glycoside hydrolases (GH2). We distinguish five topologies or domain architectures based on the presence and distribution of protein domains defined in Pfam and Interpro databases. All of them share a central TIM barrel (catalytic module) with two β-sandwich domains (non-catalytic) at the N-terminal end, but differ in the occurrence and nature of additional non-catalytic modules at the C-terminal region. Phylogenetic analysis was based on the sequence of the Pfam Glyco_hydro_2_C catalytic module present in most GH2 proteins. Our results led us to propose a model in which evolutionary diversity of GH2 enzymes is driven by the addition of different non-catalytic domains at the C-terminal region. This model accounts for the divergence of β-galactosidases from β-glucuronidases, the diversification of β-galactosidases with different transglycosylation specificities, and the emergence of bicistronic β-galactosidases. This study also allows the identification of groups of functionally uncharacterized protein sequences with potential biotechnological interest.</p></div

Analysis of Domain Architecture and Phylogenetics of Family 2 Glycoside Hydrolases (GH2) - Fig 5

<p><b>Docking of a DA type 5 (A) and a DA type 3 (B) β-galactosidase with their main transglycosylation products.</b> The figure shows the domains that compose the architecture of the enzymes, represented in different colors. The residues potentially interacting with β-D-(1,4)-galactosyl-lactose in <i>Bacillus circulans</i> β-galactosidase (A) or with β-D-(1,3)-galactosyl-lactose in <i>Thermotoga maritima</i> β-galactosidase (B) are highlighted on the right side.</p

Domain architecture of DA type 5 sequences.

<p>Colored and stripped boxes correspond to domains identified by Pfam and Interpro databases, respectively. Modules with more than 40% sequence identity compared to BIG1 domains identified by Interpro, with a coverage higher than 60%, were tagged as BIG1. Letters (a-i) on the right edge of the figure group sequences with similar DAs. Domain assignment at the I1, I2, and I3 regions (subtype a) was based on the analysis carried out with the β-galactosidase from <i>S</i>. <i>pneumoniae</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168035#pone.0168035.ref030" target="_blank">30</a>]. Numbers on top of non-identified regions indicate approximate number of residues.</p