Structural features of cold-adapted dimeric GH2 β-D-galactosidase from Arthrobacter sp. 32cB

Rutkiewicz, Maria; Bujacz, A.; Bujacz, G.

doi:10.1016/j.bbapap.2019.06.001

Cited by 21 publications

(12 citation statements)

References 60 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Domain 3 (residues 335–624) was the catalytic domain containing the typical TIM-barrel with eight-stranded α/β barrel. Glu460 and Glu536 were two highly conserved glutamic acid residues in Domain 3, which act as an acid/base catalyst and a nucleophilic site, respectively [ 35 , 36 , 37 ]. Therein, Glu460 is of great significance for binding substrates and stable transition states.…”

Section: Resultsmentioning

confidence: 99%

Cloning and Characterization of a New β-Galactosidase from Alteromonas sp. QD01 and Its Potential in Synthesis of Galacto-Oligosaccharides

et al. 2020

Marine Drugs

View full text Add to dashboard Cite

As prebiotics, galacto-oligosaccharides (GOSs) can improve the intestinal flora and have important applications in medicine. β-galactosidases could promote the synthesis of GOSs in lactose and catalyze the hydrolysis of lactose. In this study, a new β-galactosidase gene (gal2A), which belongs to the glycoside hydrolase family 2, was cloned from marine bacterium Alteromonas sp. QD01 and expressed in Escherichia coli. The molecular weight of Gal2A was 117.07 kDa. The optimal pH and temperature of Gal2A were 8.0 and 40 °C, respectively. At the same time, Gal2A showed wide pH stability in the pH range of 6.0–9.5, which is suitable for lactose hydrolysis in milk. Most metal ions promoted the activity of Gal2A, especially Mn2+ and Mg2+. Importantly, Gal2A exhibited high transglycosylation activity, which can catalyze the formation of GOS from milk and lactose. These characteristics indicated that Gal2A may be ideal for producing GOSs and lactose-reducing dairy products.

show abstract

Section: Resultsmentioning

confidence: 99%

Cloning and Characterization of a New β-Galactosidase from Alteromonas sp. QD01 and Its Potential in Synthesis of Galacto-Oligosaccharides

et al. 2020

Marine Drugs

View full text Add to dashboard Cite

show abstract

“…Thanks to comparative analysis, the structural features that may play a key part in its cold-adaptation were described. Most interesting was maximization of energy gain from the surface residue–solvent interactions, that was obtained by reduction of oligomerization state and formation of hydrophobic patches on the protein’s surface [30].…”

Section: Introductionmentioning

confidence: 99%

Active Site Architecture and Reaction Mechanism Determination of Cold Adapted β-d-galactosidase from Arthrobacter sp. 32cB

Rutkiewicz

Bujacz

Wanarska

et al. 2019

IJMS

Self Cite

View full text Add to dashboard Cite

ArthβDG is a dimeric, cold-adapted β-d-galactosidase that exhibits high hydrolytic and transglycosylation activity. A series of crystal structures of its wild form, as well as its ArthβDG_E441Q mutein complexes with ligands were obtained in order to describe the mode of its action. The ArthβDG_E441Q mutein is an inactive form of the enzyme designed to enable observation of enzyme interaction with its substrate. The resulting three-dimensional structures of complexes: ArthβDG_E441Q/LACs and ArthβDG/IPTG (ligand bound in shallow mode) and structures of complexes ArthβDG_E441Q/LACd, ArthβDG/ONPG (ligands bound in deep mode), and galactose ArthβDG/GAL and their analysis enabled structural characterization of the hydrolysis reaction mechanism. Furthermore, comparative analysis with mesophilic analogs revealed the most striking differences in catalysis mechanisms. The key role in substrate transfer from shallow to deep binding mode involves rotation of the F581 side chain. It is worth noting that the 10-aa loop restricting access to the active site in mesophilic GH2 βDGs, in ArthβDG is moved outward. This facilitates access of substrate to active site. Such a permanent exposure of the entrance to the active site may be a key factor for improved turnover rate of the cold adapted enzyme and thus a structural feature related to its cold adaptation.

show abstract

“…Results of the TSA (thermofluor shift assay) of Arth βDG_D207A did not exhibit significant differences compared with ones obtained for wild-type protein Arth βDG [ 52 ]. The highest melting temperature (44 °C) was obtained in conditions containing 50 mM sodium phosphate pH 6.0.…”

Section: Resultsmentioning

confidence: 99%

“…The question, why such a large enzyme was developed and retained by extremophilic bacteria for catalysis of relatively simple reactions, remains open. However such a strategy, to elevate the accessibility of a substrate by its weak binding on the enzyme's surface, may be one of explanation together with the maximization of the energy gain from the surface residue-solvent interactions [52]. Combining the structural data with results of activity assays suggests that the mutation of D207 effects the enzyme's activity by disturbing the hydroxyl group O4 stabilization of galactosyl moiety.…”

Section: Galactose Binds On the Enzyme's Surfacementioning

confidence: 99%

“…Furthermore, it exhibits additional transglycosylation activity, it catalyzes synthesis of GOS and HOS, for example: lactulose and alkyl glycosides. [50] Architecture of its five domains [7], structural details of hydrolysis of lactose catalyzed by this enzyme [51], and its structural features responsible for the enzyme's cold adaptation [52] were investigated and discussed previously. Upon the solution of native structure, E441 and E517 were assigned as catalytic residues based on superposition of ArthβDG structure with lacZ E. coli β-d-galactosidase [7].…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Mapping the Transglycosylation Relevant Sites of Cold-Adapted β-d-Galactosidase from Arthrobacter sp. 32cB

Rutkiewicz

Wanarska

Bujacz

2020

IJMS

Self Cite

View full text Add to dashboard Cite

β-Galactosidase from Arthrobacter sp. 32cB (ArthβDG) is a cold-adapted enzyme able to catalyze hydrolysis of β-d-galactosides and transglycosylation reaction, where galactosyl moiety is being transferred onto an acceptor larger than a water molecule. Mutants of ArthβDG: D207A and E517Q were designed to determine the significance of specific residues and to enable formation of complexes with lactulose and sucrose and to shed light onto the structural basis of the transglycosylation reaction. The catalytic assays proved loss of function mutation E517 into glutamine and a significant drop of activity for mutation of D207 into alanine. Solving crystal structures of two new mutants, and new complex structures of previously presented mutant E441Q enables description of introduced changes within active site of enzyme and determining the importance of mutated residues for active site size and character. Furthermore, usage of mutants with diminished and abolished enzymatic activity enabled solving six complex structures with galactose, lactulose or sucrose bounds. As a result, not only the galactose binding sites were mapped on the enzyme’s surface but also the mode of lactulose, product of transglycosylation reaction, and binding within the enzyme’s active site were determined and the glucopyranose binding site in the distal of active site was discovered. The latter two especially show structural details of transglycosylation, providing valuable information that may be used for engineering of ArthβDG or other analogous galactosidases belonging to GH2 family.

show abstract

Structural features of cold-adapted dimeric GH2 β-D-galactosidase from Arthrobacter sp. 32cB

Cited by 21 publications

References 60 publications

Cloning and Characterization of a New β-Galactosidase from Alteromonas sp. QD01 and Its Potential in Synthesis of Galacto-Oligosaccharides

Cloning and Characterization of a New β-Galactosidase from Alteromonas sp. QD01 and Its Potential in Synthesis of Galacto-Oligosaccharides

Active Site Architecture and Reaction Mechanism Determination of Cold Adapted β-d-galactosidase from Arthrobacter sp. 32cB

Mapping the Transglycosylation Relevant Sites of Cold-Adapted β-d-Galactosidase from Arthrobacter sp. 32cB

Contact Info

Product

Resources

About