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

Abstract

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.

Keywords: GH2; cold-adapted; complex structures; galactosidase; hydrolysis; reaction mechanism.

Figure 1

The crystals of ArthβDG: after addition of ONPG (A); after 2 h of soaking in X-gal (B). The crystals of ArthβDG_E441Q mutein soaked 24 h with mixture of lactose and galactose (C).

Figure 2

The dimer of ArthβDG_E441Q and the zoom of one of the active site cavities with lactose.

Figure 3

The surface potential visualization at the active site of ArthβDG (A), ArthβDG_E441Q (B), EcolβDG (C), and KlyvβDG (D).

Figure 4

The reaction mechanism of Koshland double displacement with the catalytic residues numbered as for ArthβDG [33].

Figure 5

Early complexes of ArthβDG with saccharide substrate and substrate analogue: the molecule of lactose (A) and IPTG (B) bound at shallow binding site.

Figure 6

Enzyme active site of shallow and deep binding of lactose. Electron density 2F_o-F_c map of lactose in deep (A) and shallow (B) binding mode (contoured at 1σ). Superposition of enzyme active site in both structures (C).

Figure 7

Late complexes of ArthβDG with substrates: the molecules of lactose (A) and ONPG (B).

Figure 8

Superposition of catalytic sites of ArthβDG with lactose bound in deep mode (green) and EcolβDG (purple).

Figure 9

Complex structure of ArthβDG with galactose in half-chair conformation bound in the active center.