Molecular Structural Basis for the Cold Adaptedness of the Psychrophilic β-Glucosidase BglU in Micrococcus antarcticus

Abstract

Psychrophilic enzymes play crucial roles in cold adaptation of microbes and provide useful models for studies of protein evolution, folding, and dynamic properties. We examined the crystal structure (2.2-Å resolution) of the psychrophilic β-glucosidase BglU, a member of the glycosyl hydrolase 1 (GH1) enzyme family found in the cold-adapted bacterium Micrococcus antarcticus. Structural comparison and sequence alignment between BglU and its mesophilic and thermophilic counterpart enzymes (BglB and GlyTn, respectively) revealed two notable features distinct to BglU: (i) a unique long-loop L3 (35 versus 7 amino acids in others) involved in substrate binding and (ii) a unique amino acid, His299 (Tyr in others), involved in the stabilization of an ordered water molecule chain. Shortening of loop L3 to 25 amino acids reduced low-temperature catalytic activity, substrate-binding ability, the optimal temperature, and the melting temperature (Tm). Mutation of His299 to Tyr increased the optimal temperature, the Tm, and the catalytic activity. Conversely, mutation of Tyr301 to His in BglB caused a reduction in catalytic activity, thermostability, and the optimal temperature (45 to 35°C). Loop L3 shortening and H299Y substitution jointly restored enzyme activity to the level of BglU, but at moderate temperatures. Our findings indicate that loop L3 controls the level of catalytic activity at low temperatures, residue His299 is responsible for thermolability (particularly heat lability of the active center), and long-loop L3 and His299 are jointly responsible for the psychrophilic properties. The described structural basis for the cold adaptedness of BglU will be helpful for structure-based engineering of new cold-adapted enzymes and for the production of mutants useful in a variety of industrial processes at different temperatures.

FIG 1

Ribbon representation of the overall structure of BglU. The α-helices and β-strands of the TIM barrel are shown by salmon and sky blue. Loops L1, L2, αB, and αC are orange. L5, βC, and βD are green/cyan. L3 is yellow. L4 and other residues are gray.

FIG 2

Sequence alignment of BglU and its mesophilic (2Z1S) and thermophilic (1NP2) homologues in the GH1 family. Sequence alignment was performed using the ClustalW (v2.0) and ESPript programs. Conserved sequences are indicated by boxes, and similar sequences are indicated by colored background. α-Helix, β-sheet, random coil, and beta turn are labeled α, β, η, and T, respectively. Secondary structures and their designations are shown. β-Strands and α-helices are represented by arrows and coils, respectively. Residues Glu170 and Glu377 are indicated by purple circles; His299 and His301 are indicated by yellow triangles; Lys163, Glu218, Asn228, Ala368, and Thr383 are indicated by cyan diamonds; Gly261 and Ala389 are indicated by blue stars.

FIG 3

(A) Structure superposition of BglU on the thermophilic counterpart GlyTn (A chain) and the mesophilic counterpart BglB. BglU, GlyTn, and BglB are colored in salmon, light blue, and pale green, whereas their loop L3 is shown in red, blue, and cyan, respectively. The cellotetraose molecule modeled into the substrate-binding pocket is shown as sticks. (B) Detail of substrate-binding pocket with cellotetraose. Residues His125, Trp126, Glu170, Tyr300, Glu377, Trp424, and Glu431, Phe440 (indicated in cyan) interact mainly with the first nonretaining monosaccharide group, whereas residues Glu183 (also with the third monosaccharide group), His184, His301, Ile177, Asn225, Trp250, Trp349, and Trp434 (indicated in green) interact mainly with the second nonretaining monosaccharide. Residues from loop L3, which is indicated in violet but with residue Ser327 emphasized in steel blue, interact with the third and fourth nonretaining monosaccharides. The figures were prepared using the PyMOL program.

FIG 4

Comparison of optimal temperatures of BglU and its mutants. (A) BglU, H299Y, delta-L3, and delta-L3/H299Y. (B) BglU, BglB, and BglB-Y301H. The maximal activity (385.6 U/mg) of BglU was defined as 100%. The experiments were performed in triplicate or quadruplicate.

FIG 5

Relative activity at various temperatures of BglU and its mutants (A), the T_m value from the derivative of the fluorescent signal (B), and the fluorescence adsorption of BglU, BglB, and their mutants (C). The calculated T_m values were 36.5°C for BglU, 43.5°C for H299Y, 34°C for delta-L3/H299Y, 47°C for BglB, and 40.5°C for BglB-Y301H. BglU and its mutant enzymes were maintained at various temperatures as shown for 40 min. For each protein, activity at 0 min was defined as 100%. The T_m was determined by differential scanning fluorimetry (DSF). The experiments were performed in triplicate or quadruplicate.

FIG 6

Substrate-binding pocket of BglU. (A) Electronic potential surface of BglU (left) showing the large, deep cavity for substrate binding, and a section of BglU showing the inner surface (right, green grid) of the substrate-binding pocket. Residues Glu170, His299, His301, and Glu377 and a Tris molecule in the pocket are indicated by sticks (right). (B) Closer inspection of the binding mode of the Tris molecule, in yellow, located at the substrate-binding pocket. Residues Gln25, His125, Glu170, Glu377, and Glu431 interacting with the Tris molecule are indicated by sticks.

FIG 7

Structure superposition of the ordered water molecule chains, as well as residues His299 and His301, of BglU (green), BglB (yellow), and GlyTn (gray).