Mutagenesis and mechanistic study of a glycoside hydrolase family 54 alpha-L-arabinofuranosidase from Trichoderma koningii

Abstract

A GH (glycoside hydrolase) family 54 alpha-L-arabinofuranosidase from Trichoderma koningii G-39 (termed Abf) was successfully expressed in Pichia pastoris and purified to near homogeneity by cation-exchange chromatography. To determine the amino acid residues essential for the catalytic activity of Abf, extensive mutagenesis of 24 conserved glutamate and aspartate residues was performed. Among the mutants, D221N, E223Q and D299N were found to decrease catalytic activity significantly. The kcat values of the D221N and D299N mutants were 7000- and 1300-fold lower respectively, than that of the wild-type Abf. E223Q was nearly inactive. These results are consistent with observations obtained from the Aspergillus kawachii alpha-L-arabinofuranosidase three-dimensional structure. This structure indicates that Asp221 of T. koningii Abf is significant for substrate binding and that Glu223 as well as Asp299 function as a nucleophile and a general acid/base catalyst for the enzymatic reaction respectively. The catalytic mechanism of wild-type Abf was further investigated by NMR spectroscopy and kinetic analysis. The results showed that Abf is a retaining enzyme. It catalyses the hydrolysis of various substrates via the formation of a common intermediate that is probably an arabinosyl-enzyme intermediate. A two-step, double-displacement mechanism involving first the formation, and then the breakdown, of an arabinosyl-enzyme intermediate was proposed. Based on the kcat values of a series of aryl-alpha-L-arabinofuranosides catalytically hydrolysed by wild-type Abf, a relatively small Brønsted constant, beta(lg)=-0.18, was obtained, suggesting that the rate-limiting step of the enzymatic reaction is the dearabinosylation step. Further kinetic studies with the D299G mutant revealed that the catalytic activity of this mutant depended largely on the pK(a) values (>6) of leaving phenols, with beta(lg)=-1.3, indicating that the rate-limiting step of the reaction becomes the arabinosylation step. This kinetic outcome supports the idea that Asp299 is the general acid/base residue. The pH activity profile of D299N provided further evidence strengthening this suggestion.

Figure 1. The proposed mechanism of a retaining α-L-arabinofuranosidase

A two-step, double displacement mechanism is proposed in which two essential amino acid residues, one functioning as the nucleophile and the other as the general acid/base, were involved.

Figure 2. Analysis of the molecular mass of the recombinant Abf by SDS/PAGE

Lanes: M, molecular mass markers; 1, the purified wild-type Abf (approx. 4 μg).

Figure 3. pH activity profiles of wild-type Abf (○) and the D299N mutant enzyme (●)

The k_cat values of wild-type (WT) and the D299N mutant were measured at the final pH values: 1.9, 2.0, 2.4, 3.3, 3.9, 4.2, 5.5 and 6.5. Data were fitted by non-linear regression analysis [35].

Figure 4. The active site of the GH54 family enzyme from A. kawachii IFO4308 (PDB number 1WD4) with arabinofuranose in place

The corresponding amino acids in the T. koningii Abf are labelled in parentheses.

Figure 5. Stereochemical properties and common intermediates of Abf catalysis

(a) Enzymatic reactions, using various substrates, in the presence of methanol. (b) A partial NMR spectrum (chemical shift 3.4–5.4 p.p.m.) of the end-products. Peak assignment is given in the text. The integrations of the signals of the C1 protons on each sugar ring were used to calculate the end-product ratio (MAF/arabinose).

Figure 6. Brønsted plots of wild-type Abf (○) and D299G (●) mutant enzyme

(a) Plots of log k_cat against pK_a of the leaving phenol. (b) Plots of log k_cat/K_m against pK_a of the leaving phenol.