Rational engineering of AA5_2 copper radical oxidases to probe the molecular determinants governing their substrate selectivity

Abstract

Fungal copper radical oxidases (CROs) from the Auxiliary Activity family 5 (AA5) constitute a group of metalloenzymes that oxidize a wide panel of natural compounds, such as galactose-containing saccharides or primary alcohols, into product derivatives exhibiting promising biotechnological interests. Despite a well-conserved first copper-coordination sphere and overall fold, some members of the AA5_2 subfamily are incapable of oxidizing galactose and galactosides but conversely efficiently catalyse the oxidation of diverse aliphatic alcohols. The objective of this study was to understand which residues dictate the substrate preferences between alcohol oxidases and galactose oxidases within the AA5_2 subfamily. Based on structural differences and molecular modelling predictions between the alcohol oxidase from Colletotrichum graminicola (CgrAlcOx) and the archetypal galactose oxidase from Fusarium graminearum (FgrGalOx), a rational mutagenesis approach was developed to target regions or residues potentially driving the substrate specificity of these enzymes. A set of 21 single and multiple CgrAlcOx variants was produced and characterized leading to the identification of six residues (W39, F138, M173, F174, T246, L302), in the vicinity of the active site, crucial for substrate recognition. Two multiple CgrAlcOx variants, i.e. M4F (W39F, F138W, M173R and T246Q) and M6 (W39F, F138W, M173R, F174Y, T246Q and L302P), exhibited a similar affinity for carbohydrate substrates when compared to FgrGalOx. In conclusion, using a rational site-directed mutagenesis approach, we identified key residues involved in the substrate selectivity of AA5_2 enzymes towards galactose-containing saccharides.

Expansion of Auxiliary Activity Family 5 sequence space via biochemical characterization of six new copper radical oxidases

“…No substrate or product complexes of AA5 members have been solved, so direct information on active-site interactions is currently lacking ( 19 , 32 , 33 , 37 , 64 ). However, site-directed mutagenesis and in silico studies have implicated several key active site residues in the modulation of substrate specificity ( 47 , 65 – 67 ). Yet, these studies also indicate that substrate specificity cannot be readily attributed to the effects of any specific amino acids in a predictable manner.…”

“…Loss of one or both glutamines is generally, but not exclusively (e.g., Efe GalOx), correlated with a loss of GalOx activity. A recent study on the engineering of Cgr AlcOx has shown that these glutamines increase affinity for galactose, likely through hydrogen bonding ( 65 ). Interestingly, Xpa GalOx, which has a twofold higher catalytic efficiency and 10-fold lower K M on galactose compared to Fgr GalOx ( Table 1 ), contains an arginine at the position corresponding to Q326 in Fgr GalOx ( Table 2 ; Fig.…”

Current Advances in the Enzyme Engineering of O2‐Dependent Enzymes – Boosting the Versatility and Applicability of Oxygenases and Oxidases

“…A variant with four (V4) and a variant with six mutations (V6) was identified, showing a comparable affinity to Dgalactose like the FgrGOx. [40] However, the variants had a 10fold lower activity towards aliphatic or aromatic alcohols. Both studies demonstrate that only a few amino acids are needed to manipulate the substrate spectrum of CROs, allowing rational engineering to change significantly the substrate preference.…”

“…Using structure‐based rational engineering, 21 Cgr AAO variants were generated with mutations in six residues (W39F, F138W, M173R, F174Y, T246Q and L302P) responsible for substrate recognition at the active site, Figure 3b. A variant with four (V4) and a variant with six mutations (V6) was identified, showing a comparable affinity to D‐galactose like the Fgr GOx [40] . However, the variants had a 10‐fold lower activity towards aliphatic or aromatic alcohols.…”