Table 1.
Amino acids and positions within characterized AA5_2 sequences that are implicated in catalysis and substrate preference.
Fig 1.
Subfamilies 1 (AA5_1) and 2 (AA5_2) are indicated. GenBank identifiers (Uniprot identifier P0CS93 in the case of the F. graminearum) are given for all sequences available in the public CAZy database [18] as of May 2018. JGI protein identifiers are given for Ascomycota homologs of PruAA5_2A. Sequences for which biochemical data is available are displayed in bold and indicated as glyoxal oxidases (GlyOx) [27–31], galactose oxidases (GalOx) [26,32], general alcohol oxidases (AlcOx) [16] and raffinose oxidase (RaOx) [6]. When available the three dimensional structures are also indicated with the corresponding PDBID. Sequences were aligned using MUSCLE and the tree was constructed using RAxML v8.2.10. The robustness of the branches was assessed by the bootstrap method with 100 replications. Bootstrap values are indicated at each branch supporting the different subgroups. Subgroups were formed by exhibiting bootstrap values > 75 and colored accordingly.
Fig 2.
Structural comparison of PruAA5_2A, FgrGaOx, CgrAlcOx and CgrRaOx.
(A) The modular structure of PruAA5_2A was created using Phyre2. Similar to FgrGaOx, PruAA5_2A consists of (1) an N-terminal CBM32 (F5_F8_type_C) domain, (2) a central catalytic β-propeller domain and (3) a C-terminal DUF1929 domain. (B) The active site of PruAA5_2A indicating conserved aromatic residues implicated in catalysis (Table 1) are shown in green; amino acids that deviate from FgrGaOx are shown in magenta. (C) The active site of FgrGaOx (PDBID 1GOG) containing the aromatic residues implicated in catalysis (Table 1), which are located within consensus sequence stretches around the active site (orange) (Fig A in S1 File). F194, F441 and F464 (green) do not lie in consensus sequences but are highly conserved in AA5_2. (D) Amino acids contributing to substrate preference or that affect FgrGaOx performance (Table 1), are shown in purple. (E) The active site of CgrAlcOx (PDB ID 5C92), which lacks all known galactose ligands in FgrGaOx. Amino acids that deviate from FgrGaOx are shown in magenta whereas conserved aromatic residues implicated in catalysis is shown in green. (F) Active site of the CgrRaOx model. CgrRaOx contains the arginine corresponding to Arg330 in FgrGaOx, but lacks galactose ligands at positions corresponding to Trp290 and Qln406 in FgrGaOx. Amino acids that deviate from FgrGaOx are shown in magenta whereas conserved aromatic residues implicated in catalysis are shown in green.
Fig 3.
Influence of pH and choice of buffer on PruAA5_2A activity.
(A) Activity as a function of pH established in 25 mM MOPS, 25 mM HEPES, or 25 mM sodium phosphate. (B) Influence of buffer type and concentration on PruAA5_2A activity on 150 mM raffinose (pH 7.5). n = 4; error bars indicate standard deviation.
Fig 4.
Lag-phase of PruAA5_2A activity.
The lag-phase (tlag) is defined as the time from initiation of the reaction (T = 00:00) to where the maximum slope crosses the x-axis. (A) Impact of buffer type and concentration on rate of product formation during oxidation of 300 mM raffinose (pH 7.5). (B) Impact of pH on reaction rate and tlag, during oxidation of 300 mM raffinose. pH was established using 25 mM sodium phosphate buffer and 25 mM MOPS. (C) Impact of substrate on reaction rate and tlag, where each substrate was prepared to 300 mM in 25 mM MOPS (pH 7.5). (D) Impact of substrate concentration on reaction rate and tlag, where raffinose was prepared in 25 mM MOPS (pH 7.5). n = 4 error bars indicate standard deviation.
Fig 5.
PruAA5_2A activity was measured using 300 mM substrate, except for glyceraldehyde, acetaldehyde and glycolaldehyde dimer, where the substrate concentration was 50 mM. Activity on Glyoxalic acid was measured at 15 mM since no activity was detected at 50 mM. In all cases, reactions were performed at 30°C in 20 mM MOPS (pH 7.5). n = 4; error bars indicate standard deviation.
Fig 6.
Kinetic analysis of PruAA5_2A on preferred substrates.
Raffinose (■), galactose (●), glycerol (▲) and a fresh solution of glycolaldehyde dimer (▼). n = 4; error bars indicate standard deviations. The data were fitted to the Michaelis-Menten or substrate-inhibition (glycolaldehyde dimer) functions using the OriginPro analysis software (iteration algorithm: Levenberberg-Marquardt); in all cases R2 values were > 0.95. For all substrates besides the glycolaldehyde dimer, saturation kinetics were not achieved below substrate solubility. Accordingly, apparent kinetic parameters are reported.