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

ABSTRACT Bacterial and fungal copper radical oxidases (CROs) from Auxiliary Activity Family 5 (AA5) are implicated in morphogenesis and pathogenesis. The unique catalytic properties of CROs also make these enzymes attractive biocatalysts for the transformation of small molecules and biopolymers. Despite a recent increase in the number of characterized AA5 members, especially from subfamily 2 (AA5_2), the catalytic diversity of the family as a whole remains underexplored. In the present study, phylogenetic analysis guided the selection of six AA5_2 members from diverse fungi for recombinant expression in Komagataella pfaffii (syn. Pichia pastoris) and biochemical characterization in vitro. Five of the targets displayed predominant galactose 6-oxidase activity (EC 1.1.3.9), and one was a broad-specificity aryl alcohol oxidase (EC 1.1.3.7) with maximum activity on the platform chemical 5-hydroxymethyl furfural (EC 1.1.3.47). Sequence alignment comparing previously characterized AA5_2 members to those from this study indicated various amino acid substitutions at active site positions implicated in the modulation of specificity. IMPORTANCE Enzyme discovery and characterization underpin advances in microbial biology and the application of biocatalysts in industrial processes. On one hand, oxidative processes are central to fungal saprotrophy and pathogenesis. On the other hand, controlled oxidation of small molecules and (bio)polymers valorizes these compounds and introduces versatile functional groups for further modification. The biochemical characterization of six new copper radical oxidases further illuminates the catalytic diversity of these enzymes, which will inform future biological studies and biotechnological applications.


Supplementary Tables
. Purified protein yields of expressed AA5_2 enzymes from 400 mL of BMMY shake-flask culture after three days of methanol feeding.

Enzyme
Yield (mg) Yield (mg/L)           was also applied to data from reactions that did not reach saturation.AstAAO displayed substrate inhibition (blue line) when assayed on veratryl alcohol and HMF.

Figure S10. Galactose oxidation by
XpaGalOx. 1 H NMR spectra for the negative control reaction (red), reaction with XpaGalOx (blue) and reaction with XpaGalOx and 0.1 mg/mL BSA added (green).A 1:1 ratio of HRP: catalase was added and reactions were carried out for 24 hours at ambient temperature with stirring at 400 rpm.A concentration of 0.7 M (50g) XpaGalOx was used in a 1 mL reaction.Supplementary References

Figure S1 .
Figure S1.Maximum likelihood phylogeny of AA5_2 members.A maximum likelihood phylogeny was generated with a curated set of 623 AA5_2 catalytic modules omitting any accessory modules, using AA5_1 sequences as an outgroup (1).Each subgroup is distinguished by different branch colors and.numbers.Subgroups containing characterized members are indicated in green.AA5_2 members with previously available biochemical data are labeled in black and.those from this work are in red.Bootstrap values indicated at each node/branch support the 38 subgroups identified.

Figure S3 .
Figure S3.pH-rate profiles.pH-rate profiles were determined using the HRP-ABTS coupled assay with 300mM galactose as the substrate for Xpa-,Bsp-,Efe, Nex-and Nha-GalOx and 10mM HMF for AstAAO.Measurements were made in triplicate at room temperature.Sodium phosphate (black square), citrate phosphate (red circle), sodium acetate (blue circle), glycine NaOH (blue triangle) and CHES (green triangle) buffers at 50mM were used to cover a pH range of 4-10.5.

Figure S4 .
Figure S4.Temperature stability profiles.Activity was determined using the couple HRP-ABTS assay with 300 mM galactose and 10 mM HMF as substrates for the galactose oxidases and alcohol oxidase, respectively.Reactions were performed in duplicate at room temperature and each enzyme was pre-incubated at each temperature and maintained by a gradient thermocycler: 30°C (purple diamond), 35.2°C (green triangle), 39.3°C (blue triangle), 44.9°C (red circle) and 51.9°C (black square).

Figure S5 .
Figure S5.Effects of BSA addition on observed activity of XpaGalOx.Due to the high activity of XpaGalOx, diluted solutions of enzyme required stabilisation by BSA.A working concentration of 5 x 10 4 mg mL -1 (7.3 nM) was used.

Figure S6 .
Figure S6.XpaGalOx Michaelis-Menten kinetics.Initial rates were measured in triplicate at each substrate concentration.The individual kcat and KM values were calculated by performing a non-linear fitting analysis of the standard Michaelis-Menten equation (red line) using OriginLab 9.85.A linear fit (black line) was applied to data from reactions that did not reach saturation.XpaGalOx displayed substrate inhibition (blue line) when assayed on melibiose.

Figure S7 .
Figure S7.BspGalOx Michaelis-Menten kinetics.Initial rate values were measured in triplicate at each substrate concentration.The individual kcat and KM values were calculated by performing a non-linear fitting analysis of the standard Michaelis-Menten equation (red line) using OriginLab 9.85.A linear fit (black line) was also applied to the data from reactions where saturation kinetics were not observed.

Figure S8 .
Figure S8.EfeGalOx, NexGalOx and NhaGalOx Michaelis-Menten kinetics.Initial rate values were measured in triplicate at each substrate concentration.The individual kcat and KM values were calculated by performing a nonlinear fitting analysis of the standard Michaelis-Menten equation (red line) using OriginLab 9.85.A linear fit (black line) was also applied to data from reactions that did not reach saturation.EfeGalOx displayed substrate inhibition (blue line) when assayed on galactose.

Figure S9 .
Figure S9.AstAAO Michaelis-Menten kinetics.Initial rate values were measured in triplicate at each substrate concentration.The individual kcat and KM values were calculated by performing a non-linear fitting analysis of the standard Michaelis-Menten equation (red line) using OriginLab 9.85.A linear fit (black line) was also applied to data from reactions that did not reach saturation.AstAAO displayed substrate inhibition (blue line) when assayed on veratryl alcohol and HMF.

Figure S11 .
Figure S11.HMF oxidation byAstAAO.1 H NMR spectra for the negative control reaction (red) and reaction with AstAAO (blue).A 1:1 ratio of HRP: catalase was added, and reactions were carried out for 24 hours at ambient temperature with stirring at 400 rpm.A concentration of 1.4 M (100 g) AstAAO was used in a 1 mL reaction.HMF, DFF, DFF hydrate and FFCA are denoted by open squares, open circles, black circles and black stars, respectively.Peaks were assigned versus reference spectra (1, 2) .

Table S2 . Specific activities of AA5_2 members.* Enzymes and Specific Activities (µmol/min/mg enzyme)
*ABTS coupled assay in 50 mM sodium phosphate buffer, pH 7.0, at room temperature with 300 mM carbohydrate or polyol, or 10 mM aryl alcohol or aldehyde and.2mg/mL polysaccharide substrates, except for NexGalOx that was assayed in 50mM sodium acetate buffer, pH 5.0.n.d.= Activity not detected at [E] up to 2 mg/mL (27M).n.t.= Not tested.

Fgr GalOx Mre GalOx Fve GalOx Fsu GalOx Fox AAO Fgr AAO Fox AlcOx Cgr AAO
Sequence identity and similarity values are highlighted in green and blue, respectively.Previously characterised AA5_2s are in bold font; see main text for literature references.Percentage coverage of the catalytic module sequences was between 98 -100%.