*3.1. Phospho-Mimetic GOX and Inhibition of Enzyme Activity*

Phospho-mimetic GOX proteins exhibited different degrees of inhibition of their glycolate oxidase activity, except in one case, AtGOX1T212D, where the recombinant protein maintained GOX1WT activity (Table 2). This inhibition was seen to correlate with reduced amounts (or the absence) of FMN (Figure 1, Table 4) for GOXT4/5D and GOXT158/158D but this was not observed for GOXS212/213D and GOXT265/266D. When the phospho-mimetic mutation affected FMN content, the degree of inhibition was not constant since the mutation of T4/T5 to aspartate dramatically decreased GOX activity without changing the KM glycolate, while T158D and T159D mutations resulted in inactive recombinant GOX proteins (Table 2). These modifications in both Arabidopsis and maize GOX kinetics parameters could be attributed to the lack of FMN in these mutated proteins since its presence is essential for GOX activity (Table 4) [30]. Recently, the 3D structure of apo-GOX (lacking FMN) and holo-GOX from *Nicotiana benthamiana* revealed that loop4 (residues 157–165; 156–164 of *At*GOX) and loop6 (residues 253–265; 252–264 of *At*GOX) together with a loop situated between residues 28–33 (27–32 for *At*GOX) formed a lid preventing the loss of FMN [30] (Figure 2A, Figure S1). Moreover, this lid had a strong interaction with a neighbouring GOX subunit of the tetrameric *Nb*GOX, with α-helix4 (next to loop4) forming H-bonds with E3, T5 and N6 (E2, T4 and N5 of *At*GOX) allowing a cooperative mechanism in FMN binding between GOX subunits of the same tetramer [30] (Figure 2B, Figure S1). Structural models of *Arabidopsis thaliana* GOX1 and GOX2 as well as *Zm*GO1 based on the structure of *Spinacia oleracea* GOX (PDB 1AL7) indicated that T4 formed H-bonds with R163, E165 and K169 of α-helix4 of a neighbouring GOX subunit.

When T4 was replaced by an aspartate in these models, only an H-bond with D163 of α-helix4 was present. Moreover, T158 is located close to a residue implicated in FMN-binding (T155) [31,32], the latter being also potentially phosphorylated [25]. Thus, T158 (present in loop4) and T4 (at the N-terminus) are located in essential domains for FMN binding; therefore, their mutation to aspartate, and probably their phosphorylation, may disturb FMN binding to holoGOX resulting in a less active or inactive enzyme. Furthermore, structural models indicated that when T158/159 was replaced by an aspartate, a new H-bond formed with Y129. This tyrosine residue normally formed an H-bond with FMN and when mutated to a phenylalanine (spinach GOXY129F) the resulting protein had only 3.5% of GOXWT activity and a kcat of only 0.74 s−<sup>1</sup> instead of 20 s−1; however, FMN content was not affected [33]. Therefore, it is possible that phosphorylation of T158/159 could alter GOX activity even if FMN was retained in the GOX protein.

The mutation of T265/266 to aspartate decreased the glycolate-dependent kcat of *At*GOX1/2T265D and *Zm*GO1T266D compared to the wild-type recombinant protein but only changed the KM glycolate of *At*GOX1T265D (Table 2). As mentioned earlier, contrary to GOXT4D/T5D and GOXT158D/T159D, the FMN content of phospho-mimetic GOXT265D/T266D was comparable to recombinant GOXWT protein (Table 4) even though T265/T266 is located just at the end of loop6 (residues 253–265; 252–264 of *At*GOX1/2) which is part of the "lid" structure involved in FMN loss in the pH sensor model [30]. Again, based on GOX structural models, T265 formed an H-bond with D285 of β-sheet7; however, when replaced by an aspartate this bond was broken and a new H-bond was formed with N253 of loop6 that is next to the active site H254 involved in proton abstraction during catalysis. We propose that GOXT265/T266D inhibited glycolate oxidase activity by bringing about a conformational change that interfered with catalysis via the displacement of H254.

**Figure 2.** Localisation of phosphorylated residues and important structures of *At*GOX1. A structural model of *At*GOX1 based on the 3D-structure of spinach GOX [31]. (**A**) *At*GOX1 monomer showing phosphorylated residues T4, T158, S212 and T265 (red), loops 4 (yellow) and 6 (blue), α-helix4 (green) and the flavin mononucleotide (FMN) (pink). The bottom structure is rotated by 90◦ with respect to the top structure. (**B**) View of two *At*GOX monomers (in grey and in pink) and the proximity between T4 (red) of a GOX subunit with α-helix4 (green) of a neighbouring GOX subunit.
