*2.2. Phospho-Mimetic AtGOX1, AtGOX2 and ZmGO1 Exhibit Altered Glycolate Oxidase Activities*

To investigate the potential regulation of *At*GOX1 and *At*GOX2 activity by protein phosphorylation and to see if this was conserved in maize photorespiratory GO1, the kinetic parameters of purified recombinant N-terminal His-tagged GOX proteins (see Figure S3) was undertaken. All selected phosphorylated residues were replaced by an aspartate to mimic a constitutive phosphorylation. To produce phospho-dead GOX proteins, S212/213 and T265/266 (Arabidopsis GOX/*Zm*GO1 numberings) were replaced by an alanine while T4/5 and T158/159 were changed to a valine to also mimic the sequence of *At*HOAX1 and *At*HOAX2 (and *Homo sapiens* HAOX2 for the T4 position) (Figure S2) so as to evaluate the role of these amino acids in substrate specificity. Using glycolate as a substrate, *At*GOX1, *At*GOX2 and *Zm*GO1 presented rather similar KM glycolate (210 μM, 279 μM and 126 μM) and kcat (11.12 s−1, 10.93 s−<sup>1</sup> and 14.45 s−1) values (Table 2). The T4/5V mutations did not have any consequences on either KM glycolate or kcat while the T4/5D phospho-mimetic mutations drastically decreased (by 10–20 fold) the kcat of the three recombinant GOX enzymes without significantly altering the Km glycolate (Table 2). Similar results were observed for the T265/266 phospho-site since its mutation to alanine did not have any effect on the calculated kinetic parameters while kcat was strongly decreased for *At*GOX1T265D, *At*GOX2T265D and *Zm*GO1T266D proteins by 99%, 81% and 95% (Table 2). *At*GOX1T158V, *At*GOX2T158V and *Zm*GO1T159V also showed altered kinetic parameters with an improved KM glycolate (2–3 fold lower) but a 4–5 fold decreased kcat compared to their wild-type GOX counterparts (Table 2). When this residue was mutated to mimic a phosphorylated GOX, the resulting recombinant proteins (*At*GOX1T158D, *At*GOX2T158D and *Zm*GO1T159D) were inactive (Table 2). Perhaps surprisingly, the mutated S212/213 phospho-site gave a differential affect amongst the three GOX proteins studied. While *At*GOX1S212A and *At*GOX1S212D did not show any differences in their kinetic parameters compared to *At*GOX1WT, both *At*GOX2S212A and *At*GOX2S212D as well as *Zm*GO1S213A and *Zm*GO1S213D exhibited an approximately 2-fold decrease of their kcat with no change in KM glycolate (Table 2).

**Table 2.** Effect of phospho-site mutations on glycolate-dependent kinetic parameters of recombinant *At*GOX1, *At*GOX2 and *Zm*GO1.


Mean values ± SD from three independent biological replicates. Statistical significance was determined by a Student's *t*-test. Values in bold and marked by an asterisk were significantly different compared to the corresponding WT protein (*p* < 0.05).
