*2.3. Phospho-site Mutations at T4*/*5 and T158*/*159 Have a Limited E*ff*ect on Substrate Specificity*

*At*HOAX1 and *At*HAOX2 proteins preferentially use long-chain hydroxy-acids as substrates although *At*HOAX1 can also quite efficiently use both lactate and glycolate [6]. Since the two Arabidopsis HAOX enzymes had a valine at the T4 and T158 positions, it was decided to test whether the mutation of these residues could give rise to substrate-specific effects. To achieve this, activity measurements were repeated with either L-lactate or 2-hydroxyoctanoate using T4/5 and T158/159 phospho-site mutated GOX proteins and their kinetic parameters were calculated and compared (Table 3). We chose to test 2-hydroxyoctanoate because it was found to be a good substrate for both Arabidopsis HAOX enzymes [6]. First, however, the substrate specificity of GOXWT proteins were compared and as previously reported, the Km L-lactate was higher than the Km glycolate [8] with an approximate 8-fold higher value for *At*GOX1 and *At*GOX2 while only a 4-fold difference was seen for *Zm*GO1, and surprisingly, the calculated kcat values for the lactate oxidase reaction were not significantly different when compared to the glycolate oxidase activities (Table 2; Table 3). When using 2-hydroxyoctanoate as a substrate, again a higher Km was observed compared to glycolate for *At*GOX1 and *At*GOX2 (3.6-fold and 1.8-fold, respectively), while the Km 2-hydroxyoctanoate for *Zm*GO1 remained unchanged (Tables 2 and 3). However, the kcat for the 2-hydroxyoctanoate reaction was halved for all recombinant GOXWT proteins when compared to glycolate oxidase activity (Tables 2 and 3). Taken together, these results showed that our recombinant GOXWT proteins were more efficient (based on kcat/Km) using glycolate as a substrate, although *Zm*GO1 appeared to be less selective.


**Table 3.** Effect of selected phospho-site mutations on L-lactate and 2-hydroxy-octanoate 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).

The effect of the selected (T4/5 and T158/159) phospho-site mutations on the calculated kinetic parameters of the lactase oxidase and 2-hydroxyoctanoate oxidase reactions was compared. It was found that T158V/D and T159V/D mutations led to similar effects on KM and kcat (T158/159V) and activity (T158/159D) using either L-lactate or 2-hydroxyoctanoate when compared to glycolate (Tables 2 and 3). For the T4V/D and T5V/D mutations, various consequences were observed (Table 3). As seen when testing the glycolate oxidase activity, the kcat of *At*GOX1T4D, *At*GOX2T4D and *Zm*GO1T5D was strongly reduced compared to GOXWT proteins when either L-lactate or 2-hydroxyoctanoate was used (Tables 2 and 3). In these conditions, some significant changes were observed also for certain KM values but this depended on the GOX protein since only the KM L-lactate of *At*GOX1T4D was slightly increased (1.4-fold) while the KM 2-hydroxyoctanoate of *At*GOX2T4D and *Zm*GO1T5D was increased by 3.5-fold and 3-fold, respectively, when compared to their corresponding GOXWT proteins (Table 3). When the T4/T5 phospho-site was replaced by a valine, only *At*GOX1T4V and *At*GOX2T4V differed from their *At*GOXWT counterparts with respect to Km 2-hydroxyoctanoate since a 2-fold decrease for *At*GOX1T4V and a 2-fold increase for *At*GOX2T4V was observed (Table 3). Therefore, in general, the T4/5 and T158/159 mutations led to similar effects on the calculated kinetic parameters and enzyme activities when either glycolate, L-lactate or 2-hydroxyoctanoate was used as substrate. The T4/5 and T158/159 mutations to valine did not appear to alter substrate specificity since enzymatic efficiency using either L-lactate or 2-hydroxyoctanoate was not improved except in the cases of *At*GOX1T4V, *At*GOX1T158V and *Zm*GO1T159V where kcat/Km ratios for the 2-hydroxyoctanoate reaction appeared to be higher.
