Structure-based directed evolution improves S. cerevisiae growth on xylose by influencing in vivo enzyme performance

Table 2 Kinetic parameters of PirXI variants

PirXI variants	Metal	K_act (µM)^c	Kinetic parameters^a
PirXI variants	Metal	K_act (µM)^c	k_cat (s⁻¹)	K_M (mM)	k_cat/K_M (s⁻¹ M⁻¹)
WT	Mg²⁺	130 ± 7	2.2 ± 0.1	7.1 ± 0.5	310
	Mn²⁺	0.44 ± 0.05	6.9 ± 0.2	6.2 ± 0.7	1110
	Ca²⁺	230 ± 20	0.8 ± 0.1	1360 ± 160	0.4^b
V270A–A273G	Mg²⁺	160 ± 14	1.8 ± 0.1	11.6 ± 1.6	155
	Mn²⁺	0.39 ± 0.05	2.8 ± 0.1	9 ± 1	310
	Ca²⁺	140 ± 12	0.28 ± 0.01	1760 ± 120	0.18^b
S141N–T142S–A143S–G174A	Mg²⁺	126 ± 10	1.6 ± 0.2	27 ± 9	60
	Mn²⁺	2.4 ± 0.5	4.9 ± 1	37 ± 1.5	132
	Ca²⁺	96.5 ± 3.5	0.06 ± 0.01	1200 ± 300	0.03^b
E15D–T142S	Mg²⁺	190 ± 10	1.9 ± 0.1	22 ± 2	86
	Mn²⁺	2.1 ± 0.3	5 ± 0.2	53 ± 4	94
	Ca²⁺	50 ± 10	> 0.02	N.A.	0.018^b
N338C	Mg²⁺	112 ± 7	4.2 ± 0.2	22 ± 2	191
	Mn²⁺	1.3 ± 0.3	7.2 ± 0.2	29 ± 1.5	248
	Ca²⁺	163 ± 6.5	0.78 ± 0.2	2800 ± 800	0.2^b

^aAll assays were performed at 30 °C in 20 mM MOPS buffer, pH 7. Values are averages from three (wild type, V270A–A273G PirXI) or two (S141N–T142S–A143S–G174A, E15D–T142S and N338C PirXI) independent measurements. The margins represent standard deviations
^bCatalytic efficiencies obtained from slopes of Michaelis–Menten plots at [S] ≪ K_M because of the high K_M for xylose in the presence of Ca²⁺ as activating metal
^cThe K_act value is defined as the metal concentration giving half-maximal activity, as measured with a saturating concentration of xylose (100 mM in case of Mg⁺ and Mn²⁺, 400 mM in case of Ca²⁺)

ISSN: 2731-3654