Table 1.

Enzymes of the DC/HB cycle characterized in this study

Enzyme/Substrate	Gene	Assay temp. [°C]	Vmax [U ⋅ mg−1 protein]		KM [µM]	kcat/KM [s−1 ⋅ µM−1] *
			Measured	extrapolated at 90 °C
Malate dehydrogenase	Igni_1263	45
Oxaloacetate			628 ± 20	14,200 ± 450	270 ± 28	32.2
NADPH			621 ± 61	14,000 ± 1,380	110 ± 26	77.9
NADH			109 ± 6	2,460 ± 140	157 ± 21	9.6
Succinic semialdehyde reductase	Igni_0132	65
Succinic semialdehyde			504 ± 21	2,850 ± 120	75 ± 8	30.5
NADPH			14 ± 1	80 ± 6	41 ± 10	1.6
NADH			497 ± 44	2,810 ± 250	246 ± 43	9.2
Crotonyl-CoA hydratase/ (S)-3-hydroxybutyryl-CoA dehydrogenase	Igni_1058	50
Crotonyl-CoA (NAD reduction)			6.1 ± 0.2	98 ± 3	71 ± 8	1.8
(S)-3-Hydroxybutyryl-CoA (NAD reduction)			7.3 ± 0.3	117 ± 5	86 ± 13	1.8
NAD [with (S)-3-hydroxybutyryl-CoA]			5.5 ± 0.1	88 ± 2	15 ± 2	7.6
PEP carboxylase†	Igni_0341†	NA†	NA†	NA†	NA†	NA†

NA, not applicable. The V_max values are normalized to 90 °C based on the assumption that a 10 °C rise in temperature doubles the reaction rate. The determination of K_M and V_max values was at least performed two times.

^*k_cat /K_M was calculated for the activities at 90 °C.

^†PEP carboxylase was produced in the insoluble form and thus was not active.