Table 1.

Biochemical characterization of CntB wild-type and variant proteins^a

Mutation	Function	Relative FMN content (%)	Relative l-carnitine depletion activity (%)	Relative TMA formation (%)	Relative NADH depletion activity (%)	Iron content (nmol protein−1)	Sulfur content (nmol protein−1)	EPR signal for [2Fe-2S]
D75K	[FMN] coordinating	78 ± 5	70 ± 8	92	69 ± 3	1.85 ± 0.19	1.84 ± 0.16	WT
S82A	[FMN] coordinating	15 ± 4	35 ± 6	56	23 ± 2	2.12 ± 0.18	1.85 ± .019	WT
C265A	No catalytic function	96 ± 4	99 ± 11	70	90 ± 4	2.05 ± 0.17	1.95 ± 0.29	WT
C267A	[2Fe-2S] coordinating	–	<5	–	<3	0.10 ± 0.04	0.25 ± 0.17	ND
C272A	[2Fe-2S] coordinating	–	<5	–	<3	0.14 ± 0.05	0.05 ± 0.04	ND
C275A	[2Fe-2S] coordinating	–	<5	–	<3	0.23 ± 0.11	0.28 ± 0.09	ND
C305A	[2Fe-2S] coordinating	–	<5	–	<3	0.18 ± 0.09	0.24 ± 0.12	ND

^a Table summarizes biochemical and spectroscopic properties of CntB variants. The FMN content (∼0.6 ± 0.1 mol per mol CntB), the specific activity of the l-carnitine depletion assay (771 ± 67 nmol min⁻¹ mg⁻¹), enzymatic TMA formation (determined by gas chromatography), and NADH depletion activity (407 ± 11 nmol min⁻¹ mg⁻¹) of the wild type were set as 100% (compared to experimental procedures). –, experiment not performed. <5 or <3, enzymatic activity below the detection limit of the respective assay. ND, an EPR signal was not detectable.