TABLE 2.

Enzyme and Mo-bisPGD cofactor quantifications and enzyme activities determined by in vitro reduction assays and in vivo growth experiments

Enzyme	[DmsABC]a	Mo-bisPGD contentb	Specific activityc		Growthd
			BV	LPCH2
	nmol/mg
pBR322	0	0	11.8	0.44	None
DmsABC	0.94	100	141	7.72	High
R61K	0.99	90	125	2.63	High
ΔN21	0.57	0	5.91	0.07	None
CS1	0.62	13	5.22	0.14	None
CS1 + R61K	0.58	44	7.97	0.33	None

^a The concentrations of wild-type and mutant DmsABC in each membrane sample were determine by HOQNO fluorescence quench titration. pBR322 is an empty vector, so no DmsABC could be detected.

^b The Mo-bisPGD cofactor content determined by the Form A fluorescence is qualitative and reported in terms of relative fluorescence intensities. The peak emission fluorescence for wild-type DmsABC was normalized to 100, and the relative fluorescence for the mutant enzymes was calculated using their peak emission fluorescence. The relative cofactor occupancies were then calculated using relative cofactor content and DmsABC concentrations and are shown in Fig. 5B.

^c Specific enzyme activities of wild-type and mutant DmsABC were assayed by BV- or LPCH₂-dependent reductions of trimethylamine N-oxide. Assays were carried out in triplicate, and average enzyme specific activities are reported. The specific enzyme activities are given in micromoles of BV or LPCH₂/min/mg of total protein. The turnover rates (s⁻¹) were then calculated using the specific activities and DmsABC concentrations, and the relative rates with the wild type normalized to 100 are reported in Fig. 5B.

^d DSS301 cells transformed with plasmid encoding wild-type DmsABC and its variants were grown anaerobically on glycerol/dimethyl sulfoxide minimum medium.