Table S3.

Thermodynamic parameters for the interaction of CaFADS WT and mutants with flavins determined by ITC. Data obtained at 25 °C in 20 mM PIPES, pH 7.0, at the indicated MgCl₂ concentration. Errors in ΔG, ΔH and −TΔS where ±0.3 kcal/mol, in general larger than the standard deviation between replicates and the numerical error after fitting the analysis.

	FADS:FMN 10 mM Mg2+			FADS:FAD 10 mM Mg2+			FADS:ATP 10 mM Mg2+			FADS:ATP 0 mM Mg2+
	ΔG (kcal/mol)	ΔH (kcal/mol)	−TΔS (kcal/mol)	ΔG (kcal/mol)	ΔH (kcal/mol)	−TΔS (kcal/mol)	ΔG (kcal/mol)	ΔH (kcal/mol)	−TΔS (kcal/mol)	ΔG (kcal/mol)	ΔH (kcal/mol)	−TΔS (kcal/mol)
WT	−7.0	−35	28	−8.4	−26	18	−6.2	−30	23	−7.2	−31	24
H28A	−6.7	−1.4	−5.3	−5.9	−3.5	−2.4	−4.3	−8.3	4.0	---	---	---
H28D	−7.0	−43	36	−7.8	−42	35	−5.3	−23	18	−6.0	−6.1	0.1
H31A	−6.7	−2.0	−4.7	−6.5	−1.2	−5.3	−5.9	−2.6	−3.3	---	---	---
H31D	−7.1	−1.4	−5.7	−6.7	−2.1	−4.6	−5.9	−4.6	−1.3	---	---	---
N125A	−7.2	−40	33	−7.6	−29	22	−5.3	−25	20	−7.1	−16	8.7
N125D	−6.0	−39	33	−6.7	−24	17	−5.7	−42	36	−5.5	−30	25
R161A	−6.5	−42	36	−8.4	−30	22	−6.2	−34	27	−6.2	−29	23
R161D	−6.6	−5.2	−1.4	−8.3	−4.9	−3.4	−5.7	−7.2	1.5	−5.2	−38	33
S164A	−8.0	−1.1	−6.9	−6.3	−17	11	−5.4	−24	19	−5.5	−40	35
S164D	−7.2	−2.6	−4.6	−6.2	−64	58	−5.4	−25	20	−5.4	−15	9.6
T165A	−5.7	−26	20	−6.7	−18	11	−5.4	−21	16	−6.1	−31	25
T165D	−7.8	−37	29	−8.8	−22	13	−5.4	−55	50	−6.7	−6.9	0.2