Table 1. Pyocyanin complementation of a P. aeruginosa PA14 pqsE⁻ strain transformed with the PqsE variants and the predicted free energy variation caused by each point mutation.

	PqsE Variant	Pyocyanin Production (mg/L) a	ΔΔG 0 (kcal/mol)b	SA
	WT	6.2 (0.2)	–	–
Active Site [AS]	H69A	0.1 (0.1)	2.9	0.0
	H71A	2.7 (0.6)	1.5	0.2
	D73A	4.1 (0.8)	0.9	0.1
	H74A	3.5 (0.6)	2.3	0.0
	H159A	0.1 (0.0)	2.3	0.0
	D178A	0.1 (0.1)	1.4	0.0
	E182A	1.2 (0.4)	1.5	0.1
	L193A	4.7 (1.2)	2.3	0.1
	F195A	0.8 (0.3)	2.7	0.1
	H221A	1.1 (0.2)	2.3	0.1
	Q272A	1.4 (0.4)	0.9	0.2
	S273A	0.1 (0.1)	0.9	0.0
	F276A	0.1 (0.0)	2.3	0.0
	L277A	4.5 (1.1)	1.8	0.1
	S285A	5.0 (0.6)	0.3	0.1
	S285W	5.6 (0.6)	0.1	0.1
	R288A	5.2 (1.0)	0.5	0.5
C-terminal α-helix motif [α8/α9]	L248A	4.1 (1.1)	2.2	0.0
	L261A	0.2 (0.1)	2.0	0.0
	W269A	0.1 (0.0)	3.1	0.1
	H282A	0.4 (0.2)	2.0	0.1
	M286A	0.1 (0.1)	1.9	0.0
	L290A	0.1 (0.1)	2.5	0.0
KH-like motif [KH]	R95A	5.1 (0.5)	0.6	0.5
	K101A	4.4 (0.4)	0.6	0.6
	R107A	4.0 (0.8)	0.1	0.6
	R111A	0.3 (0.2)	0.2	0.6
	D130A	0.2 (0.1)	0.2	0.6

^aPyocyanin production (mg/L) was measured in triplicate as described in the experimental procedures. The mean value (± standard deviation) is shown. ^b Free energy variations caused by point mutants of PqsE, as predicted by the PoPMuSiC software (v.2). ^c Calculated solvent accessibility for each WT residue, ranging from 0 (fully buried in the protein core) to 1 (fully accessible to the solvent).