TABLE 1.

Summary of site-directed mutagenesis analysis of the transmembrane domain of OafA^a

Mutant	O:5 signal intensity compared to WT (% ± SEM)	Position	Reason for mutation
R14A	0.07 ± 0.04	TMH1	Specifically conserved in AT3-SGNH proteins
H25A	0.33 ± 0.18	TMH1	Conserved in TMH1 across all aligned proteins
S32A	105.25 ± 30.89	Periplasmic loop and TMH2	XGG-F/Y-XGV-D/P/V-X motif found to be conserved in AT3-SGNH fused acyltransferases. In the first periplasmic loop between TMH1-2
G33A	119.17 ± 18.72	Periplasmic loop and TMH2
G34A	1.36 ± 0.88b	Periplasmic loop and TMH2
F35A	19.24 ± 2.70	Periplasmic loop and TMH2
I36A	101.47 ± 22.72	Periplasmic loop and TMH2
G37A	118.13 ± 22.11	Periplasmic loop and TMH2
V38A	86.38 ± 12.73	Periplasmic loop and TMH2
D39A	0.31 ± 0.07	Periplasmic loop and TMH2
V40A	121.28 ± 23.82	Periplasmic loop and TMH2
S45A	98.18 ± 24.30	TMH2	Conserved in SG in TMH2
G46A	99.59 ± 22.01	TMH2
R69A	0.10 ± 0.04	TMH3	RXXR motif previously identified as critical for function
R72A	0.07 ± 0.02	TMH3
S112A	0.24 ± 0.09	TMH3-4 Periplasmic loop	Conserved in periplasmic loop between TMH3-4 in AT3-SGNH fused proteins
N113A	93.79 ± 14.92	TMH3-4 Periplasmic loop
Y122A	85.76 ± 7.58	TMH3-4 Periplasmic loop
G202A	74.14 ± 10.70	TMH6	Conserved transmembrane glycine
E325A (linker)	4.84 ± 1.13	TMH10-11 cytoplasmic loop	Conserved after TMH10 in all AT3-SGNH fused proteins

^aDark gray shading, point mutants with <1% O:5 signal intensity; Light gray shading, point mutants with <50% O:5 signal intensity.

^bNo OafA protein expression detected. Values represent the average of 2 biological repeats with 3 technical replicates.