Supplementary Table 1. Properties of lysates of E. coli strains expressing CcLpxI point mutants Species UDP-2,3- diacylglucosamine hydrolase specific activity (nmol min -1 mg -1 ) Fold vectorcontrol specific activity Within H-bonding distance of substrate in D225A structure a UDP-2,3- diacylglucosamine accumulates in cells b Lipid X accumulates in cells b Vector control 6.02 1 N.A. c No No CcLpxI 13,100 2,180 N.A. No Yes Q169A Did not overexpress Did not overexpress Yes No No E182A E185A T187A R193A Q220A D225A 6.79 ~1 No No No 31.8 5.3 No No Yes 74.7 12.4 Yes No Yes N.D. 3 ~2,000 3 No N.D. d N.D. d 12.6 2.1 No No No 15.1 2.5 N.A. Yes Yes a. See Fig. 3 and Fig. 4 b. See Supplementary Fig. 5 c. Not applicable (N.A) d. Not determined (N.D.)
Supplementary Figure 1. Chromatography of CcLpxI and D225A, and purification benchmarks for D225A a), The overlay of two absorbance traces, both monitored at A262, from two separate size-exclusion separations performed on both wild-type and D225A. The red trace corresponds to ~ 40 mg of CcLpxID225A, while the blue trace follows ~ 60 mg of wild-type CcLpxI. The chromatography for both proteins was carried out in matched buffer, and using the same Superdex S200 sizing column. Note that the D225A peak, normalized for injection volume, is positioned at a slightly higher elution volume than the peak of wild-type CcLpxI, consistent with a smaller hydrodynamic radius expected relative to wild-type CcLpxI. Also note that the large difference in the samplesʼ A262, peak amplitude is due to the presence of the uridine moiety in the UDP-2,3-diacylglucosamine bound to D225A. The protein itself has very little absorbance at A262, as it lacks any tryptophan residues. b), An SDS-PAGE analysis of the sizing column fractions from a purification of the D225A mutant. Each lane contains 10 µl of a 5 ml sizingcolumn fraction. c), 5 µl portions of the same fractions are spotted, separated, and detected on a silica TLC plate following A UDP-2,3charring with H2SO4. diacygucosamine standard is present at the far left side of the plate.
Supplementary Figure 2. Supplemental Structural Data a), The 2Fo-Fc map (light blue mesh) corresponding to the LXD (dark blue) of CcLpxI. Two orientations of lipid X, shown in teal, are modeled into the density, which here were refined using partial occupancies. b), The same model, but with 2Fo-Fc density of a simulated-annealing OMIT map (orange mesh). c), The same data as (a), but viewing the entire molecule of CcLpxI. d), A stereo rendering of the CcLpxA backbone trace rendered as a ribbon.
Supplementary Figure 3. Co-purification of ligands with CcLpxI. a), The total ion current for lipid X isolated from purified CcLpxI. b), The mass spectrum corresponding to this peak. c). The MS/MS confirming the identity of the lipid X. d), The total ion current for UDP-2,3- diacylglucosamine isolated from purified D225A. e), The singly-charged mass spectrum corresponding to this peak. f), The MS/MS confirming the identity of the singly-charged UDP-2,3- diacylglucosamine species.
Supplementary Figure 4. Expression of CcLpxI point mutants Each lane of this 12% polyacrylamide gel is loaded with 15 µg of membrane-free lysate. VC denotes strain VC-21b and WT denotes CcI-21b. Q169A, E182A, E185A, T187A, Q220A, and D225A label membrane-free lysate from strains CcI-Q169A, CcI-E182A, CcI-E185A, CcI-T187A, CcI-Q220A, and CcI- D225A. Note that E185A seems to be expressed, but partially proteolyzed in vivo.
Supplementary Figure 5. Semi-quantitative assay of CcLpxI point mutants to determine R193A activity Crude lysates of E. coli expressing an empty vector, wild-type (W.T.) CcLpxI, and point mutants D225A, E185A, and R193A were assayed semi-quantitatively as follows: Each 25 µl reaction contained the following components: 500 µm UDP-2,3-diacyglucosamine, 0.5 mg/ml bovine serum albumin (BSA), 0.05% w/v Triton X-100, 20 mm HEPES ph 8.0, and 2 mm MgCl 2, and crude lysate at various concentrations. All assays were conducted at 30ºC, and quenched by spotting portions of the reaction onto silica TLC plates, which were resolved in CHCl 3 /MeOH/water/acetate 25/15/4/2 v/v/v/v, dried, sprayed with 10% v/v H 2 SO 4 in EtOH, and visualized by charring on a hot plate at 200 C. Lysate from E. coli over-expressing R193A has a specific activity similar to that of wild-type CcLpxI.
Supplementary Figure 6. Accumulation of lipid X or UDP-2,3-diacylglucosamine in E. coli over-expressing various CcLpxI constructs. a,b,c), For E. coli cells expressing an empty vector, regions of the total negative-ion LC/MS spectrum corresponding to the singly-charged lipid X region, the doubly-charged UDP-2,3-diacyglucosamine region, and the singly-charged UDP-2,3-diacyglucosamine region, respectively. d,e,f), These data when CcLpxI is over-expressed. g,h,i), The same data for E. coli over-expressing D225A.