Purification and characterization of the bacterial UDP-GlcNAc:undecaprenyl-phosphate GlcNAc-1-phosphate transferase WecA

Abstract

To date, the structural and functional characterization of proteins belonging to the polyprenyl-phosphate N-acetylhexosamine-1-phosphate transferase superfamily has been relentlessly held back by problems encountered with their overexpression and purification. In the present work and for the first time, the integral membrane protein WecA that catalyzes the transfer of the GlcNAc-1-phosphate moiety from UDP-GlcNAc onto the carrier lipid undecaprenyl phosphate, yielding undecaprenyl-pyrophosphoryl-GlcNAc, the lipid intermediate involved in the synthesis of various bacterial cell envelope components, was overproduced and purified to near homogeneity in milligram quantities. An enzymatic assay was developed, and the kinetic parameters of WecA as well as the effects of pH, salts, cations, detergents, and temperature on the enzyme activity were determined. A minimal length of 35 carbons was required for the lipid substrate, and tunicamycin was shown to inhibit the enzyme at submicromolar concentrations.

FIG. 1.

SDS-PAGE of purified T. maritima WecA protein. The WecA protein was overproduced in E. coli cells in the His₆-tagged form. The one-step purification on Ni²⁺-NTA agarose was performed as described in the text, and aliquots were analyzed by SDS-PAGE. Lane 2, DDM extract; lane 3, flowthrough; lane 4, 200 mM imidazole-containing fraction. Molecular mass standards indicated on the left (lane 1) are as follows: phosphorylase b, 97 kDa; bovine serum albumin, 66 kDa; ovalbumin, 45 kDa; carbonic anhydrase, 31 kDa; trypsin inhibitor, 21.5 kDa; and lysozyme, 14 kDa. The arrow points to the purified WecA protein. Staining was performed with Coomassie brilliant blue R250 (Merck).

FIG. 2.

Biochemical properties of the pure WecA enzyme. The effects of different parameters on WecA enzyme activity were investigated: pH (A); the salts NaCl (⧫) and KCl (▪) (B); MgCl₂ (C); the detergents Triton X-100 (▪), C₁₂E₆ (□), n-octyl-β-d-glucopyranoside (⧫), Tween 20 (▴), DDM (Δ), N-lauroyl sarcosine (○) and n-decyl-β-d-maltoside (•) (D); and temperature (E). A standard assay was carried out as described in the text with the following modifications: in panel A, bis-Tris-HCl buffer was used for pH 6.0 to 7.0, and Tris-HCl buffer was used for pH 7.2 to 9.4. Activity values are expressed in nmol/min/mg of protein. Each data point represents the mean of three independent experiments, and the standard deviation was in all cases less than 10%.

FIG. 3.

Specificity of the WecA enzyme for the lipid substrate. The importance of the carbon chain length of the lipid substrate for the activity of WecA was determined. A standard assay was carried out as detailed in the text using a 1.1 mM concentration of the tested lipid substrate. An activity value of 100% corresponds to 306 nmol/min/mg of protein. Data represent the mean of independent triplicate data sets, and the standard deviation was less than 10%.

FIG. 4.

Inhibition of WecA activity by tunicamycin. Incubation was performed as described in Materials and Methods except that various amounts of tunicamycin were added in the reaction mixture. Data represent the mean of independent triplicate data sets, and the standard deviation was less than 7%.