Journal of Molecular Biology
The Structural Basis of Chain Length Control in Rv1086
Introduction
Pathogenic bacteria synthesize complex sugars containing polymers that they utilize in their cell wall as structural components including, in some cases, polymers that shield the organism from the human immune system. The biosynthesis of these complex carbohydrate molecules often utilizes sugars that are linked to long-chain lipid (polyprenyl phosphate, Pol-P) molecules. The most important example of this involves peptidoglycan biosynthesis where Pol-P is an essential carrier for its biosynthesis. Hence, these long-chain lipid carrier molecules are an essential component of cell wall biosynthesis and, as such, they have attracted considerable attention as potential drug targets.
The most common prenol (and therefore Pol-P) structures are generally confined to four main groups: (i) all-E-prenol, (ii) di-E, poly-Z-prenol, (iii) tri-E, poly-Z-prenol, and (iv) all-Z-prenol.1 Eubacteria typically contain a single predominant Pol-P composed of 11 isoprene units (55 carbon atoms), with the di-E, poly-Z configuration as seen in undecaprenyl phosphate (bactoprenyl phosphate).2, 3 The enzymes that carry out the synthesis of these molecules constitute the Z-prenyl synthases,4 and in bacteria, these enzymes are highly conserved, as shown in the sequence comparisons illustrated in Fig. 1a. Generally there is a single Z-prenyl synthase found in each organism. Thus, undecaprenyl phosphate is made using single-enzyme undecaprenyl diphosphate synthase (UDPS) starting from a 15-carbon ω,E,E-farnesyl diphosphate (EE-FPP) substrate. UDPS sequentially adds nine isoprene units.5 In contrast, Mycobacterium spp. contain structurally unusual Pol-P molecules, which have 10 isoprene units (50 carbon atoms) with a mono E and poly-Z configuration.3, 6, 7, 8 Thus, the human pathogen Mycobacterium tuberculosis relies on an unusual combination of two related Z-prenyl synthetase enzymes, Rv2361c and Rv1086 (Fig. 1a), to synthesize this 50-carbon molecule decaprenyl diphosphate,6, 7, 8 which is subsequently dephosphorylated, generating an activated carrier molecule for sugars. Rv1086 makes only a (relatively) short C15 ω,E,Z-farnesyl diphosphate (EZ-FPP). Although Rv2361c can use EE-FFP and even the 10-carbon geranyl diphosphate (GPP) as substrates, it has a strong substrate preference for EZ-FPP to which it adds seven isoprene units6 (Fig. 1b shows these isoprene structures). Since the discussion of substrate and product binding sites can be confusing for an enzyme that further elongates its products (i.e., a processive enzyme), we designate the binding sites as the isopentenyl diphosphate (IPP) binding site and as the isoprene polymer (polymer) binding site as illustrated in Fig. 1c.
The first crystal structure of a Z-prenyl synthase was reported for UDPS from Micrococcus luteus and the IPP and isoprene polymer binding sites were described.9 The structural characterization of the enzyme from Escherichia coli with substrate and substrate analogue complexes identified the key residues involved in recognition and catalysis.10 The mechanism proposed for UDPS (and, by extension, all Z-prenyl transferases)9, 10 requires binding both IPP and EE-FPP. An essential Mg2+ ion catalyzes the nucleophilic attack of IPP at the C1 of EE-FPP, displacing diphosphate and making the 20-carbon isoprene polymer (Fig. 1c) that now occupies the IPP site. The nascent isoprene polymer then moves from the IPP site to the isoprene polymer site allowing reloading of IPP for the next condensation (Fig. 1c). Recent studies on the M. luteus UDPS enzyme have identified several residues that are important in regulating the length of the C55 product.11 Mutant UDPS enzymes that make both longer and shorter product chain lengths have been reported.11
Rv1086, unlike all other characterized Z-prenyl diphosphate synthases, is not a processive enzyme; instead, it condenses IPP and GPP to synthesize only EZ-FPP. The molecular basis for this tight regulation of chain length is currently unknown. We now report the crystal structures of Rv1086 and Rv2361c, both complexed with an inhibiting substrate analogue, (S)-(−)-β-citronellyl diphosphate (CITPP). In addition, for Rv1086, we report an EE-FPP complex (product mimic), and for Rv2361c, we report an IPP complex. These structures have allowed detailed molecular insight into chain-length regulation in Rv1086. We have engineered a mutant of Rv1086 that is no longer limited to a 15-carbon product.
Section snippets
Overall structure
Rv1086 has a six-stranded β sheet that has four α helices on one face (Fig. 2a). Rv2361c has a structure very similar to that of Rv1086 (Fig. 2b) and the rmsd for 203 overlapping Cα atoms is 1.4 Å. A comparison of the structure is shown in Supplementary Fig. 1. Gel-filtration studies and the crystal structures suggest both Rv1086 and Rv2361c are dimers. The dimer interface is reminiscent of a four-helix bundle with two helices from each monomer. In addition to the helical bundle, there are
Discussion
The crystal structures of Rv2361c and Rv1086 confirm these proteins are related and belong to the Z-prenyl synthetase superfamily. The structural data support the UDPS study, which concluded9, 10 that N89 of Rv1086 (N124 in Rv2361c) acts as the catalytic base removing a proton from C2 of IPP. In all complexes where the IPP site is occupied, the OD1 atom of this Asn residue is within 4.3 Å of the C2 of IPP where the pro S proton has to be abstracted (Fig. 1c). The basicity of the Asn is enhanced
Materials
[1-14C]Isopentenyl diphosphate ([14C]IPP, 55 mCi/mmol) was purchased from GE Healthcare Life Sciences. Kanamycin, (S)-(−)-β-citronellol, E,E-farnesol, geraniol and E,E,E-geranylgeraniol were purchased from Sigma-Aldrich. E,E-FPP, CITPP and GPP were synthesized as described by Davisson et al.13 Authentic long-chain polyprenols were obtained from the Institute of Biochemistry and Biophysics, Polish Academy of Sciences (Warsaw, Poland).
LKC18F reverse-phase thin-layer chromatography (TLC) plates
Acknowledgements
We thank Robert Eales and Casie Johnson for technical assistance. The work was supported by National Institutes of Health grants AI057836, AI049151, AI065357 and AI018357 and Wellcome Trust. We acknowledge use of European Synchrotron Radiation Facility beamlines.
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