Expression and characterization of soluble 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase from bacterial pathogens

Abstract

Many bacterial pathogens utilize the 2-C-methyl-D-erythritol 4-phosphate pathway for biosynthesizing isoprenoid precursors, a pathway that is vital for bacterial survival and absent from human cells, providing a potential source of drug targets. However, the characterization of 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME) kinase (IspE) has been hindered due to a lack of enantiopure CDP-ME and difficulty in obtaining pure IspE. Here, enantiopure CDP-ME was chemically synthesized and recombinant IspE from bacterial pathogens were purified and characterized. Although gene disruption was not possible in Mycobacterium tuberculosis, IspE is essential in Mycobacterium smegmatis. The biochemical and kinetic characteristics of IspE provide the basis for development of a high throughput screen and structural characterization.

Fig. 1

The MEP pathway and the reaction catalyzed by IspE. Starting with pyruvate and glyceraldehyde 3-phosphate (GAP), the sequential activities of DXS, IspC, and IspD produce CDP-ME, the substrate of IspE. IspE phosphorylates the tertiary hydroxyl group of CDP-ME forming CDP-ME2P. The reaction catalyzed by IspE is indicated by the dotted box. Abbreviations; DXS, 1-deoxy-D-xylulose 5-phosphate synthase; IspC, 1-deoxy-D-xylulose 5-phosphate reductoisomerase; IspD, 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase; IspE, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase; IspF, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; IspG, 1-hydroxyl-2-methyl-2(E)-butenyl 4-diphosphate synthase; IspH, 1-hydroxy-2-methyl-2(E)-butenyl 4-diphosphate reductase; CDP-ME, 4-diphosphocytidyl-2-C-methyl-D-erythritol; CDP-ME2P, CDP-ME 2 phosphate; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate.

Fig. 2

Essentiality of M. smegmatis IspE for the bacterial growth. Panel a: Schematic diagram of showing recombination in M. smegmatis mc²155. Black boxes indicate coding sequence of IspE (MSMEG_5436). Hatched regions represent the intragenic DNA fragment replaced with a Kanamycin (Kan) resistant cassette (Kan^R) from pUC4K. For generation of MSMEG_5436 disrupted Kan^R construct, Kan^R was cloned into RsrII sites of MSMEG_5436 and ligated with pPR27 yielding pPR27∷Sm ispE∷Kan^R. After homologous recombination, selection for Kan resistance, sucrose resistance and xylE phenotype resulted in colonies with a disrupted MSMEG_5436, only in the presence of rescue vector pCG76∷Sm ispE, which contains temperature sensitive origin of replication and heat shock protein 60 promoter (P_hsp60). Panel b: Growth curves of M. smegmatis mc²155 strains at 32 and 42°C. M. smegmatis mc²155 containing a rescue plasmid pCG76∷Sm ispE at 32°C (solid circles) or at 42°C (open circles), M. smegmatis mc²155 wild-type at 32°C (solid triangles) or at 42°C (open triangles). All cultures were grown in LB broth containing appropriate antibiotics (See Experimental Procedures). Panel c: Growth patterns of M. smegmatis mc²155 double-crossover strains containing pCG76∷Sm ispE at 32°C (left) and 42°C (right) on LB plates with appropriate antibiotics.

Fig. 3

Identification of M. tuberculosis IspE and purification strategy. Panel a: Alignment of the amino acid sequence of E. coli IspE (ECO) and the putative IspE amino acids of M. tuberculosis (Rv1011) (MTB), B. mallei (BMA), S. Typhi (STY), V. cholerae (VCH), and previously characterized Aquifex aeolicus (AAE) (Sgraja et al., 2008). Identities are highlighted in black and similarities in gray. The amino acids reported to be involved in CDP-ME binding (I) and ATP-binding (II + III) reported in E. coli IspE (Miallau et al., 2003) are underlined. Panel b: Rv1011 contains two predicted kinase motifs, a homoserine (ThrB) kinase (a.a. 36-299) and a GHMP Kinase (a.a. 88-268)]. A series of eight fragments were engineered as indicated. Panel c: Characteristics of the eight recombinant Rv1011 fragments. The primers used to amplify the fragments are listed in Supplemental Data S3. The solubility was determined by using SDS-PAGE analysis and the activities were determined using the radioisotope based in vitro IspE assay. − no; +, low; ++, intermediate; +++, good.

Fig. 4

Biochemical properties of M. tuberculosis IspE. a. The optimal pH for catalytic activity was determined using MES (pH 5.5 - pH 7.0), MOPS (pH 7.0 - pH 7.5), Tris (pH 7.5 - pH 8.5), TAPS (pH 8.5 - pH 9.0), and CAPS (pH 9.0 - pH 10.5) using appropriate counter ions. b. Divalent cations (Mg²⁺, Mn²⁺, Zn²⁺, or Ca²⁺) were added to the reaction mixtures at the indicated concentrations. The reaction mixtures were as described in Experimental Procedures.

Fig. 5

TLC analysis of the product generated by Rv1011 I. a. The reaction mixtures containing CDP-ME, MgCl₂, and [γ-³²P]ATP without Rv1011 I (lane 1) and with Rv1011 I (lane 2). b. Co-migration experiment with a reaction containing [γ-³²P]ATP, CDP-ME, and Rv1011 I in the presence of MnCl₂ (lane 2) and a reaction containing [γ-³²P]ATP, CDP-ME, Rv1011 I, and M. tuberculosis IspF (Eoh et al., 2009) in the presence of MgCl₂ (lane 3). c. Reaction mixture containing CDP-ME and different amount of wild type M. tuberculosis IspE. No (lane 1), 20 μg (lane 2), 40 μg (lane 3), and 60 μg (lane 4) of M. tuberculosis IspE were used. The migration positions of ATP, CDP-ME2P, and MECPP are indicated by the arrows.

Scheme 1

Synthetic scheme of enantiopure CDP-ME. Enantiopure 2-C-methyl-D-erythritol 4-phosphate (MEP) was synthesized from commercially available 1,2-O-isopropylidene-α-D-xylofuranose 3. Subsequently, MEP is coupled with CMP to give a 45% yield of pure CDP-ME.