A 1-deoxy-D-xylulose 5-phosphate reductoisomerase catalyzing the formation of 2-C-methyl-D-erythritol 4-phosphate in an alternative nonmevalonate pathway for terpenoid biosynthesis

Abstract

Several eubacteria including Esherichia coli use an alternative nonmevalonate pathway for the biosynthesis of isopentenyl diphosphate instead of the ubiquitous mevalonate pathway. In the alternative pathway, 2-C-methyl-D-erythritol or its 4-phosphate, which is proposed to be formed from 1-deoxy-D-xylulose 5-phosphate via intramolecular rearrangement followed by reduction process, is one of the biosynthetic precursors of isopentenyl diphosphate. To clone the gene(s) responsible for synthesis of 2-C-methyl-D-erythritol 4-phosphate, we prepared and selected E. coli mutants with an obligatory requirement for 2-C-methylerythritol for growth and survival. All the DNA fragments that complemented the defect in synthesizing 2-C-methyl-D-erythritol 4-phosphate of these mutants contained the yaeM gene, which is located at 4.2 min on the chromosomal map of E. coli. The gene product showed significant homologies to hypothetical proteins with unknown functions present in Haemophilus influenzae, Synechocystis sp. PCC6803, Mycobacterium tuberculosis, Helicobacter pyroli, and Bacillus subtilis. The purified recombinant yaeM gene product was overexpressed in E. coli and found to catalyze the formation of 2-C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate in the presence of NADPH. Replacement of NADPH with NADH decreased the reaction rate to about 1% of the original rate. The enzyme required Mn2+, Co2+, or Mg2+ as well. These data clearly show that the yaeM gene encodes an enzyme, designated 1-deoxy-D-xylulose 5-phosphate reductoisomerase, that synthesizes 2-C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate, in a single step by intramolecular rearrangement and reduction and that this gene is responsible for terpenoid biosynthesis in E. coli.

Figure 1

Hypothetical alternative nonmevalonate pathway for IPP biosynthesis (A) and ketol acid reductoisomerase reaction (B).

Figure 2

Phenotypes of the isolated mutants and the parent strain W3110. The three mutants designated ME1, ME2, and ME3 cannot grow on M9 and LB plates but do grow on M9 plates supplemented 0.1% ME (M9+ME). In addition, these mutants transformed with pMEW73 (Fig. 3) become able to grow on M9 without ME (M9) (Lower Right).

Figure 3

Restriction map around the yaeM gene and DNA fragments that complemented IPP biosynthesis coding region of the isolated mutants. The arrows indicate the extents and directions of the ORFs found in the database of E. coli genome sequence. smb, frr, and cdsA have been identified as MukB suppressor protein, ribosome recycling factor, and phosphatidate cytidylyltransferase, respectively. Both yaeM and yaeS have been reported as hypothetical proteins. pMEW73 contains only the yaeM gene in its inserted DNA. The plasmid overcame requirement of ME for growth of all the mutants.

Figure 4

Multiple alignment of the amino acid sequences of E. coli yaeM gene and other yaeM gene homologs. Identical amino acids among six proteins are marked by asterisks. The putative NADPH binding motif is marked by #. Dashes indicate gaps introduced for the optimization of the alignment. Ec, E. coli (accession no. AB013300); Bs, Bacillus subtilis (accession no. B69881); Hi, Haemophilus influenzae (accession no. P44055); Hp, Helicobacter pyroli, (accession no. P56139); Mycobacterium tuberculosis (accession no. Q10798), Sy, Synechocystis sp. PCC6803 (accession no. Q55663).

Figure 5

Expression and purification of the recombinant DXP reductoisomerase. SDS/PAGE of the enzyme. Lanes: 1, low molecular mass marker; 2, the extract of uninduced E. coli M15 (pREP4, pQEDXR) culture; 3, the same extract after induction with isopropyl β-d-thiogalactoside; 4, purified enzyme after Ni-nitrilotriacetic acid agarose chromatography. Native PAGE of the purified enzyme. Lanes: 5, purified enzyme with 10 mM DTT; 6 and 8, high molecular mass markers; 7, purified enzyme without DTT. Positions of DXP reductoisomerase are indicated by arrows.

Figure 6

DXP reductoisomerase reaction.