Studies on the nonmevalonate terpene biosynthetic pathway: metabolic role of IspH (LytB) protein

Abstract

Isopentenyl diphosphate and dimethylallyl diphosphate serve as the universal precursors for the biosynthesis of terpenes. Although their biosynthesis by means of mevalonate has been studied in detail, a second biosynthetic pathway for their formation by means of 1-deoxy-D-xylulose 5-phosphate has been discovered only recently in plants and certain eubacteria. Earlier in vivo experiments with recombinant Escherichia coli strains showed that exogenous 1-deoxy-D-xylulose can be converted into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate by the consecutive action of enzymes specified by the xylB and ispCDEFG genes. This article describes the transformation of exogenous [U-(13)C(5)]1-deoxy-D-xylulose into a 5:1 mixture of [U-(13)C(5)]isopentenyl diphosphate and [U-(13)C(5)]dimethylallyl diphosphate by an E. coli strain engineered for the expression of the ispH (lytB) gene in addition to recombinant xylB and ispCDEFG genes.

Figure 1

The nonmevalonate pathway of isoprenoid biosynthesis.

Figure 2

Expression vectors comprising synthetic operons of xylB together with genes of the nonmevalonate pathway used for the in vivo production of intermediates in the nonmevalonate pathway. ¹³C-labeled compounds detected in cell extracts obtained from E. coli cells overexpressing the indexed genes and supplied with [U-¹³C₅]1-deoxy-d-xylulose are indicated. Genes and their respective products are shown in the same colors.

Figure 3

¹³C NMR spectra of crude extracts obtained from the feeding of [U-¹³C₅]1-deoxy-d-xylulose to recombinant cells of E. coli overexpressing (a) the xylB, ispC-F genes, (b) the xylB, ispC-ispG genes, and (c) the xylB, ispC-ispH genes.

Figure 4

¹³C NMR signals of IPP (in red) and isopentenol (in light blue) (a) of the crude extract obtained from the feeding of [U-¹³C₅]1-deoxy-d-xylulose to recombinant cells of E. coli overexpressing the xylB, ispC-H genes (cf. Fig. 3) (b and c) of reference samples with natural ¹³C abundances.

Figure 5

¹³C NMR signals of DMAPP (in magenta) (b) of the crude extract obtained from the feeding of [U-¹³C₅]1-deoxy-d-xylulose to recombinant cells of E. coli overexpressing the xylB, ispC-H genes (cf. Fig. 3) (a) of a reference sample with natural ¹³C abundance.

Figure 6

A generalized structure for the hypothetical intermediate(s) at the branching point of the IspH-mediated reaction. * is related to the unknown number of electrons (2 to 4) in the delocalized allyl system.