LytB, a novel gene of the 2‐C‐methyl‐<scp>D</scp>‐erythritol 4‐phosphate pathway of isoprenoid biosynthesis in Escherichia coli

315

231

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-Dxylulose 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 C5]1-deoxy-D-xylulose into a 5:1 mixture of [U-13 C5]isopentenyl diphosphate and [U-13 C5]dimethylallyl diphosphate by an E. coli strain engineered for the expression of the ispH (lytB) gene in addition to recombinant xylB and ispCDEFG genes.T erpenes are one of the largest groups of natural products comprising numerous medically relevant compounds (e.g., vitamins, hormones, and antitumor agents such as Taxol) (1). Bloch, Lynen, Cornforth, and their coworkers showed that the universal terpenoid precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), are biosynthesized by means of the mevalonate pathway in yeasts and animals (for review see refs. 2-5). These studies served as the basis for the development of metabolic inhibitors that are widely used for the treatment of hypercholesterolemia.For a period of several decades, these milestone achievements completely eclipsed the existence of a second terpenoid pathway for the biosynthesis of IPP and DMAPP. Recently, however, knowledge on that pathway has been unfolding rapidly on the basis of seminal discoveries by the research groups of . Briefly, the mevalonate independent pathway starts from 1-deoxy-D-xylulose 5-phosphate, which is assembled from pyruvate and D-glyceraldehyde 3-phosphate (13, 14) and was already known to serve as a biosynthetic precursor of vitamins B 1 (thiamine) and B 6 (pyridoxal) (Fig. 1) (15-17). 1-Deoxy-D-xylulose 5-phosphate is converted into 2C-methyl-D-erythritol 4-phosphate, the first committed intermediate of the nonmevalonate pathway, by isomerization followed by a two-electron reduction step catalyzed by 2C-methyl-D-erythritol 4-phosphate synthase specified by the ispC gene (formerly designated yaeM and then dxr) (18) (Fig. 1).In the next step, 2C-methyl-D-erythritol 4-phosphate is converted into the 2C-methyl-D-erythritol 2,4-cyclodiphosphate by the sequential action of three enzymes specified by the ispD, ispE, and ispF genes (19-24). The last known step of the sequence, the reductive transformation of the cyclic diphosphate into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate has been identified recently in work with a recombinant Escherichia coli strain engineered for hyperexpression of the ispG gene (previously designated gcpE) (25). Comparative genomics suggest that the protein specified by the lytB gene is involved in the conversion of ...

mentioning

confidence: 88%

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

Rohdich

Hecht²,

Gärtner³

et al. 2002

315

231

“…In vivo experiments showed that the reductive ring opening of 7 affording 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate 8 requires a protein specified by the ispG (gcpE) gene (18,19). The same compound was shown also to accumulate in an ispH-deficient Escherichia coli strain (20), and its formation from 14 C-labeled 7 was observed in cell extracts from E. coli overexpressing the ispG (gcpE) gene (21). Subsequent in vivo studies with a recombinant E. coli strain showed that the conversion of 8 into 9 and 10 requires a protein specified by the ispH (lytB) gene (22).…”

Section: Earlier In Vivo Studies Showed the Involvement Of Isph Protementioning

confidence: 99%

Biosynthesis of terpenes: Studies on 1-hydroxy-2-methyl-2-( E )-butenyl 4-diphosphate reductase

Adam

Hecht²,

Eisenreich³

et al. 2002

152

Earlier in vivo studies showed the involvement of IspH protein in the conversion of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate into isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). We have demonstrated now that cell extract of an Escherichia coli strain engineered for hyperexpression of the ispH (lytB) gene catalyzes the in vitro conversion of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate into IPP and DMAPP. The reaction requires NADH, FAD, divalent cations (preferably Co 2؉ ), and probably one or more as-yet-unidentified proteins. The low intrinsic catalytic activities of wild-type E. coli cell extract and isolated chromoplasts of red pepper (Capsicum annuum) are enhanced by the addition of purified recombinant IspH protein.

“…The additional implementation of a recombinant ispG gene resulted in the in vivo formation of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (6) (16), which was also isolated from an ispH-deficient E. coli mutant (17). Subsequently, the in vitro formation of 6 from 5 by crude cell extracts of E. coli overexpressing ispG was confirmed by radiochemical methods (18).…”

mentioning

confidence: 99%

The deoxyxylulose phosphate pathway of isoprenoid biosynthesis: Studies on the mechanisms of the reactions catalyzed by IspG and IspH protein

Rohdich

Zepeck

Adam

et al. 2003

210

234

Earlier in vivo studies have shown that the sequential action of the IspG and IspH proteins is essential for the reductive transformation of 2C-methyl-D-erythritol 2,4-cyclodiphosphate into dimethylallyl diphosphate and isopentenyl diphosphate via 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate. A recombinant fusion protein comprising maltose binding protein and IspG protein domains was purified from a recombinant Escherichia coli strain. The purified protein failed to transform 2C-methyl-D-erythritol 2,4-cyclodiphosphate into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate, but catalytic activity could be restored by the addition of crude cell extract from an ispG-deficient E. coli mutant. This indicates that auxiliary proteins are required, probably as shuttles for redox equivalents. On activation by photoreduced 10-methyl-5-deazaisoalloxazine, the recombinant protein catalyzed the formation of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate from 2C-methyl-D-erythritol 2,4-cyclodiphosphate at a rate of 1 nmol⅐min ؊1 ⅐mg ؊1 . Similarly, activation by photoreduced 10-methyl-5-deaza-isoalloxazine enabled purified IspH protein to catalyze the conversion of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate into a 6:1 mixture of isopentenyl diphosphate and dimethylallyl diphosphate at a rate of 0.4 mol⅐min ؊1 ⅐mg ؊1 . IspH protein could also be activated by a mixture of flavodoxin, flavodoxin reductase, and NADPH at a rate of 3 nmol⅐min ؊1 ⅐mg ؊1 . The striking similarities of IspG and IspH protein are discussed, and plausible mechanistic schemes are proposed for the two reactions.