Duilio Arigoni

Biosynthesis of isoprenoids via the non-mevalonate pathway

The mevalonate pathway for the biosynthesis of the universal terpenoid precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), is known in considerable detail. Only recently, the existence of a second mevalonate-independent pathway for the biosynthesis of IPP and DMAPP was detected in plants and certain eubacteria. Experiments with 13C and/or 2H-labelled precursors were crucial in the elucidation of this novel route. The pathway is essential in plants, many eubacteria and apicomplexan parasites, but not in archaea and animals. The genes, enzymes and intermediates of this pathway were rapidly unravelled over the past few years. Detailed knowledge about the mechanisms of this novel route may benefit the development of novel antibiotics, antimalarials and herbicides.

The deoxyxylulose phosphate pathway of terpenoid biosynthesis in plants and microorganisms

Recent studies have uncovered the existence of an alternative, non-mevalonate pathway for the formation of isopentenyl pyrophosphate and dimethylallyl pyrophosphate, the two building blocks of terpene biosynthesis.

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

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-13C5]1-deoxy-d-xylulose into a 5:1 mixture of [U-13C5]isopentenyl diphosphate and [U-13C5]dimethylallyl diphosphate by an E. coli strain engineered for the expression of the ispH (lytB) gene in addition to recombinant xylB and ispCDEFG genes.

Studies on the nonmevalonate pathway to terpenes: the role of the GcpE (IspG) protein.

Recombinant Escherichia coli cells engineered for the expression of the xylB gene in conjunction with genes of the nonmevalonate pathway were supplied with 13C-labeled 1-deoxy-d-xylulose. Cell extracts were analyzed directly by NMR spectroscopy. 13C-labeled 2C-methyl-d-erythritol 2,4-cyclodiphosphate was detected at high levels in cells expressing xylB, ispC, ispD, ispE, and ispF. The additional expression of the gcpE gene afforded 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate as an intermediate of the nonmevalonate pathway. Hypothetical mechanisms involving conserved cysteine residues are proposed for the enzymatic conversion of 2C-methyl-d-erythritol 2,4-cyclodiphosphate into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate catalyzed by the GcpE protein.

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

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-deaza-isoalloxazine, 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 nmol⋅min−1⋅mg−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 μmol⋅min−1⋅mg−1. IspH protein could also be activated by a mixture of flavodoxin, flavodoxin reductase, and NADPH at a rate of 3 nmol⋅min−1⋅mg−1. The striking similarities of IspG and IspH protein are discussed, and plausible mechanistic schemes are proposed for the two reactions.

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

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 Co2+), 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.

Quantitative assessment of crosstalk between the two isoprenoid biosynthesis pathways in plants by NMR spectroscopy

Plants have been shown to use the mevalonate pathway for the biosynthesis of sterols and triterpenes in the cytoplasm and the recently discovered deoxyxylulose phosphate pathway for the biosynthesis of a variety of hemiterpenes, monoterpenes, diterpenes, as well as for the biosynthesis of carotenoids and the phytol side chain of chlorophyll in plastids. Despite the compartmental separation, at least one terpene precursor can be exchanged between the two pathways. In order to assess quantitatively the crosstalk between the two isoprenoid pathways, [2-13C1]mevalonolactone or [U-13C6]glucose were supplied to cell cultures of Catharanthus roseus grown under illumination or in darkness. Sitosterol, lutein and phytol were isolated and analysed by NMR spectroscopy. The incorporations of exogenous [2-13C1]mevalonolactone were 48% and 7% into the DMAPP and IPP precursors of sitosterol and lutein, respectively. With [U-13C6]glucose as precursor, at least 95% of sitosterol precursors were obtained via the mevalonate pathway, whereas phytol appeared to be biosynthesised via the deoxyxylulose phosphate pathway (approximately 60%) as well via the mevalonate pathway (approximately 40%). The apparent ratios for the contribution of the two pathways depend on the nature of the precursor supplied as well as the nature of the target compound. Thus, crosstalk between the two terpenoid pathways cannot be explained in detail by a simple two compartment model and requires an additional in depth study of complex regulatory mechanisms.

Zum Mechanismus der Propandioldehydrase-Reaktion

Investigation of the propanediol dehydrase reaction with18O-labelled substrates indicates that the conversion of propane-1,2-diol to propionaldehyde involves transfer of the oxygen atom from C-2 to C-1. The dehydration of the so formed propane-1,1-diol is sterically controlled by the enzyme.

Vitamin B1 biosynthesis in plants requires the essential iron–sulfur cluster protein, THIC

Vitamin B1 (thiamin) is an essential compound in all organisms acting as a cofactor in key metabolic reactions and has furthermore been implicated in responses to DNA damage and pathogen attack in plants. Despite the fact that it was discovered almost a century ago and deficiency is a widespread health problem, much remains to be deciphered about its biosynthesis. The vitamin is composed of a thiazole and pyrimidine heterocycle, which can be synthesized by prokaryotes, fungi, and plants. Plants are the major source of the vitamin in the human diet, yet little is known about the biosynthesis of the compound therein. In particular, it has never been verified whether the pyrimidine heterocycle is derived from purine biosynthesis through the action of the THIC protein as in bacteria, rather than vitamin B6 and histidine as demonstrated for fungi. Here, we identify a homolog of THIC in Arabidopsis and demonstrate its essentiality not only for vitamin B1 biosynthesis, but also plant viability. This step takes place in the chloroplast and appears to be regulated at several levels, including through the presence of a riboswitch in the 3′-untranslated region of THIC. Strong evidence is provided for the involvement of an iron–sulfur cluster in the remarkable chemical rearrangement reaction catalyzed by the THIC protein for which there is no chemical precedent. The results suggest that vitamin B1 biosynthesis in plants is in fact more similar to prokaryotic counterparts and that the THIC protein is likely to be the key regulatory protein in the pathway.

Reaction Mechanism of Pyridoxal 5′-Phosphate Synthase DETECTION OF AN ENZYME-BOUND CHROMOPHORIC INTERMEDIATE

Vitamin B6 is an essential metabolite in all organisms. De novo synthesis of the vitamin can occur through either of two mutually exclusive pathways referred to as deoxyxylulose 5-phosphate-dependent and deoxyxylulose 5-phosphate-independent. The latter pathway has only recently been discovered and is distinguished by the presence of two genes, Pdx1 and Pdx2, encoding the synthase and glutaminase subunit of PLP synthase, respectively. In the presence of ammonia, the synthase alone displays an exceptional polymorphic synthetic ability in carrying out a complex set of reactions, including pentose and triose isomerization, imine formation, ammonia addition, aldol-type condensation, cyclization, and aromatization, that convert C3 and C5 precursors into the cofactor B6 vitamer, pyridoxal 5′-phosphate. Here, employing the Bacillus subtilis proteins, we demonstrate key features along the catalytic path. We show that ribose 5-phosphate is the preferred C5 substrate and provide unequivocal evidence that the pent(ul)ose phosphate imine occurs at lysine 81 rather than lysine 149 as previously postulated. While this study was under review, corroborative crystallographic evidence has been provided for imine formation with the corresponding lysine group in the enzyme from Thermotoga maritima (Zein, F., Zhang, Y., Kang, Y.-N., Burns, K., Begley, T. P., and Ealick, S. E. (2006) Biochemistry 45, 14609–14620). We have detected an unanticipated covalent reaction intermediate that occurs subsequent to imine formation and is dependent on the presence of Pdx2 and glutamine. This step most likely primes the enzyme for acceptance of the triose sugar, ultimately leading to formation of the pyridine ring. Two alternative structures are proposed for the chromophoric intermediate, both of which require substantial modifications of the proposed mechanism.