Effects of producing high levels of hyperthermophile-specific C25,C25-archaeal membrane lipids in Escherichia coli.

A hyperthermophilic archaeon, Aeropyrum pernix, synthesizes C25,C25-archaeal membrane lipids, or extended archaeal membrane lipids, which contain two C25 isoprenoid chains that are linked to glycerol-1-phosphate via ether bonds and are longer than the usual C20,C20-archaeal membrane lipids. The C25,C25-archaeal membrane lipids are believed to allow the archaeon to survive under harsh conditions, because they are able to form lipid membranes that are impermeable at temperatures approaching the boiling point. The effect that C25,C25-archaeal membrane lipids exert on living cells, however, remains unproven along with an explanation for why the hyperthermophilic archaeon synthesizes these specific lipids instead of the more common C20,C20-archaeal lipids or double-headed tetraether lipids. To shed light on the effects that these hyperthermophile-specific membrane lipids exert on living cells, we have constructed an E. coli strain that produces C25,C25-archaeal membrane lipids. However, a resultant low level of productivity would not allow us to assess the effects of their production in E. coli cells. Herein, we report an enhancement of the productivity of C25,C25-archaeal membrane lipids in engineered E. coli strains via the introduction of metabolic pathways such as an artificial isoprenol utilization pathway where the precursors of isoprenoids are synthesized via a two-step phosphorylation of prenol and isoprenol supplemented to a growth medium. In the strain with the highest titer, a major component of C25,C25-archaeal membrane lipids reached ∼11 % of total lipids of E. coli. It is noteworthy that the high production of the extended archaeal lipids did not significantly affect the growth of the bacterial cells. The permeability of the cell membrane of the strain became slightly lower in the presence of the exogenous membrane lipids with longer hydrocarbon chains, which demonstrated the possibility to enhance bacterial cell membranes by the hyperthermophile-specific lipids, along with the surprising robustness of the E. coli cell membrane.

Crystal structure of mevalonate 3,5-bisphosphate decarboxylase reveals insight into the evolution of decarboxylases in the mevalonate metabolic pathways.

Mevalonate 3,5-bisphosphate decarboxylase is involved in the recently discovered Thermoplasma-type mevalonate pathway. The enzyme catalyzes the elimination of the 3-phosphate group from mevalonate 3,5-bisphosphate as well as concomitant decarboxylation of the substrate. This entire reaction of the enzyme resembles the latter half-reactions of its homologs, diphosphomevalonate decarboxylase and phosphomevalonate decarboxylase, which also catalyze ATP-dependent phosphorylation of the 3-hydroxyl group of their substrates. However, the crystal structure of mevalonate 3,5-bisphosphate decarboxylase and the structural reasons of the difference between reactions catalyzed by the enzyme and its homologs are unknown. In this study, we determined the X-ray crystal structure of mevalonate 3,5-bisphosphate decarboxylase from Picrophilus torridus, a thermoacidophilic archaeon of the order Thermoplasmatales. Structural and mutational analysis demonstrated the importance of a conserved aspartate residue for enzyme activity. In addition, although crystallization was performed in the absence of substrate or ligands, residual electron density having the shape of a fatty acid was observed at a position overlapping the ATP-binding site of the homologous enzyme, diphosphomevalonate decarboxylase. This finding is in agreement with the expected evolutionary route from phosphomevalonate decarboxylase (ATP-dependent) to mevalonate 3,5-bisphosphate decarboxylase (ATP-independent) through the loss of kinase activity. We found that the binding of geranylgeranyl diphosphate, an intermediate of the archeal isoprenoid biosynthesis pathway, evoked significant activation of mevalonate 3,5-bisphosphate decarboxylase, and several mutations at the putative geranylgeranyl diphosphate-binding site impaired this activation, suggesting the physiological importance of ligand binding as well as a possible novel regulatory system employed by the Thermoplasma-type mevalonate pathway.

Conversion of Mevalonate 3-Kinase into 5-Phosphomevalonate 3-Kinase by Single Amino Acid Mutations.

Mevalonate 3-kinase plays a key role in a recently discovered modified mevalonate pathway specific to thermophilic archaea of the order Thermoplasmatales The enzyme is homologous to diphosphomevalonate decarboxylase, which is involved in the widely distributed classical mevalonate pathway, and to phosphomevalonate decarboxylase, which is possessed by halophilic archaea and some Chloroflexi bacteria. Mevalonate 3-kinase catalyzes the ATP-dependent 3-phosphorylation of mevalonate but does not catalyze the subsequent decarboxylation as related decarboxylases do. In this study, a substrate-interacting glutamate residue of Thermoplasma acidophilum mevalonate 3-kinase was replaced by smaller amino acids, including its counterparts in diphosphomevalonate decarboxylase and phosphomevalonate decarboxylase, with the aim of altering substrate specificity. These single amino acid mutations resulted in the conversion of mevalonate 3-kinase into 5-phosphomevalonate 3-kinase, which can synthesize 3,5-bisphosphomevalonate from 5-phosphomevalonate. The mutants catalyzing the hitherto undiscovered reaction enabled the construction of an artificial mevalonate pathway in Escherichia coli cells, as was demonstrated by the accumulation of lycopene, a red carotenoid pigment.IMPORTANCE Isoprenoid is the largest family of natural compounds, including important bioactive molecules such as vitamins, hormones, and natural medicines. The mevalonate pathway is a target for metabolic engineering because it supplies precursors for isoprenoid biosynthesis. Mevalonate 3-kinase is an enzyme involved in the modified mevalonate pathway specific to limited species of thermophilic archaea. Replacement of a single amino acid residue in the active site of the enzyme changed its substrate preference and allowed the mutant enzymes to catalyze a previously undiscovered reaction. Using the genes encoding the mutant enzymes and other archaeal enzymes, we constructed an artificial mevalonate pathway, which can produce the precursor of isoprenoid through an unexplored route, in bacterial cells.

Modified mevalonate pathway of the archaeon Aeropyrum pernix proceeds via trans-anhydromevalonate 5-phosphate.

The modified mevalonate pathway is believed to be the upstream biosynthetic route for isoprenoids in general archaea. The partially identified pathway has been proposed to explain a mystery surrounding the lack of phosphomevalonate kinase and diphosphomevalonate decarboxylase by the discovery of a conserved enzyme, isopentenyl phosphate kinase. Phosphomevalonate decarboxylase was considered to be the missing link that would fill the vacancy in the pathway between mevalonate 5-phosphate and isopentenyl phosphate. This enzyme was recently discovered from haloarchaea and certain Chroloflexi bacteria, but their enzymes are close homologs of diphosphomevalonate decarboxylase, which are absent in most archaea. In this study, we used comparative genomic analysis to find two enzymes from a hyperthermophilic archaeon, Aeropyrum pernix, that can replace phosphomevalonate decarboxylase. One enzyme, which has been annotated as putative aconitase, catalyzes the dehydration of mevalonate 5-phosphate to form a previously unknown intermediate, trans-anhydromevalonate 5-phosphate. Then, another enzyme belonging to the UbiD-decarboxylase family, which likely requires a UbiX-like partner, converts the intermediate into isopentenyl phosphate. Their activities were confirmed by in vitro assay with recombinant enzymes and were also detected in cell-free extract from A. pernix These data distinguish the modified mevalonate pathway of A. pernix and likely, of the majority of archaea from all known mevalonate pathways, such as the eukaryote-type classical pathway, the haloarchaea-type modified pathway, and another modified pathway recently discovered from Thermoplasma acidophilum.

Identification of enzymes involved in the mevalonate pathway of Flavobacterium johnsoniae.

The mevalonate pathway is prevalent in eukaryotes, archaea, and a limited number of bacteria. This pathway yields the fundamental precursors for isoprenoid biosynthesis, i.e., isopentenyl diphosphate and dimethylally diphosphate. In the downstream part of the general eukaryote-type mevalonate pathway, mevalonate is converted into isopentenyl diphosphate by the sequential actions of mevalonate kinase, phosphomevalonate kinase, and diphosphomevalonte decarboxylase, while a partial lack of the putative genes of these enzymes is sometimes observed in archaeal and bacterial genomes. The absence of these genes has led to the recent discovery of modified mevalonate pathways. Therefore, we decided to investigate the mevalonate pathway of Flavobacterium johnsoniae, a bacterium of the phylum Bacteroidetes, which is reported to lack the genes of mevalonate kinase and phosphomevalonate kinase. This study provides proof of the existence of the general mevalonate pathway in F. johnsoniae, although the pathway involves the kinases that are distantly related to the known enzymes.

A Single Amino Acid Mutation Converts (R)-5-Diphosphomevalonate Decarboxylase into a Kinase.

The biosynthesis of isopentenyl diphosphate, a fundamental precursor for isoprenoids, via the mevalonate pathway is completed by diphosphomevalonate decarboxylase. This enzyme catalyzes the formation of isopentenyl diphosphate through the ATP-dependent phosphorylation of the 3-hydroxyl group of (R)-5-diphosphomevalonate followed by decarboxylation coupled with the elimination of the 3-phosphate group. In this reaction, a conserved aspartate residue has been proposed to be involved in the phosphorylation step as the general base catalyst that abstracts a proton from the 3-hydroxyl group. In this study, the catalytic mechanism of this rare type of decarboxylase is re-investigated by structural and mutagenic studies on the enzyme from a thermoacidophilic archaeon Sulfolobus solfataricus The crystal structures of the archaeal enzyme in complex with (R)-5-diphosphomevalonate and adenosine 5'-O-(3-thio)triphosphate or with (R)-5-diphosphomevalonate and ADP are newly solved, and theoretical analysis based on the structure suggests the inability of proton abstraction by the conserved aspartate residue, Asp-281. Site-directed mutagenesis on Asp-281 creates mutants that only show diphosphomevalonate 3-kinase activity, demonstrating that the residue is required in the process of phosphate elimination/decarboxylation, rather than in the preceding phosphorylation step. These results enable discussion of the catalytic roles of the aspartate residue and provide clear proof of the involvement of a long predicted intermediate, (R)-3-phospho-5-diphosphomevalonate, in the reaction of the enzyme.

In Vivo Formation of the Protein Disulfide Bond That Enhances the Thermostability of Diphosphomevalonate Decarboxylase, an Intracellular Enzyme from the Hyperthermophilic Archaeon Sulfolobus solfataricus.

UNLABELLED: In the present study, the crystal structure of recombinant diphosphomevalonate decarboxylase from the hyperthermophilic archaeon Sulfolobus solfataricus was solved as the first example of an archaeal and thermophile-derived diphosphomevalonate decarboxylase. The enzyme forms a homodimer, as expected for most eukaryotic and bacterial orthologs. Interestingly, the subunits of the homodimer are connected via an intersubunit disulfide bond, which presumably formed during the purification process of the recombinant enzyme expressed in Escherichia coli. When mutagenesis replaced the disulfide-forming cysteine residue with serine, however, the thermostability of the enzyme was significantly lowered. In the presence of ß-mercaptoethanol at a concentration where the disulfide bond was completely reduced, the wild-type enzyme was less stable to heat. Moreover, Western blot analysis combined with nonreducing SDS-PAGE of the whole cells of S. solfataricus proved that the disulfide bond was predominantly formed in the cells. These results suggest that the disulfide bond is required for the cytosolic enzyme to acquire further thermostability and to exert activity at the growth temperature of S. solfataricus. IMPORTANCE: This study is the first report to describe the crystal structures of archaeal diphosphomevalonate decarboxylase, an enzyme involved in the classical mevalonate pathway. A stability-conferring intersubunit disulfide bond is a remarkable feature that is not found in eukaryotic and bacterial orthologs. The evidence that the disulfide bond also is formed in S. solfataricus cells suggests its physiological importance.