Escherichia coli YggS is a member of the highly conserved uncharacterized protein family that binds pyridoxal 5=-phosphate (PLP). To assist with the functional assignment of the YggS family, in vivo and in vitro analyses were performed using a yggSdeficient E. coli strain (⌬yggS) and a purified form of YggS, respectively. In the stationary phase, the ⌬yggS strain exhibited a completely different intracellular pool of amino acids and produced a significant amount of L-Val in the culture medium. The log-phase ⌬yggS strain accumulated 2-ketobutyrate, its aminated compound 2-aminobutyrate, and, to a lesser extent, L-Val. It also exhibited a 1.3-to 2.6-fold increase in the levels of Ile and Val metabolic enzymes. The fact that similar phenotypes were induced in wild-type E. coli by the exogenous addition of 2-ketobutyrate and 2-aminobutyrate indicates that the 2 compounds contribute to the ⌬yggS phenotypes. We showed that the initial cause of the keto acid imbalance was the reduced availability of coenzyme A (CoA); supplementation with pantothenate, which is a CoA precursor, fully reversed phenotypes conferred by the yggS mutation. The plasmid-borne expression of YggS and orthologs from Bacillus subtilis, Saccharomyces cerevisiae, and humans fully rescued the ⌬yggS phenotypes. Expression of a mutant YggS lacking PLP-binding ability, however, did not reverse the ⌬yggS phenotypes. These results demonstrate for the first time that YggS controls Ile and Val metabolism by modulating 2-ketobutyrate and CoA availability. Its function depends on PLP, and it is highly conserved in a wide range species, from bacteria to humans.

Prenyltransferases catalyze the consecutive condensation of isopentenyl diphosphate (IPP) with allylic diphosphates to produce prenyl diphosphates whose chain lengths are absolutely determined by each enzyme. In order to investigate the mechanisms of the consecutive reaction and of the determination of ultimate chain length, a random mutational approach was planned. The farnesyl diphosphate (FPP) synthase gene of Bacillus stearothermophilus was subjected to random mutagenesis by NaNO2 treatment to construct libraries of mutated FPP synthase genes on a high-copy plasmid. From the libraries, the mutants that showed the activity of geranylgeranyl diphosphate (GGPP) synthase were selected by the red-white screening method (Ohnuma, S.-i., Suzuki, M., and Nishino, T. (1994) J. Biol. Chem. 268, 14792-14797), which utilized carotenoid synthetic genes, phytoene synthase, and phytoene desaturase, to visualize the formation of GGPP in vivo. Eleven red positive clones were identified from about 24,300 mutants, and four (mutant 1, 2, 3, and 4) of them were analyzed for the enzyme activities. Results of in vitro assays demonstrated that all these mutants produced (all-E)-GGPP although the amounts were different. Each mutant was found to contain a few amino acid substitutions: mutant 1, Y81H and L275S; mutant 2, L34V and R59Q; mutant 3, V157A and H182Y; mutant 4, Y81H, P239R, and A265T. Site-directed mutagenesis showed that Y81H, L34V, or V157A was essential for the expression of the activity of GGPP synthase. Especially, the replacement of tyrosine 81 by histidine is the most effective because the production ratios of GGPP to FPP in mutant 1 and 4 are the largest. Based on prediction of the secondary structure, it is revealed that the tyrosine 81 situates on a point 11 approximately 12 A apart from the first DDXXD motif, whose distance is similar to the length of hydrocarbon moiety of FPP. These data might suggest that the aromatic ring of tyrosine 81 blocks the chain elongation longer than FPP. Comparisons of kinetic parameters of the mutated and wild type enzymes revealed several phenomena that may relate with the change of the ultimate chain length. They are a decrease of the total reaction rate, increase of Kmfor dimethylallyl diphosphate, decrease of Vmax for dimethylallyl diphosphate, and allylic substrate dependence of Km for IPP.

Using FMN and a reducing agent such as NAD(P)H, type 2 isopentenyl-diphosphate isomerase catalyzes isomerization between isopentenyl diphosphate and dimethylallyl diphosphate, both of which are elemental units for the biosynthesis of highly diverse isoprenoid compounds. Although the flavin cofactor is expected to be integrally involved in catalysis, its exact role remains controversial. Here we report the crystal structures of the substrate-free and complex forms of type 2 isopentenyl-diphosphate isomerase from the thermoacidophilic archaeon Sulfolobus shibatae, not only in the oxidized state but also in the reduced state. Based on the active-site structures of the reduced FMN-substrate-enzyme ternary complexes, which are in the active state, and on the data from site-directed mutagenesis at highly conserved charged or polar amino acid residues around the active site, we demonstrate that only reduced FMN, not amino acid residues, can catalyze proton addition/elimination required for the isomerase reaction. This discovery is the first evidence for this long suspected, but previously unobserved, role of flavins just as a general acid-base catalyst without playing any redox roles, and thereby expands the known functions of these versatile coenzymes.Flavins are generally regarded as redox coenzymes because their primary function in redox-catalyzing flavoenzymes is donation and/or acceptance of electrons (1). As summarized in a review article (2), the redox activities of flavins also take part in the flavoenzymes that catalyze reactions with no net redox change. Most of these enzymes are thought to have redoxbased mechanisms, whereas flavins have only structural or stabilizing roles in a few exceptions. Recently, however, a report on UDP-galactopyranose mutase unexpectedly showed that flavin can act as a nucleophilic catalyst (3, 4). In UDP-galactopyranose mutase, a sugar carbon undergoes nucleophilic attack by the N-5 nitrogen of reduced FMN, concomitantly with the dissociation of UDP, forming an adduct intermediate. In this reaction, the flavin cofactor has no redox function because it is continuously in the reduced state.Type 2 isopentenyl-diphosphate isomerase (IDI) 4 is the flavoenzyme that catalyzes the interconversion between isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) that occurs with no net change in redox status (5). Both compounds are fundamental units for the biosynthesis of isoprenoids, a diverse family of Ͼ50,000 metabolites (6). Type 2 IDI requires FMN, NAD(P)H, and Mg 2ϩ to be active; however, NAD(P)H is used only for the reduction of FMN and can be replaced with Na 2 S 2 O 4 (7-9). The observation that reduced FMN is required for type 2 IDI activity allowed for development of various plausible reaction mechanisms, including redoxbased mechanisms. Based on the traditional interpretation of the results from the experiments that used cofactor analogues such as 5-deaza-FMN, radical-mediated mechanisms were proposed at first, negating both the hydride transfer mechanism and the mer...

Geranylgeranyl reductase from Sulfolobus acidocaldarius was shown to catalyze the reduction of geranylgeranyl groups in the precursors of archaeal membrane lipids, generally reducing all four double bonds. However, when geranylgeranyl diphosphate was subjected to the reductase reaction, only three of the four double bonds were reduced. Mass spectrometry and acid hydrolysis indicated that the allylic double bond was preserved in the partially reduced product derived from geranylgeranyl diphosphate. Thus, the reaction product was shown to be phytyl diphosphate, which is a substrate for archaeal prenyltransferases, unlike the completely reduced compound phytanyl diphosphate.

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