Phenyl- and benzylurea cytokinins as competitive inhibitors of cytokinin oxidase/dehydrogenase: A structural study

Cytokinin oxidase/dehydrogenase (CKO) is a flavoenzyme, which irreversibly degrades the plant hormones cytokinins and thereby participates in their homeostasis. Several synthetic cytokinins including urea derivatives are known CKO inhibitors but structural data explaining enzyme–inhibitor interactions are lacking. Thus, an inhibitory study with numerous urea derivatives was undertaken using the maize enzyme (ZmCKO1) and the crystal structure of ZmCKO1 in a complex with N-(2-chloro-pyridin-4-yl)-N′-phenylurea (CPPU) was solved. CPPU binds in a planar conformation and competes for the same binding site with natural substrates like N⁶-(2-isopentenyl)adenine (iP) and zeatin (Z). Nitrogens at the urea backbone are hydrogen bonded to the putative active site base Asp169. Subsequently, site-directed mutagenesis of L492 and E381 residues involved in the inhibitor binding was performed. The crystal structures of L492A mutant in a complex with CPPU and N-(2-chloro-pyridin-4-yl)-N′-benzylurea (CPBU) were solved and confirm the importance of a stacking interaction between the 2-chloro-4-pyridinyl ring of the inhibitor and the isoalloxazine ring of the FAD cofactor. Amino derivatives like N-(2-amino-pyridin-4-yl)-N′-phenylurea (APPU) inhibited ZmCKO1 more efficiently than CPPU, as opposed to the inhibition of E381A/S mutants, emphasizing the importance of this residue for inhibitor binding. As highly specific CKO inhibitors without undesired side effects are of major interest for physiological studies, all studied compounds were further analyzed for cytokinin activity in the Amaranthus bioassay and for binding to the Arabidopsis cytokinin receptors AHK3 and AHK4. By contrast to CPPU itself, APPU and several benzylureas bind only negligibly to the receptors and exhibit weak cytokinin activity.     

The effect of aluminum on cytokinins in the mycelia of Amanita muscaria

High performance liquid chromatography analysis of immunoaffinity-purified extracts of mycelia of Amanita muscaria, and the Amaranthus bioassay of the eluted fractions, revealed the following seven cytokinins: zeatin, zeatin riboside, zeatin N-9-glucoside, dihydrozeatin, dihydrozeatin riboside, isopentenyl adenine, and isopentenyl adenosine. The decreased growth of aluminum-treated mycelia correlated with a 35% decrease in the total amount of the cytokinins. Among individual cytokinins, zeatin was the most affected, exhibiting a reduction of about 90%. The results are compared with previous investigations of aluminum effects on cytokinins in the mycelia of Lactarius piperatus, whose growth is stimulated by aluminum.Abbreviations ZR zeatin riboside - iPA isopentenyl adenosine - Z zeatin - DHZ dihydrozeatin - iP isopentenyl adenine - DHZR dihydrozeatin riboside - Z-9G zeatin N-9-glucoside - iP-9G isopentenyl N-9-glucoside - HPLC high performance liquid chromatography - DHZRMP dihydrozeatin riboside monophosphate - ZRMP zeatin riboside monophosphate     

Dynamics of endogenous cytokinin pools in tobacco seedlings: a modelling approach

Recent advances in cytokinin analysis have made it possible to measure the content of 22 cytokinin metabolites in the tissue of developing tobacco seedlings. Individual types of cytokinins in plants are interconverted to their respective forms by several enzymatic activities (5'-AMP-isopentenyltransferase, adenosine nucleosidase, 5'-nucleotidase, adenosine phosphorylase, adenosine kinase, trans-hydroxylase, zeatin reductase, beta-glucosidase, O-glucosyl transferase, N-glucosyl transferase, cytokinin oxidase). This paper reports modelling and measuring of the dynamics of endogenous cytokinins in tobacco plants grown on media supplemented with isopentenyl adenine (IP), zeatin (Z) and dihydrozeatin riboside (DHZR). Differences in phenotypes generated by the three cytokinins are shown and discussed, and the assumption that substrate concentration drives enzyme kinetics underpinned the construction of a simple mathematical model of cytokinin metabolism in developing seedlings. The model was tested on data obtained from liquid chromatography/tandem mass spectrometry cytokinin measurements on tobacco seedlings grown on Murashige and Skoog agar nutrient medium, and on plants grown in the presence of IP, Z and DHZR. A close match was found between measured and simulated data, especially after a series of iterative parameter searches, in which the parameters were set to obtain the best fit with one of the data sets.     

A novel target of lithium therapy

Phosphatases converting 3'-phosphoadenosine 5'-phosphate (PAP) into adenosine 5'-phosphate are of fundamental importance in living cells as the accumulation of PAP is toxic to several cellular systems. These enzymes are lithium-sensitive and we have characterized a human PAP phosphatase as a potential target of lithium therapy. A cDNA encoding a human enzyme was identified by data base screening, expressed in Escherichia coli and the 33 kDa protein purified to homogeneity. The enzyme exhibits high affinity for PAP (K(m)<1 microM) and is sensitive to subtherapeutic concentrations of lithium (IC(50)=0.3 mM). The human enzyme also hydrolyzes inositol-1, 4-bisphosphate with high affinity (K(m)=0.4 microM), therefore it can be considered as a dual specificity enzyme with high affinity (microM range) for both PAP and inositol-1,4-bisphosphate. Hydrolysis of inositol-1,4-bisphosphate was also inhibited by lithium (IC(50)=0.6 mM). Thus, we present experimental evidence for a novel target of lithium therapy, which could explain some of the side effects of this therapy.     

Endogenous cytokinin accumulation and cytokinin oxidase activity during shoot organogenesis of Petunia hybrida

Changes in endogenous cytokinin content and cytokinin oxidase activity were characterized in leaf explants from two Petunia hybrida Vilm. genetic lines which differed in their shoot organogenic response to exogenous N⁶-benzyladenine (BA). Endogenous cytokinin content in leaf explants of the highly shoot organogenic line, St40, increased 1.7-fold during the shoot induction phase (days 6–10) and had an additional 2.6-fold cytokinin increase correlated with the shift from induction to the shoot development phase. The cytokinins isopentenyl adenine (iP) and isopentenyl adenosine (iPAR) increased, while the cytokinins zeatin, zeatin riboside and dihydrozeatin remained at consistently low levels. In contrast, isoprenoid cytokinins did not accumulate in petunia TLV1 leaf explants which were incapable of shoot induction during 12 days of culture with BA. Cytokinin oxidase activity continuously increased in leaf explants of both petunia genotypes in response to BA, with a larger increase in St40. These results suggest that the differences in organogenic response in the two petunia genotypes may be the result of differences in BA uptake and metabolism which subsequently affects the accumulation of isoprenoid cytokinins and the activity of cytokinin oxidase in the early stages of shoot development.     

Mechanism-based inhibitors of cytokinin oxidase/dehydrogenase attack FAD cofactor

Cytokinin oxidases/dehydrogenases (CKOs) mediate catabolic regulation of cytokinin levels in plants. Several substrate analogs containing an unsaturated side chain were studied for their possible inhibitory effect on maize CKO (ZmCKO1) by use of various bioanalytical methods. Two allenic derivatives, N⁶-(buta-2,3-dienyl)adenine (HA-8) and N⁶-(penta-2,3-dienyl)adenine (HA-1), were identified as strong mechanism-based inhibitors of the enzyme. Despite exhaustive dialysis, the enzyme remained inhibited. Conversely, substrate analogs with a triple bond in the side chain were much weaker inactivators. The crystal structures of recombinant ZmCKO1 complexed with HA-1 or HA-8 were solved to 1.95 Å resolution. Together with Raman spectra of the inactivated enzyme, it was revealed that reactive imine intermediates generated by oxidation of the allenic inhibitors covalently bind to the flavin adenine dinucleotide (FAD) cofactor. The binding occurs at the C4a atom of the isoalloxazine ring of FAD, the planarity of which is consequently disrupted. All the compounds under study were also analyzed for binding to the Arabidopsis cytokinin receptors AHK3 and AHK4 in a bacterial receptor assay and for cytokinin activity in the Amaranthus bioassay. HA-1 and HA-8 were found to be good receptor ligands with a significant cytokinin activity. Nevertheless, due to their ability to inactivate CKO in the desired time intervals or developmental stages, they both represent attractive compounds for physiological studies, as the inhibition mechanism of HA-1 and HA-8 is mainly FAD dependent.     

Molecular and biochemical characterization of a cytokinin oxidase from maize

It is generally accepted that cytokinin oxidases, which oxidatively remove cytokinin side chains to produce adenine and the corresponding isopentenyl aldehyde, play a major role in regulating cytokinin levels in planta. Partially purified fractions of cytokinin oxidase from various species have been studied for many years, but have yet to clearly reveal the properties of the enzyme or to define its biological significance. Details of the genomic organization of the recently isolated maize (Zea mays) cytokinin oxidase gene (ckx1) and some of its Arabidopsis homologs are now presented. Expression of an intronless ckx1 in Pichia pastoris allowed production of large amounts of recombinant cytokinin oxidase and facilitated detailed kinetic and cofactor analysis and comparison with the native enzyme. The enzyme is a flavoprotein containing covalently bound flavin adenine dinucleotide, but no detectable heavy metals. Expression of the oxidase in maize tissues is described.     

Partial purification of a farnesyl diphosphate synthase from whole-body Manduca sexta

Farnesyl diphosphate synthase (FPP synthase) is a ubiquitous enzyme that is required for the biosynthesis of sesquiterpenes, dolichols ubiquinones, and prenylated proteins in insects. We report on the partial purification and characterization of an FPP synthase, obtained from whole-body preparations of the lepidopteran insect, Manduca sexta. The larval enzyme was separated from isopentenyl diphosphate (IPP) isomerase, phosphatase, and GGPP synthase by preparative isoelectric focusing, and was further purified by DEAE Sepharose, hydroxyapatite, and size exclusion chromatography. Whole-body M. sexta FPP synthase has a native molecular weight of 60.5+/-3.5 kDa and consists of two subunits of 28.5+/-0.5 kDa. As seen with other prenyltransferases, the enzyme has an absolute requirement for divalent cation and both Mn(2+) and Mg(2+) stimulated activity, although the former was inhibitory at higher concentrations. Insect FPP synthase catalyzes the condensation of IPP (K(m)=2.9+/-1.2 microM) with both dimethylallyl diphosphate and geranyl diphosphate (K(m)=0.8+/-0.4 microM). The enzyme requires the presence of detergent, glycerol, and non-specific protein-protein interactions for stability and maximum catalytic activity.     

Calcium-stimulated guanosine--inosine nucleosidase from yellow lupin (Lupinus luteus)

Guanosine-inosine-preferring nucleoside N-ribohydrolase has been purified to homogeneity from yellow lupin (Lupinus luteus) seeds by ammonium sulfate fractionation, ion-exchange chromatography and gel filtration. The enzyme functions as a monomeric, 80kDa polypeptide, most effectively between pH 4.7 and 5.5. Of various mono- and divalent cations tested, Ca(2+) appeared to stimulate enzyme activity. The nucleosidase was activated 6-fold by 2mM exogenous CaCl(2) or Ca(NO(3))(2), with K(a)=0.5mM (estimated for CaCl(2)). The K(m) values estimated for guanosine and inosine were 2.7+/-0.3 microM. Guanosine was hydrolyzed 12% faster than inosine while adenosine and xanthosine were poor substrates. 2'-Deoxyguanosine, 2'-deoxyinosine, 2'-methylguanosine, pyrimidine nucleosides and 5'-GMP were not hydrolyzed. However, the enzyme efficiently liberated the corresponding bases from synthetic nucleosides, such as 1-methylguanosine, 7-methylguanosine, 1-N(2)-ethenoguanosine and 1-N(2)-isopropenoguanosine, but hydrolyzed poorly the ribosides of 6-methylaminopurine and 2,6-diaminopurine. MnCl(2) or ZnCl(2) inhibited the hydrolysis of guanosine with I(50) approximately 60 microM. Whereas 2'-deoxyguanosine, 2'-methylguanosine, adenosine, as well as guanine were competitive inhibitors of this reaction (K(i) values were 1.5, 3.6, 21 and 9.7 microM, respectively), hypoxanthine was a weaker inhibitor (K(i)=64 microM). Adenine, ribose, 2-deoxyribose, 5'-GMP and pyrimidine nucleosides did not inhibit the enzyme. The guanosine-inosine hydrolase activity occurred in all parts of lupin seedlings and in cotyledons it increased up to 5-fold during seed germination, reaching maximum in the third/fourth day. The lupin nucleosidase has been compared with other nucleosidases.     

Analysis of N-glycosylation in maize cytokinin oxidase/dehydrogenase 1 using a manual microgradient chromatographic separation coupled offline to MALDI-TOF/TOF mass spectrometry

Cytokinin oxidase/dehydrogenase (CKO; EC 1.5.99.12) irreversibly degrades the plant hormones cytokinins. A recombinant maize isoenzyme 1 (ZmCKO1) produced in the yeast Yarrowia lipolytica was subjected to enzymatic deglycosylation by endoglycosidase H. Spectrophotometric assays showed that both activity and thermostability of the enzyme decreased after the treatment at non-denaturing conditions indicating the biological importance of ZmCKO1 glycosylation. The released N-glycans were purified with graphitized carbon sorbent and analyzed by MALDI-TOF MS. The structure of the measured high-mannose type N-glycans was confirmed by tandem mass spectrometry (MS/MS) on a Q-TOF instrument with electrospray ionization. Further experiments were focused on direct analysis of sugar binding. Peptides and glycopeptides purified from tryptic digests of recombinant ZmCKO1 were separated by reversed-phase chromatography using a manual microgradient device; the latter were then subjected to offline-coupled analysis on a MALDI-TOF/TOF instrument. Glycopeptide sequencing by MALDI-TOF/TOF MS/MS demonstrated N-glycosylation at Asn52, 63, 134, 294, 323 and 338. The bound glycans contained 3-14 mannose residues. Interestingly, Asn134 was found only partially glycosylated. Asn338 was the sole site to carry large glycan chains exceeding 25 mannose residues. This observation demonstrates that contrary to a previous belief, the heterologous expression in Y. lipolytica may lead to locally hyperglycosylated proteins.     

Purification and partial characterization of camel (Camelus Dromedarius) ceruloplasmin

Adult and young camel ceruloplasmin (Cp) were isolated and purified using the single-step chromatography on amino ethyl-activated sepharose. There are no differences between the adult and the young camel protein. The molecular mass of the protein, as estimated by SDS-PAGE (denaturant conditions), was approximately 130000 Da. The electrophoretic mobility of camel Cp is slightly higher as compared to human and sheep protein suggesting that the camel Cp is homogeneous, compact and more acid. The copper content was estimated to be 5.8+/-0.3 atoms per molecule. The spectroscopic feature includes an absorption maximum at 610 nm, which could be attributed to type 1 copper. The EPR spectrum was completely devoid of any typical signal of the type 2 copper. The kinetic parameters of the adult camel Cp for the specific activity as p-phenylendiamine oxidase were determined as K(m)=0.42 mM and V(max)=0.93 microM NADH/mn/mg Cp. The optimum pH for the activity was 5.7.     

Crystal structures of a type-1 ribosome inactivating protein from Momordica balsamina in the bound and unbound states

The ribosome inactivating proteins (RIPs) of type 1 are plant toxins that eliminate adenine base selectively from the single stranded loop of rRNA. We report six crystal structures, type 1 RIP from Momordica balsamina (A), three in complexed states with ribose (B), guanine (C) and adenine (D) and two structures of MbRIP-1 when crystallized with adenosine triphosphate (ATP) (E) and 2'-deoxyadenosine triphosphate (2'-dATP) (F). These were determined at 1.67?, 1.60?, 2.20?, 1.70?, 2.07? and 1.90? resolutions respectively. The structures contained, (A) unbound protein molecule, (B) one protein molecule and one ribose sugar, (C) one protein molecule and one guanine base, (D) one protein molecule and one adenine base, (E) one protein molecule and one ATP-product adenine molecule and (F) one protein molecule and one 2'-dATP-product adenine molecule. Three distinct conformations of the side chain of Tyr70 were observed with (i) χ(1)=-66°and χ(2)=165° in structures (A) and (B); (ii) χ(1)=-95° and χ(2)=70° in structures (C), (D) and (E); and (iii) χ(1)=-163° and χ(2)=87° in structure (F). The conformation of Tyr70 in (F) corresponds to the structure of a conformational intermediate. This is the first structure which demonstrates that the slow conversion of DNA substrates by RIPs can be trapped during crystallization.     

Properties of purified recombinant human polyamine oxidase,PAOh1/SMO

The discovery of an inducible oxidase whose apparent substrate preference is spermine indicates that polyamine catabolism is more complex than that originally proposed. To facilitate the study of this enzyme, the purification and characterization of the recombinant human PAOh1/SMO polyamine oxidase are reported. Purified PAOh1/SMO oxidizes both spermine (K(m)=1.6 microM) and N(1)-acetylspermine (K(m)=51 microM), but does not oxidize spermidine. The purified human enzyme also does not oxidize eight representative antitumor polyamine analogues; however, specific oligamine analogues were found to be potent inhibitors of the oxidation of spermine by PAOh1/SMO. The results of these studies are consistent with the hypothesis that PAOh1/SMO represents a new addition to the polyamine metabolic pathway that may represent a new target for antineoplastic drug development.     

Isolation and characterization of mitochondrial F(1)-ATPase from crayfish (Orconectes virilis) gills

A soluble F(1)-ATPase was isolated from the mitochondria of crayfish (Orconectes virilis) gill tissue. The maximal mitochondrial disruption rate (95%) was obtained by sonicating for 4 min at pH 8.6. A 15-fold purification was estimated. The properties for both soluble and membrane-bound enzyme were studied. Both enzyme forms were stable at 4 to -70 degrees C when kept in 20% glycerol. Soluble F(1)-ATPase was more stable at room temperature than membrane-bound enzyme. It displayed a narrower pH profile (pK(1) =6.58, pK(2)=7.68) and more acid pH optimum (7.13) than membrane-bound enzyme (pK(1)=6.42, pK(2)=8.55, optimum pH 7.49). The anion-stimulated activities were in the order HCO(3)(-)>SO(4)(2-)>Cl(-). The apparent K(a) values for soluble enzyme were 11.4, 11.2, and 10.9 mM, respectively, but the K(a) of HCO(3)(-) for membrane-bound enzyme (14.9 mM) was higher than for soluble enzyme. Oligomycin and DCCD inhibited membrane-bound F(1)-ATPase with I(50) of 18.6 ng/ml and 2.2 microM, respectively, but were ineffective in inhibiting soluble enzyme. Both enzyme forms shared identical sensitivity to DIDS (I(50)=12.5 microM) and vanadate (I(50)=9.0 mM). Soluble ATPase was significantly more sensitive to pCMB (I(50)=0.15 microM) and NO(3)(-) (I(50)=28.6 mM) than membrane-bound enzyme (I(50)=1.04 microM pCMB and 81.5 mM NO(3)(-)). In addition, soluble F(1)-ATPase was slightly more sensitive to azide (I(50)=91.8 microM) and NBD-Cl (I(50)=9.18 microM) than membrane-bound enzyme (I(50)=111.6 microM azide and 12.88 microM NBD-Cl). These data suggest a conformational change transmission between F(0) and F(1) sectors and slight conformational differences between soluble F(1) and membrane-bound F(1). In addition, an unmodified F(0) stabilizes F(1) and decreases F(1) sensitivities to inhibitors and modulators.     

Changes in Concentrations of Cytokinins (CKs) in Root and Axillary Bud Tissue of Miniature Rose Suggest that Local CK Biosynthesis and Zeatin-Type CKs Play Important Roles in Axillary Bud Growth

Involvement of cytokinins (CKs) in axillary bud growth of miniature rose was studied. Variation in root formation and axillary bud growth was induced by two indole 3-butyric acid (IBA) pretreatments in two cutting sizes. At six physiological developmental stages around the onset of axillary bud growth, concentrations of CKs were determined in both root and axillary bud tissue by liquid chromatography combined with electrospray tandem mass spectrometry (LC-ESP-MS/MS). Chronological early onset of axillary bud growth occurred in long cuttings pretreated at low IBA concentration, whereas physiological early root formation was associated with long cuttings and high IBA concentration. The CKs zeatin (Z), isopentenyl adenine (iP), zeatin riboside (ZR), dihydrozeatin riboside (DHZR), isopentenyl adenosine (iPA), zeatin O-glucoside (ZOG), zeatin riboside O-glucoside (ZROG), zeatin riboside 5′-monophosphate (ZRMP), and isopentenyl adenosine 5′-monophosphate (iPAMP) were detected. Concentrations of CKs in axillary bud tissue far exceeded those in root tissue. Indole 3-butyric acid pretreatment influenced the concentration of CKs in axillary bud tissue more than did cutting size, whereas pretreatments only slightly affected CKs in root tissue. The dominant CKs found were iPAMP and ZR. An early and large increase in iPAMP indicated rapid CK biosynthesis in rootless cuttings, suggesting that green parts, including the axillary bud, can synthesize CKs. At the onset of axillary bud growth an increase in concentration of Z, ZR, ZRMP, ZOG, and ZROG was largely coincident with a decrease in iPAMP, iPA, iP, and DHZR. After the onset of axillary bud growth, CK content largely decreased. These results strongly indicate a positive role for CKs in axillary bud growth, and presumably ZRMP, ZR, and Z are active in miniature rose.     

Endogenous Cytokinins in Bougainvillea ;San Diego Red': I. OCCURRENCE OF CYTOKININ GLUCOSIDES IN THE ROOT SAP

Using chromatographic, chemical, and enzymic techniques, 11 compounds capable of stimulating the division of soybean callus cells were tentatively identified in the root sap of Bougainvillea `San Diego Red.' These cytokinin-like compounds included phosphorylated and glucosylated forms of zeatin, ribosylzeatin, and their dihydro derivatives. In addition, isopentenyl adenosine and isopentenyl adenine were apparently also present. The occurrence of glucosylated derivatives in the root sap of plants has not been substantiated previously.     

Conversion of 4-hydroxyacetophenone into 4-phenyl acetate by a flavin adenine dinucleotide-containing Baeyer-Villiger-type monooxygenase

An arylketone monooxygenase was purified from Pseudomonas putida JD1 by ion exchange and affinity chromatography. It had the characteristics of a Baeyer-Villiger-type monooxygenase and converted its substrate, 4-hydroxyacetophenone, into 4-hydroxyphenyl acetate with the consumption of one molecule of oxygen and oxidation of one molecule of NADPH per molecule of substrate. The enzyme was a monomer with an M(r) of about 70,000 and contained one molecule of flavin adenine dinucleotide (FAD). The enzyme was specific for NADPH as the electron donor, and spectral studies showed rapid reduction of the FAD by NADPH but not by NADH. Other arylketones were substrates, including acetophenone and 4-hydroxypropiophenone, which were converted into phenyl acetate and 4-hydroxyphenyl propionate, respectively. The enzyme displayed Michaelis-Menten kinetics with apparent K(m) values of 47 microM for 4-hydroxyacetophenone, 384 microM for acetophenone, and 23 microM for 4-hydroxypropiophenone. The apparent K(m) value for NADPH with 4-hydroxyacetophenone as substrate was 17.5 microM. The N-terminal sequence did not show any similarity to other proteins, but an internal sequence was very similar to part of the proposed NADPH binding site in the Baeyer-Villiger monooxygenase cyclohexanone monooxygenase from an Acinetobacter sp.     

Osmotic pretreatment promotes axillary shooting from cauliflower curd pieces by acting through internal cytokinin level modifications

In vitro propagation of cauliflower has generally been achieved through axillary shoot proliferation of curd explants on Murashige and Skoog (MS) medium supplemented with an auxin and a cytokinin. Recently, it has been shown (Vandemoortele 1999) that a soaking in sucrose (-2 MPa for 24 h) of cauliflower curd explants, before culture without any growth regulator, also induced axillary branching. The later procedure avoids the phenomenon of hyperhydricity in the shoots formed. Axillary shooting obtained by the two methods appears to be mediated by modifications of internal cytokinin levels. The osmotic pretreatment did not influence auxin levels, but induced a zeatin and a zeatin riboside levels increase. Curd explants cultured with the usual procedure (on MS medium supplemented with 5 μmol/L BA and 0.5 μmol/L NAA) showed a zeatin and zeatin riboside levels increase of the same magnitude and a higher one for isopentenyl adenine and isopentenyl adenosine. The modification of the cytokinin status in the curd explants subjected to a short osmotic pretreatment thus should be less favourable for hyperhydricity.     

Type II isopentenyl diphosphate isomerase from Synechocystis sp. strain PCC 6803

Open reading frame sll1556 in the cyanobacterium Synechocystis sp. strain 6803 encodes a putative type II isopentenyl diphosphate (IPP) isomerase. The His(6)-tagged protein was produced in Escherichia coli and purified by Ni(2+) chromatography. The homotetrameric enzyme required NADPH, flavin mononucleotide, and Mg(2+) for activity; K(m)(IPP) was 52 microM, and k(cat)(IPP) was 0.23 s(-1).