New Role of Flavin as a General Acid-Base Catalyst with No Redox Function in Type 2 Isopentenyl-diphosphate Isomerase
2009; Elsevier BV; Volume: 284; Issue: 14 Linguagem: Inglês
10.1074/jbc.m808438200
ISSN1083-351X
AutoresHideaki Unno, Satoshi Yamashita, Yosuke Ikeda, Shin-ya Sekiguchi, Norie Yoshida, Tohru Yoshimura, Masami Kusunoki, Tôru Nakayama, Tokuzo Nishino, Hisashi Hemmi,
Tópico(s)Plant biochemistry and biosynthesis
ResumoUsing 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. 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 (1Massey V. Biochem. Soc. Trans. 2000; 28: 283-296Crossref PubMed Google Scholar). As summarized in a review article (2Bornemann S. Nat. Prod. Rep. 2002; 19: 761-772Crossref PubMed Scopus (118) Google Scholar), 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 redox-based 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 (3Soltero-Higgin M. Carlson E.E. Gruber T.D. Kiessling L.L. Nat. Struct. Mol. Biol. 2004; 11: 539-543Crossref PubMed Scopus (125) Google Scholar, 4Miller S.M. Nat. Struct. Mol. Biol. 2004; 11: 497-498Crossref PubMed Scopus (9) Google Scholar). 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) 4The abbreviations used are: IDI, isopentenyl diphosphate:dimethylallyl-diphosphate isomerase; DMAPP, dimethylallyl diphosphate; eIPP, 3,4-epoxy-3-methylbutyl diphosphate; IPP, isopentenyl diphosphate. is the flavoenzyme that catalyzes the interconversion between isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) that occurs with no net change in redox status (5Kaneda K. Kuzuyama T. Takagi M. Hayakawa Y. Seto H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 932-937Crossref PubMed Scopus (179) Google Scholar). Both compounds are fundamental units for the biosynthesis of isoprenoids, a diverse family of >50,000 metabolites (6Christianson D.W. Science. 2007; 316: 60-61Crossref PubMed Scopus (65) Google Scholar). Type 2 IDI requires FMN, NAD(P)H, and Mg2+ to be active; however, NAD(P)H is used only for the reduction of FMN and can be replaced with Na2S2O4 (7Hemmi H. Ikeda Y. Yamashita S. Nakayama T. Nishino T. Biochem. Biophys. Res. Commun. 2004; 322: 905-910Crossref PubMed Scopus (41) Google Scholar, 8Kittleman W. Thibodeaux C.J. Liu Y.N. Zhang H. Liu H.W. Biochemistry. 2007; 46: 8401-8413Crossref PubMed Scopus (33) Google Scholar, 9Rothman S.C. Helm T.R. Poulter C.D. Biochemistry. 2007; 46: 5437-5445Crossref PubMed Scopus (45) Google Scholar). The observation that reduced FMN is required for type 2 IDI activity allowed for development of various plausible reaction mechanisms, including redox-based 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 merely structural role of FMN (7Hemmi H. Ikeda Y. Yamashita S. Nakayama T. Nishino T. Biochem. Biophys. Res. Commun. 2004; 322: 905-910Crossref PubMed Scopus (41) Google Scholar, 8Kittleman W. Thibodeaux C.J. Liu Y.N. Zhang H. Liu H.W. Biochemistry. 2007; 46: 8401-8413Crossref PubMed Scopus (33) Google Scholar). However, the study using "radical clock" substrate analogues (10Johnston J.B. Walker J.R. Rothman S.C. Poulter C.D. J. Am. Chem. Soc. 2007; 129: 7740-7741Crossref PubMed Scopus (29) Google Scholar) disproved the radical mechanisms, suggesting that the inactivation of type 2 IDI reconstituted with 5-deaza-FMN should be reconsidered with a modern interpretation. Moreover, the measurement of deuterium kinetic isotope effects (11Thibodeaux C.J. Mansoorabadi S.O. Kittleman W. Chang W.C. Liu H.W. Biochemistry. 2008; 47: 2547-2558Crossref PubMed Scopus (32) Google Scholar) strongly supported the non-redox protonation-deprotonation mechanism, which is also utilized by type 1 IDI (12Ramos-Valdivia A.C. van der Heijden R. Verpoorte R. Nat. Prod. Rep. 1997; 14: 591-603Crossref PubMed Scopus (133) Google Scholar). Type 1 enzymes, which are present in nearly all eukaryotes and some bacteria, have no sequential homology with type 2 IDI, which is found in almost all archaea and many bacteria (13Kuzuyama T. Seto H. Nat. Prod. Rep. 2003; 20: 171-183Crossref PubMed Scopus (253) Google Scholar). In type 1 IDI, isomerization proceeds via formation of a 3° carbocation intermediate, and conserved cysteine and glutamate residues act as the general acid or base to mediate (1,3)-antarafacial proton addition/elimination. Hence, there are two possible roles of reduced FMN in type 2 IDI: 1) reduced FMN itself acts as the general acid-base catalyst; and 2) protonation and deprotonation are catalyzed by amino acid residues, and reduced FMN stabilizes the intermediate. The N-5 nitrogen of FMN must be integrally involved in both mechanisms. Although the pKa values of reduced flavins are within the physiological pH range (14Miura R. Chem. Rec. 2001; 1: 183-194Crossref PubMed Scopus (96) Google Scholar, 15Macheroux P. Ghisla S. Sanner C. Ruterjans H. Muller F. BMC Biochem. 2005; 6: 26Crossref PubMed Scopus (44) Google Scholar), there has been no definitive report on the flavoenzymes in which flavin only acts as a general acid-base catalyst without having any redox function. Recent studies of type 2 IDI using substrate analogues such as 3,4-epoxy-3-methylbutyl diphosphate (eIPP), which form adducts with FMN, suggest the possibility that FMN N-5 may act as a general acid or base (10Johnston J.B. Walker J.R. Rothman S.C. Poulter C.D. J. Am. Chem. Soc. 2007; 129: 7740-7741Crossref PubMed Scopus (29) Google Scholar, 16Hoshino T. Tamegai H. Kakinuma K. Eguchi T. Bioorg. Med. Chem. 2006; 14: 6555-6559Crossref PubMed Scopus (33) Google Scholar, 17Rothman S.C. Johnston J.B. Lee S. Walker J.R. Poulter C.D. J. Am. Chem. Soc. 2008; 130: 4906-4913Crossref PubMed Scopus (37) Google Scholar). Based on what is known about the mechanism of UDP-galactopyranose mutase, this theory seems plausible. However, the absence of crystal structures of enzyme-substrate complexes inhibits complete understanding of the role of reduced FMN in type 2 IDI reactions. Here, we report the crystal structures of the substrate-enzyme complexes of type 2 IDI isolated from a thermoacidphilic archaeon, Sulfolobus shibatae, in both the oxidized and reduced state. Combined with results from mutagenic studies that targeted the highly conserved residues, the structural data showed that reduced FMN acts as the general acid-base catalyst. Enzyme Purification-The plasmid for the expression of (His)6-tagged S. shibatae IDI, pET-idi (18Yamashita S. Hemmi H. Ikeda Y. Nakayama T. Nishino T. Eur. J. Biochem. 2004; 271: 1087-1093Crossref PubMed Scopus (36) Google Scholar), was introduced into the Escherichia coli BL21(DE3) strain. The transformant was cultivated in L medium containing 50 mg/liter of ampicillin until cells reached the early stationary phase. The recombinant enzyme was purified with heat treatment at 55 °C for 30 min and a HisTrap column (GE Healthcare) as described previously (18Yamashita S. Hemmi H. Ikeda Y. Nakayama T. Nishino T. Eur. J. Biochem. 2004; 271: 1087-1093Crossref PubMed Scopus (36) Google Scholar). For crystallization, the enzyme was loaded on a HiLoad 16/60 Superdex 200 column (GE Healthcare) and eluted with 10 mm Tris-HCl (pH 7.7), containing 1 mm EDTA, 10 mm β-mercaptoethanol, and 0.15 m NaCl. Crystallization-IDI crystals were grown at 20 °C using the sitting-drop vapor-diffusion method with a reservoir solution containing 0.1 m Tris-HCl (pH 8.0), 0.2 m sodium citrate, and 30% (v/v) polyethylene glycol (PEG) 400. The native crystals were used in the analysis of the substrate-free structure. Crystals for the analysis of the reduced substrate-free structure were obtained by soaking the native crystals in the reservoir solution containing 32% (v/v) PEG 400 and 10 mm NADH. Substrate-complex structures were obtained from the crystals that had been soaked for 1 h in the reservoir solution containing 32% (v/v) PEG 400 and 5 mm IPP or 10 mm DMAPP. Crystals for the analysis of the reduced substrate-complex structures were obtained by the addition of an appropriate amount of Na2S2O4 to the soaking solution containing the substrate. X-ray Data Collection, Structure Solution, and Refinement-All data sets were collected on beamline BL-5A at the photon factory (KEK, Tsukuba, Japan). Complete data sets were collected over contiguous rotation ranges at a given wavelength before proceeding to the next wavelength, and were processed and scaled with HKL2000 (19Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38605) Google Scholar). Data collection statistics are summarized in Table 1. All data sets belonged to space group P43212 with four molecules per asymmetric unit. The IDI structure was solved with Os-derivative crystals using the multi-wavelength anomalous diffraction method. Os-derivative crystals were obtained by soaking the native crystals for 12 h in the reservoir solution containing 1 mm OsCl3. Initial phases were determined using the SHARP program (20de la Fortelle E. Bricogne G. Methods Enzymol. 1997; 276: 472-494Crossref PubMed Scopus (1797) Google Scholar). Phase improvement by density modification was performed using the program, DM (21Cowtan K. Joint CCP4 and ESF-EACBM Newsletter on Protein Crystallography. 1994; 31: 34-38Google Scholar). The structure was built using Coot (22Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23597) Google Scholar) and refined using Refmac (23Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13910) Google Scholar), with 5% of the data set aside as a free set. During subsequent refinement, the Os-derivative data set was replaced with native data sets. NCS restraints were applied to 4 subunits through refinement. FMN, IPP, and DMAPP models were fitted into the substrate-binding sites based on the difference electron density map (supplemental Fig. S1).TABLE 1Data collection and refinement statistics Numbers in parentheses are for the highest shell.Crystal typeFreeIDIFreeIDIredIPP-IDIIPP-IDIredDMAPP-IDIDMAPP-IDIredOs-derivativePeakEdgeRemoteData collection and processing statisticsSpace groupP43212P43212P43212P43212P43212P43212P43212Unit cell dimension (Å)a = b (Å)100.745101.203100.444100.359101.071100.879100.770c (Å)336.829336.501333.914334.607333.430334.864334.320α = β = γ (°)90,00090,00090,00090,00090,00090,00090,000Wavelength (Å)1.00001.00001.00001.00001.00001.00001.139801.140221.04000Resolution (Å)50.00-1.99 (2.06-1.99)50.00-2.30 (2.38-2.30)50.0-2.39 (2.48-2.39)50.0-2.64 (2.74-2.64)50.0-3.00 (3.11-3.00)50.0-2.90 (3.00-2.90)50.00-3.0 (3.11-3.00)50.00-3.1 (3.21-3.10)50.00-3.1 (3.21-3.10)Measured1,001,560699,031469,313427,040155,709467,679363,518209,356253,878I/σI24.6 (8.3)22.7 (2.5)23.6 (3.2)18.9 (2.2)13.2 (2.1)22.1 (2.5)23.3 (5.5)20.8 (6.4)23.7 (6.2)Redundancy8.4 (8.1)9.0 (5.7)6.8 (5.0)8.8 (4.4)4.5 (3.7)11.9 (6.1)5.5 (4.9)6.5 (5.6)7.8 (6.7)Completeness (%)99.4 (96.9)98.7 (89.1)99.3 (96.8)93.6 (68.2)95.9 (90.9)99.6 (96.9)100.0 (100.0)98.8 (98.3)99.9 (100.0)RmergeaRmerge = 100Σ|I – 〈I〉|/ΣI, where I is the observed intensity and 〈I〉 is the average intensity of multiple observations of symmetry-related reflections (%)8.7 (26.8)8.4 (39.0)8.0 (37.7)9.0 (33.8)9.2 (38.8)9.0 (41.2)6.4 (25.4)8.3 (23.8)7.2 (26.6)Phasing statisticsNo. of sites12Figure of meritAcentric0.479Centric0.316Refinement statisticsResolution37.50-2.0049.03-2.3049.69-2.4049.63-2.6445.22-3.0048.85-2.90Protein atoms10,99611,15211,23211,23211,23211,232Ligand atoms124124184184184184Water molecule728581578404175172Rwork/Rfree (%)18.5/22.218.0/21.618.7/21.918.2/22.218.4/21.419.2/21.3Root mean square deviationsBond lengths (Å)0.0140.0180.0130.0140.0120.012Bond angles (°)1.4621.7001.4891.5411.4721.572a Rmerge = 100Σ|I – 〈I〉|/ΣI, where I is the observed intensity and 〈I〉 is the average intensity of multiple observations of symmetry-related reflections Open table in a new tab Mutagenesis-Alanine-substitution mutations were introduced into pET-idi using a QuikChange Mutagenesis Kit (Stratagene) and oligonucleotide primers indicated in supplemental Table S1. The IDI assay for the mutants was performed as previously described (7Hemmi H. Ikeda Y. Yamashita S. Nakayama T. Nishino T. Biochem. Biophys. Res. Commun. 2004; 322: 905-910Crossref PubMed Scopus (41) Google Scholar). CD Spectroscopy-CD spectra of the enzymes were analyzed using a J-720WI spectropolarimeter (JASCO, Japan). NADH Oxidase Assay-NADH oxidase activity of wild-type and mutated S. shibatae IDIs were measured using previously described methods (7Hemmi H. Ikeda Y. Yamashita S. Nakayama T. Nishino T. Biochem. Biophys. Res. Commun. 2004; 322: 905-910Crossref PubMed Scopus (41) Google Scholar). Preparation of Apoenzymes-Preparation of apo forms of wild-type and mutated S. shibatae IDIs was performed using overnight dialysis under the conditions described previously (7Hemmi H. Ikeda Y. Yamashita S. Nakayama T. Nishino T. Biochem. Biophys. Res. Commun. 2004; 322: 905-910Crossref PubMed Scopus (41) Google Scholar). If IDI activity remained after the first dialysis step, the enzyme was dialyzed again for 48 h. After complete loss of activity, the dialysis buffer was changed to 10 mm Tris-HCl (pH 7.7), and dialysis was continued overnight. Complete removal of FMN also was confirmed by the disappearance of the peak at ∼450 nm from the absorption spectrum of the enzyme solution measured with a UV-2450 UV-visible spectrophotometer (Shimadzu, Japan). Measurement of Dissociation Constants for IPP and FMN-The Kd for IPP was determined from the change in fluorescence intensity resulting from intrinsic tryptophan residues, which results from binding of the substrate to the enzyme. The fluorescence excitation and emission wavelengths were 295 and 350 nm, respectively. Five hundred μl of purified enzyme solution containing 25 μmol of sodium succinate buffer (pH 6.0), 0.5 nmol of the purified enzyme, 5 nmol of FMN, and 1.25 μmol of MgCl2 was titrated with 10 mm IPP, and the fluorescence intensity measured at each point of titration with a F-4500 fluorometer (Shimadzu, Japan). The fluorescence intensity (F) and the concentration of IPP ([L]) were corrected for the increase in the volume of the solution. The data were plotted (F versus [L]) and fitted using the equation below. The hypothetical final fluorescence intensity is the value to which F would converge if unlimited amounts of IPP were added to the reaction. Kd and ΔF, the difference between the initial fluorescence intensity (Fi) and the hypothetical final fluorescence intensity, were set as variable parameters. Kaleidagraph (Synergy software) was utilized for data plotting and equation fitting.F=Fi+ΔF[L]/([L]+Kd)Eq. 1 To measure the Kd for FMN, apoenzyme that had been prepared as described above was used. Five hundred μl of enzyme solution containing 25 μmol of sodium succinate buffer (pH 6.0) and 0.5 nmol of the apoenzyme was titrated with 100 μm FMN. Data were corrected and fitted to Equation 1, substituting the FMN concentration for [L]. Overall Structure-We obtained six distinct types of S. shibatae type 2 IDI crystals: substrate-free IDI (termed "FreeIDI"), complexes with IPP ("IPP-IDI") and DMAPP ("DMAPP-IDI"), the substrate-free reduced form ("FreeIDIred"), the IPP-complex in the reduced state ("IPP-IDIred"), and the DMAPP-complex in the reduced state ("DMAPP-IDIred"). The structure of FreeIDI was solved using the multiple wavelength anomalous diffraction method with the Os-derivative, and then the structures of each type of crystal were refined. The refinement statistics and model quality parameters are listed in Table 1. Of 368 residues, the following were not visible in the electron density and were probably disordered: 12 residues (N-terminal, 8 residues; C-terminal, 1 residue; and residues Gly69 to Arg71) in the FreeIDI form; 7 residues (N-terminal, 6 residues; and C-terminal, 1 residue) in the FreeIDIred form; 4 residues (N-terminal, 2 residues; and C-terminal, 2 residues) in the substrate-complex forms. In all crystal types, the asymmetric unit contains four monomers that are related by a non-crystallographic 4-fold rotation, which is regarded as the tetrameric state of S. shibatae IDI in solution (18Yamashita S. Hemmi H. Ikeda Y. Nakayama T. Nishino T. Eur. J. Biochem. 2004; 271: 1087-1093Crossref PubMed Scopus (36) Google Scholar). As shown in Fig. 1, A and B, each monomer contains a regular triose-phosphate isomerase barrel structure (or α8β8 barrel: α3, α5, α7, α9, α10, α13-α14, α15, α16, β3, β4, β5, β6, β7, β8, β9, β10), like previously reported substrate-free structures of bacterial type 2 IDIs (24Steinbacher S. Kaiser J. Gerhardt S. Eisenreich W. Huber R. Bacher A. Rohdich F. J. Mol. Biol. 2003; 329: 973-982Crossref PubMed Scopus (51) Google Scholar, 25de Ruyck J. Rothman S.C. Poulter C.D. Wouters J. Biochem. Biophys. Res. Commun. 2005; 338: 1515-1518Crossref PubMed Scopus (28) Google Scholar). A notable structural aspect is that α1, α8, α11, and α12 are located on the top face of the triose-phosphate isomerase barrel, forming a portion of the active site (Fig. 1C). Large inter-subunit surfaces (3380 Å2 per monomer) were observed among the four monomers (Fig. 1D). The subunit interaction consists of β7, α8, α12, α16, α18, and the loop regions, β1-α1, α12-α13, α8-α9, and β7-α10, the structures of which are stabilized by the interaction. α8 and the loop region α8–α9 also contribute to formation of the active site; therefore, tetramer formation is required for construction of the active site. Binding of the Substrate and Cofactor-The S. shibatae IDI tetramer contains four active sites, which are located inside the triose-phosphate isomerase barrel structure. The electron densities of FMN, and of IPP/DMAPP in the substrate-complex structures, were found in all substrate-binding sites in a tetramer (supplemental Fig. S1). However, apparent electron density for NADH was not observed in FreeIDIred; although the structure was determined based on analysis of IDI crystals that had been soaked in NADH solution. In all structures, well defined FMN electron densities were identified in a pocket surrounded by β-strands (β3, β4, β5, β6, β7, β8, β9, and β10) that composed the triose-phosphate isomerase barrel structure and by α-helices (α1 and α11). In the FreeIDI structure, the FMN molecule interacts with Thr65, Gly66, Thr68, Ser96, Asn125, His155, Lys193, Ser218, Thr223, Trp225, Gly275, Arg277, Ala296, and Leu297 of S. shibatae IDI (Fig. 2A). Among these residues, Gly275, Ala296, and Leu297, which interact with the phosphate moiety of FMN via backbone amides, are located at the C-terminal end of β-strands 9 and 10. These common phosphate-binding sites have been termed the "standard phosphate binding motif" (26Nagano N. Orengo C.A. Thornton J.M. J. Mol. Biol. 2002; 321: 741-765Crossref PubMed Scopus (508) Google Scholar). The substrate-binding sites are located on the top of the triose-phosphate isomerase barrel and surrounded by helices (α1, α4, and α8), loops (β4-α4, α8-α9), and the isoalloxazine ring of FMN in the four complex structures. The substrates, IPP and DMAPP, directly interact with the side chains of Arg7, Lys8, Ser96, Arg98, His155, Gln160, Trp225 and the isoalloxazine ring of FMN in nearly the same manner via hydrophobic and electrostatic interactions (Fig. 2C). Additionally, a Mg2+ ion, which is supported by His155, via a water molecule, and by Glu161, is also coordinated to the diphosphate moiety of the substrates. The ionic and hydrogen bonds that form among the diphosphate group, Mg2+ ion, water molecules, and surrounding amino acid resides, seem to contribute significantly to substrate binding. The tight binding confronts the isopentenyl or dimethylallyl plane of the substrate with the si-face of the isoalloxazine ring of FMN. Differences in Active Site Structures-Although the overall structures of the six forms of S. shibatae IDI are similar, significant differences exist among the structures of the active site. One difference accompanies substrate binding: the active site is open to bulk solvents in the FreeIDI and FreeIDIred structures, whereas it is closed in the other structures that are complexed with substrates (supplemental Fig. S2). This difference arises from changes in the orientation of α4 and loop region α8–α9. In addition to the interactions that result from substrate and Mg2+ binding, hydrogen bond formation between Arg98 (on α4) and Glu168 (on loop α8–α9) would lead the conformational change that closes the aperture of the active site. Another structural difference is due to the redox states of the enzyme. In the FreeIDI structure, the backbone nitrogen atom of Thr68 is close enough to form hydrogen bonds with FMN N-5. Meanwhile, in the FreeIDIred structure, the main chain nitrogen of Thr68 is positioned in the opposite direction, and the backbone carbonyl oxygen of Met67 is within hydrogen-bond formation distance from FMN N-5 (Figs. 2B and 3A). Conformational changes in the residues, which interact with FMN N-5, which were derived from differences in redox states were also observed in several flavoproteins, such as flavodoxin from Clostridium beijerinckii (27Ludwig M.L. Pattridge K.A. Metzger A.L. Dixon M.M. Eren M. Feng Y. Swenson R.P. Biochemistry. 1997; 36: 1259-1280Crossref PubMed Scopus (158) Google Scholar). However, in all substrate-complex structures, the arrangements of the residues that were coordinated with FMN N-5 were similar to that in the FreeIDIred structure, even in the oxidized state (data not shown). It should be noted that isomerase reaction would proceed in the reduced substrate-complexes. Thus the substrates bound in the IPP-IDIred and DMAPP-IDIred structures are considered to be a mixture of IPP, DMAPP, and probably a reaction intermediate. However, both substrate conformation and interaction with surrounding residues in the substrate-complex structures were nearly identical, in both the oxidized and reduced states (Fig. 3B). Moreover, distortion of the isoalloxazine ring was observed in FreeIDIred, whereas the ring was more planar in FreeIDI (Fig. S3). In structural studies of flavoenzymes, reduction of flavin sometimes results in distortion (28Lennon B.W. Williams Jr., C.H. Ludwig M.L. Protein Sci. 1999; 8: 2366-2379Crossref PubMed Scopus (110) Google Scholar). Therefore, the conformational change in FMN and the loss of yellow color of the crystals (supplemental Fig. S4) are considered proof that FMN is in the reduced state in S. shibatae type 2 IDI. The reduction-induced conformational change in the isoalloxazine ring was not as obvious in the substrate-complex structures with lower resolution. When reduced, however, both the isoalloxazine ring of FMN and the isopentenyl or dimethylallyl plane of the substrates also twisted slightly, bringing them closer together (Fig. 3B). Alanine-scanning Mutagenesis-Concurrent with the structural investigations, mutagenic studies of type 2 IDI were also performed. Based on the previously reported structures of type 2 IDIs from Bacillus subtilis (Protein Data Band codes 1P0N and 1P0K (24Steinbacher S. Kaiser J. Gerhardt S. Eisenreich W. Huber R. Bacher A. Rohdich F. J. Mol. Biol. 2003; 329: 973-982Crossref PubMed Scopus (51) Google Scholar)) and Thermus thermophilus (1VCF, 1VCG, and 3DH7 (29de Ruyck J. Pouyez J. Rothman S.C. Poulter D. Wouters J. Biochemistry. 2008; 47: 9051-9053Crossref PubMed Scopus (21) Google Scholar)), 15 charged or polar amino acid residues, which are highly conserved among known type 2 IDIs and likely to be in the active site, were selected for mutagenesis (Fig. 4A). Each amino acid residue was replaced with alanine to determine the function of its side chain. In the solved structures of S. shibatae type 2 IDI, all of the mutated residues appeared to be in proximity of FMN and/or the substrates (Fig. 4B). Wild-type IDI and all mutants, except for K193A, were purified as holoenzymes that tightly bound FMN. These results indicate that the mutations did not affect the proper folding of the enzyme. Only K193A was obtained as an apoenzyme that bound FMN with very low affinity; however, the CD spectra of K193A and wild-type IDI were nearly identical, indicating that the global structure of the mutant had not been altered by the mutation (data not shown). As shown in Fig. 4C (see also supplemental Table S2), IDI activity was significantly reduced in most mutants compared with the wild-type enzyme. In particular, R7A, K8A, N157A, Q160A, E161A, and K193A showed significant loss of activity (less than 1% of wild-type enzyme activity), suggesting that the mutated residues are important for the enzyme reaction. In contrast, all mutants, except for K193A, retained more than ∼20% NADH oxidase activity of wild-type S. shibatae IDI (supplemental Table S2), which is a subfunction of the enzyme observed in the absence of the substrate (18Yamashita S. Hemmi H. Ikeda Y. Nakayama T. Nishino T. Eur. J. Biochem. 2004; 271: 1087-1093Crossref PubMed Scopus (36) Google Scholar). This result indicates that the significant inactivation of some enzymes for IDI reaction is not derived from the deficiency in the reduction of FMN by NADH. The inactivity of K193A is due to its inability to bind FMN. We also examined the Kd values of each enzyme for FMN and IPP by measuring the change in tryptophan fluorescence intensity through titration (supplemental Table S2). None of the mutations, except for K193A, affected the parameters, indicating that, other than Lys193, the mutated amino acids were not critically involved in binding of either FMN or IPP. Candidates for General Acid-Base Catalysts-In the substrate-enzyme complexes, IPP and DMAPP are bound very closely on the si-face of the FMN isoalloxazine ring, which means that rotation of their C-2–C-3 bonds during reaction is unlikely. During isomerization between IPP and DMAPP, proton exchange occurs selectively at C-2 and C-4 of IPP (the latter corresponds to E-methyl of DMAPP) (30Barkley S.J. Desai S.B. Poulter C.D. Org. Lett. 2004; 6: 5019-5021Crossref PubMed Scopus (15) Google Scholar, 31Laupitz R. Grawert T. Rieder C. Zepeck F. Bacher A. Arigoni D. Rohdich F. Eisenreich W. Chem. Biodivers. 2004; 1: 1367-1376Crossref PubMed Scopus (30) Google Scholar). In addition, deprotonation and protonation at C-2 are reportedly pro-R stereospecific (31Laupitz R. Grawert T. Rieder C. Zepeck F. Bacher A. Arigoni D. Rohdich F. Eisenreich W. Chem. Biodivers. 2004; 1: 1367-1376Crossref PubMed Scopus (30) Google Scholar, 32Kao C.L. Kittleman W. Zhang H. Seto H. Liu H.W. Org. Lett. 2005; 7: 5677-5680Crossref PubMed Scopus (22) Google Scholar). These facts and the coordination of carbon atoms of the substrates justify the assignment shown in Fig. 3B. Thus, possible proton donors to the C-4 of IPP, and proton acceptors from the E-methyl carbon of DMAPP in the active site were explored. Based on the active-state IPP-IDIred structure, the N-5 nitrogen of FMN, which is located 3.60 ± 0.10 Å from the C4 carbo
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