Structural Role for Tyr-104 in Escherichia coli Isopentenyl-diphosphate Isomerase

Isopentenyl-diphosphate (IPP):dimethylallyl diphosphate isomerase is a key enzyme in the biosynthesis of isoprenoids. The mechanism of the isomerization reaction involves protonation of the unactivated carbon-carbon double bond in the substrate, but identity of the acidic moiety providing the proton is still not clear. Multiple sequence alignments and geometrical features observed in crystal structures of complexes with IPP isomerase suggest that Tyr-104 could play an important role during catalysis. A series of mutants was constructed by directed mutagenesis and characterized by enzymology. Crystallographic and thermal denaturation data for Y104A and Y104F mutants were obtained. Those data demonstrate the importance of residue Tyr-104 for proper folding of Escherichia coli type I IPP isomerase. Isopentenyl-diphosphate (IPP):dimethylallyl diphosphate isomerase is a key enzyme in the biosynthesis of isoprenoids. The mechanism of the isomerization reaction involves protonation of the unactivated carbon-carbon double bond in the substrate, but identity of the acidic moiety providing the proton is still not clear. Multiple sequence alignments and geometrical features observed in crystal structures of complexes with IPP isomerase suggest that Tyr-104 could play an important role during catalysis. A series of mutants was constructed by directed mutagenesis and characterized by enzymology. Crystallographic and thermal denaturation data for Y104A and Y104F mutants were obtained. Those data demonstrate the importance of residue Tyr-104 for proper folding of Escherichia coli type I IPP isomerase. Isoprenoids play important roles in all living organisms; they function as steroid hormones in mammals, carotenoids in plants, and ubiquinone or menaquinone in bacteria (1Sacchettini J.C. Poulter C.D. Science. 1997; 277: 1788-1789Crossref PubMed Scopus (460) Google Scholar). All isoprenoid compounds are characterized by the basic isoprene unit. Nature uses two activated isoprene units to build these prenoid compounds as follows: isopentenyl diphosphate (IPP) 3The abbreviations used are: IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; IDI, IPP isomerase; PEG, polyethyleneglycol; EIPP, 3,4-epoxy-3-methyl-1-butyl diphosphate; mme, monomethyl ether; PDB, Protein Data Bank; W T, wild type. and its electrophilic allylic isomer dimethylallyl diphosphate (DMAPP) (Scheme 1). Isopentenyl diphosphate:dimethylallyl diphosphate (IPP:DMAPP) isomerase (IDI; EC 5.3.3.2) is a key enzyme involved in the biosynthesis of isoprenoids and catalyzes the isomerization of IPP into DMAPP (2Hahn F.M. Poulter C.D. J. Biol. Chem. 1995; 270: 11298-11303Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). DMAPP then condenses with additional molecules of IPP to form farnesyl diphosphate, which is required for protein prenylation, cholesterol biosynthesis, and synthesis of a variety of higher molecular weight isoprenoids. Although the synthesis of IPP can occur through the mevalonate pathway in which acetyl-CoA and mevalonate are substrates, a mevalonate-independent pathway producing IPP and DMAPP was found in plant chloroplasts, algae, cyanobacteria, and some bacteria. This quite new pathway, known as the deoxyxylulose 5-phosphate pathway, begins with pyruvate and glyceraldehyde 3-phosphate as the initial substrates. Two types of IDI are reported. They show no sequence similarity but catalyze the same reaction. Type I enzyme is widely distributed in microorganisms. Type II IPP isomerase is a flavoprotein that belongs to the FMN-dependent α-hydroxyacid dehydrogenase family and displays a classical TIM barrel fold (3Kaneda 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, 4Steinbacher 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). Type I IPP isomerase is composed of about 200 amino acids and folds into a compact globular protein that belongs to the class of α/β proteins. It is a metalloprotein containing divalent cations, Zn2+ and Mg2+, as cofactors (5Carrigan C.N. Poulter C.D. J. Am. Chem. Soc. 2003; 125: 9008-9009Crossref PubMed Scopus (36) Google Scholar, 6Bonnano J.B. Edo C. Eswar N. Pieper U. Romanowski M.J. Ilyin V. Gerchman S.E. Kycia H. Studier F.W. Sali A. Burley S.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12896-12901Crossref PubMed Scopus (112) Google Scholar). Crystal structures of free and metal-bound Escherichia coli IDI-1 show that the first divalent cation is involved in the active conformation folding, with the metal occupying a first coordination site composed of three histidines and two glutamates (7Durbecq V. Sainz G. Oudjama Y. Clantin B. Bompard-Gilles C. Tricot C. Caillet J. Stalon V. Droogmans L. Villeret V. EMBO J. 2001; 20: 1530-1537Crossref PubMed Scopus (83) Google Scholar). The structures of E. coli IPP isomerase in complex with diphosphate substrate analogues (N,N-dimethyl-2-amino-1-ethyl diphosphate (8Wouters J. Oudjama Y. Barkley S.J. Tricot C. Stalon V. Droogmans L. Poulter C.D. J. Biol. Chem. 2003; 278: 11903-11908Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), 3,4-epoxy-3-methyl-1-butyl diphosphate (8Wouters J. Oudjama Y. Barkley S.J. Tricot C. Stalon V. Droogmans L. Poulter C.D. J. Biol. Chem. 2003; 278: 11903-11908Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 9Wouters J. Oudjama Y. Stalon V. Droogmans L. Poulter C.D. Proteins. 2004; 54: 216-221Crossref PubMed Scopus (28) Google Scholar), and the bromohydrin of isopentenyl diphosphate (10Wouters J. Oudjama Y. Ghosh S. Stalon V. Droogmans L. Oldfield E. J. Am. Chem. Soc. 2003; 125: 3198-3199Crossref PubMed Scopus (42) Google Scholar)) show the presence of the second metal site occupied by Mg2+ cation. Carbonyl oxygen of residue 67, side chain oxygen of Glu-87, two water molecules, and two nonbridging oxygens of the diphosphate moiety of the inhibitors coordinate this metal. Resolution of these crystallographic structures helped to clarify the protonation/deprotonation catalytic mechanism. IPP would be initially protonated by an acidic residue that is not currently known. DMAPP would then be formed by deprotonation via Cys-67. The importance of this last residue in the catalytic process was proved by site-directed mutagenesis. No residual activity is observed when the thiol group of cysteine is changed by a methyl moiety (C67A). Tryptophan 161 is optimally positioned deep in the active site and would stabilize, through quadrupole-charge interactions, the highly reactive carbocation intermediate, formed upon the protonation of IPP (supplemental Scheme S1) (7Durbecq V. Sainz G. Oudjama Y. Clantin B. Bompard-Gilles C. Tricot C. Caillet J. Stalon V. Droogmans L. Villeret V. EMBO J. 2001; 20: 1530-1537Crossref PubMed Scopus (83) Google Scholar). However, these studies have not yet conclusively established the origin of the proton activating the double bond in IPP. Glu-116 is likely the critical acidic residue that drives protonation by lowering the energy of the carbocation as demonstrated by the crystallographic structure of the transition-state analogue N,N-dimethyl-2-amino-1-ethyl diphosphate in complex with the enzyme (8Wouters J. Oudjama Y. Barkley S.J. Tricot C. Stalon V. Droogmans L. Poulter C.D. J. Biol. Chem. 2003; 278: 11903-11908Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). However, Glu-116 is probably not protonated because it is directly coordinated to the catalytic metal cation. Therefore, it could not be the proton donor. Tyr-104 is a possible candidate for proton donation in the reaction catalyzed by IDI-1. Indeed, in the crystal structures of IDI-1, Tyr-104 is located in the active site of the enzyme and is directly hydrogen-bonded to Glu-116. The precise role of Tyr-104 during catalysis is not clear and deserves special attention. The present work was designed in an effort to clarify that question. Site-directed Mutagenesis—Plasmid pYL20, which contains the idi gene from E. coli with an engineered C-terminal His tag (LEHHHHHH), was available from previous studies (11Oudjama Y. Durbecq V. Sainz G. Clantin B. Tricot C. Stalon V. Villeret V. Droogmans L. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 287-288Crossref PubMed Scopus (7) Google Scholar). Mutations Y104A and Y104F were introduced using the QuickChange site-directed mutagenesis kit (Stratagene). The pYL20 plasmid was used as a template in all experiments. Purification—E. coli BL21(DE3) pLysS cells (Novagen) transformed by the mutated pYL20 plasmid were grown to an OD660 of 0.4–0.5 (from OD660 of 0.2) at 37 °C. Expression of the recombinant enzymes was induced for 3 h at 37 °C using 1 mm isopropyl-β-d-thiogalactoside in the medium. Cells were resuspended in 50 mm Tris-HCl, pH 7.4. Cell disruption has been achieved by sonication for 10 min in a Raytheon sonic oscillator (250 watts, 10 kHz). In order to remove the wild-type chromosomic IPP isomerase from recombinant enzymes, purification was performed using nickel-ion affinity chromatography with resin (chelating Sepharose supplied by Amersham Biosciences) pre-equilibrated with 50 mm Tris-HCl, pH 7.4. Bound proteins were eluted by a linear gradient of 0–0.5 m imidazole in 50 mm Tris-HCl, pH 7.4. An SDS-polyacrylamide gel was useful to check the purity of IDI mutant proteins. The fractions from elution containing active recombinant IPP isomerase were pooled and dialyzed three times against 2 liters of 50 mm Tris-HCl, pH 7.4, and 0.1 mm EDTA. The three mutant protein solutions were concentrated by ultrafiltration (YM10, Amicon) prior to crystallization trials. In contrast to WT IPP isomerase, for which an ideal concentration to obtain crystals was about 5 mg/ml, protein solutions of the mutants could not be concentrated higher than 2.0 and 3.1 mg/ml for Y104A and Y104F, respectively, based on protein concentration estimated by UV absorption. Enzymatic Activity—The principle of the IPP isomerase assay is based on the lability of DMAPP, which is transformed into the corresponding alcohol under acidic conditions, although IPP remains stable under the conditions of the assay (12Satterwhite D.M. Methods Enzymol. 1985; 110: 92-99Crossref PubMed Google Scholar). The reaction was initiated by adding the enzyme to a mixture in 50 mm Tris-HCl buffer, pH 7.4, containing the divalent metal cofactors (1.5 mm Mn2+ and Mg2+) and [1-14C]IPP 18 μm in a final volume of 50 μl. After incubation at 37 °C, the reaction was stopped by addition of 400 μl of methanol/concentrated HCl (4:1, v/v). The mixture was subsequently incubated for 10 min at 37 °C, and the radioactive products were extracted with chloroform. The extraction was performed by vigorous vortexing followed by centrifugation at 10,000 × g for 2 min. A 200-μl portion of the organic layer was mixed with 5 ml of a scintillation mixture (ICN Biomedicals). The radioactivity was counted as a measure for the conversion of IPP into DMAPP. Crystallization Experiments—Crystallization trials were achieved by the hanging drop vapor-diffusion method at 20 °C. Each drop suspended on the top of a reservoir solution (0.6 ml) was a mixture of 4 μl of protein solution (2.0 and 3.1 mg/ml for Y104A and Y104F, respectively) with the same volume of the reservoir solution. E. coli IDI-1 mutants precipitated when the same crystallization conditions for WT IPP isomerase were used. Crystallization parameters were therefore adapted by screening precipitant concentration (10–16% (w/v) PEG 2000 monomethyl ether (mme)), pH (4.5–6.5), and additives (ammonium sulfate, MnCl2, and MgCl2). Small hexagonal crystals (maximum size 0.15 mm) were obtained for the Y104A mutant in 14% (w/v) PEG 2000 mme, 100 mm Tris maleate buffer, pH 4.5, in the presence of 100 mm ammonium sulfate, 20 mm MnCl2, and MgCl2. Larger crystals were obtained for the Y104F mutant under similar conditions (14% (w/v) PEG 2000 mme, 100 mm Tris maleate buffer, pH 5.0, in the presence of 100 mm ammonium sulfate and 20 mm MnCl2 and MgCl2). Complex was obtained by soaking crystals of the Y104F mutant with 3,4-epoxy-3-methyl-1-butyl diphosphate (EIPP), a mechanism-based irreversible inhibitor (8Wouters J. Oudjama Y. Barkley S.J. Tricot C. Stalon V. Droogmans L. Poulter C.D. J. Biol. Chem. 2003; 278: 11903-11908Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Solution of the inhibitor (25 mm) in Tris maleate (100 mm, pH 4.5), PEG 2000 mme (14%), ammonium sulfate (100 mm), MnCl2 and MgCl2 (20 mm), and glycerol (25%) was replaced every 3 h for at least 24 h. Data Collection and Structure Determination—For the small Y104A mutant crystals (approximate size of about 0.15 mm) a complete data set to 1.76 Å was collected, after flash-freezing, at beam line BM30A (European Synchrotron Radiation Facility, Grenoble, France) on a MarResearch CCD. Data were processed with the HKL suite (13Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38570) Google Scholar). The Y104A mutant crystallizes in space group P3121 with cell parameters a = b = and = is in the unit. the Y104F a complete data set to Å was with a from MarResearch with and on an on a Data were processed with the The Y104F mutant crystallizes in space group with cell parameters a = b = and = are two molecules in the by data of the complex were to The cell parameters are a = b = and = which to a space are two molecules in the by The structure used for the of Y104F and to the metal-bound form of E. coli IDI The Y104A mutant was from the form of IDI-1 was performed with the Methods Enzymol. 1997; 277: PubMed Scopus Google Scholar). were with the A. C. Scholar), and the of the was with the J. Crystallogr. Google Scholar). of the moiety in was by of and Data and are in have been at the Protein Data for for Y104F, and for Y104A and of the E. coli IDI-1 mutants and the complex Y104F mutant and data Cell set for the are in for the are in Resolution of protein of of water of All protein All All water molecules by and for the are in of by and in a new of the proteins was to the to and method J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, R. G. C. L. G. PubMed Scopus Google Scholar). The at were obtained using an from The denaturation studies were in the °C using a of 1 were by protein solutions with 50 mm Tris-HCl buffer, pH 7.4, containing the divalent metal cofactors (1.5 mm Mn2+ and Mg2+) in order to obtain a final protein concentration of The solutions were then for 10 min prior to the thermal denaturation A of the was as a the first of with to was in order to the of protein This is to the thermal of the protein 2003; PubMed Scopus Google Scholar). and of Tyr-104 order to the importance of residue Tyr-104 in the mechanism of IDI-1, two mutants were constructed by site-directed mutagenesis and Y104A and In the first the chain of Tyr-104 is replaced by a side In the second mutant the is but the group is The corresponding C-terminal proteins were in E. coli and by affinity This mutants in a form for activity and protein The affinity purification was introduced in order to the recombinant enzymes from the chromosomic E. coli IDI-1 and therefore to in the Enzymatic activity of the two Tyr-104 mutants is when with the wild-type enzyme as in of the chain of Tyr-104 by an or a in a of with the mutants than residual of the mutants is by a of 100 = for the WT to = for the WT This activity that Tyr-104 is a key amino for the isomerization of IPP into DMAPP. In of that affinity of Tyr-104 mutants to the substrate is by the of the moiety and Y104F, and μm for the WT It is therefore that Tyr-104 a role in the substrate and the proper folding of the parameters of wild-type and mutant E. coli activity in a new Crystallographic Structure of Y104A and Y104F of from the two mutants were grown using the hanging drop of the Y104F mutant were obtained in the presence of divalent is similar to the obtained with the WT enzyme which belongs to the space group = b = and = of the Y104A mutant in similar crystallization In contrast to the Y104F they a and to space group = b = = as the crystallographic structure of WT enzyme cation D and the crystallographic structure of Y104F protein to The fold into a small of two as in This folding to the of the first metal site that and and to and Glu-116. A second metal site the group of the highly the side chain oxygen of Glu-87, and water molecules to form an coordination the structure of Y104A was to 1.76 Å No is with the chain of residue with of the by an introduced by site-directed mutagenesis. The site is in this but Mg2+ is still present and is to Glu-87, and water molecules A of the two mutants against the WT enzyme is in of the fold of wild-type and E. coli IDI-1 against the mutated proteins and in in and and Glu-87, and 4 water Glu-87, and 4 water Glu-87, and 4 water Glu-87, and 4 water molecules in a new a crystal of Y104F was in order to obtain a complex the mutated protein and EIPP, a mechanism-based irreversible Structure was to The fold of the enzyme is similar to the observed for the WT Two oxygens in the diphosphate the group of the highly the side chain oxygen of Glu-87, and two water molecules coordinate the second involved in the of IPP, to form the same coordination of the denaturation of the enzymes was in order to the of the WT enzyme the mutated proteins. The thermal of the WT enzyme and the mutants were under the conditions used. of denaturation of the enzyme by 20 °C when Tyr-104 is mutated into and °C for WT and Y104A enzymes, of the enzyme is when the moiety is and °C for WT and Y104F enzymes, respectively) the importance of the group of this residue Multiple sequence alignments obtained by J. PubMed Scopus Google show that a series of amino acids is type 1 the catalytic cysteine in E. and in E. a amino acids are in all (supplemental and Glu-116 are of the first metal site J.B. Edo C. Eswar N. Pieper U. Romanowski M.J. Ilyin V. Gerchman S.E. Kycia H. Studier F.W. Sali A. Burley S.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12896-12901Crossref PubMed Scopus (112) Google Scholar, V. Sainz G. Oudjama Y. Clantin B. Bompard-Gilles C. Tricot C. Caillet J. Stalon V. Droogmans L. Villeret V. EMBO J. 2001; 20: 1530-1537Crossref PubMed Scopus (83) Google Scholar). and are involved in the of the diphosphate moiety of the Tyr-104 is highly residue (8Wouters J. Oudjama Y. Barkley S.J. Tricot C. Stalon V. Droogmans L. Poulter C.D. J. Biol. Chem. 2003; 278: 11903-11908Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 9Wouters J. Oudjama Y. Stalon V. Droogmans L. Poulter C.D. Proteins. 2004; 54: 216-221Crossref PubMed Scopus (28) Google Scholar, J. Oudjama Y. Ghosh S. Stalon V. Droogmans L. Oldfield E. J. Am. Chem. Soc. 2003; 125: 3198-3199Crossref PubMed Scopus (42) Google Scholar). In the crystal structures for E. coli IPP isomerase, the side chain of Tyr-104 is hydrogen-bonded to the catalytic Glu-116 J.B. Edo C. Eswar N. Pieper U. Romanowski M.J. Ilyin V. Gerchman S.E. Kycia H. Studier F.W. Sali A. Burley S.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12896-12901Crossref PubMed Scopus (112) Google Scholar, V. Sainz G. Oudjama Y. Clantin B. Bompard-Gilles C. Tricot C. Caillet J. Stalon V. Droogmans L. Villeret V. EMBO J. 2001; 20: 1530-1537Crossref PubMed Scopus (83) Google Scholar, J. Oudjama Y. Barkley S.J. Tricot C. Stalon V. Droogmans L. Poulter C.D. J. Biol. Chem. 2003; 278: 11903-11908Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 9Wouters J. Oudjama Y. Stalon V. Droogmans L. Poulter C.D. Proteins. 2004; 54: 216-221Crossref PubMed Scopus (28) Google Scholar, J. Oudjama Y. Ghosh S. Stalon V. Droogmans L. Oldfield E. J. Am. Chem. Soc. 2003; 125: 3198-3199Crossref PubMed Scopus (42) Google Scholar). After of Tyr-104 into and observed a activity and affinity that Tyr-104 is a key amino for the isomerization of IPP into DMAPP and could play a role in the of the substrate into the a second to this by the crystallographic structure of the mutated proteins. The Y104F mutant a fold similar to the wild-type enzyme in the presence of metal Therefore, not to the conformation of the a crystal form of the Y104A although obtained in the presence of divalent cations, is similar to the obtained for the wild-type E. coli IDI-1 (7Durbecq V. Sainz G. Oudjama Y. Clantin B. Bompard-Gilles C. Tricot C. Caillet J. Stalon V. Droogmans L. Villeret V. EMBO J. 2001; 20: 1530-1537Crossref PubMed Scopus (83) Google Scholar). In this the first amino acids are not in the of this when the catalytic site is not A similar of the in the structure of the Y104A Therefore, of Tyr-104 into an to the of the catalytic in the presence of metal in the solution (supplemental measure the of the mutants in to the WT thermal denaturation studies were by UV The of denaturation for Y104A and Y104F is with the WT and °C for Y104F, and Y104A enzymes, that Tyr-104 is in the of IDI-1 folding as by the crystallographic structure of on this the role of Tyr-104 to be In order to check the of the catalytic data were on a complex Y104F mutant and a mechanism-based irreversible The a bond oxygen of Glu-116 in the active site and of the inhibitor the in with a proton in order to be This was then by Glu-116 (Scheme the crystal structure is with a of of Glu-116 on of the same the of this reaction was The of two and was to and for molecules in the (supplemental This that the first of the is the of a The diphosphate group of the inhibitor is by with and of the the diphosphate Therefore, the structure of the Y104F mutant of E. coli IDI-1 in complex with that the protonation is still when the protein is Indeed, in the of the function at residue the of is activated by the enzyme to with Glu-116. This that Tyr-104 is not the proton in the active site of the enzyme and the residual activity observed with the Tyr-104 In the crystal structure of the Y104F mutant that not the group of Tyr-104 the active site of IDI-1 by to Glu-116 but that additional the The acidic moiety providing the proton during isomerization of IPP into DMAPP catalyzed by IDI-1 is not yet Multiple sequence alignments and geometrical features observed in crystal structures of complexes with E. coli IDI-1 suggest that Tyr-104 could play this important role during catalysis. Therefore, two mutants and were constructed by site-directed mutagenesis and Enzymatic of mutants that of activity is when with the wild-type affinity for the substrate is for and of the mutants is by a of 100 to