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Roles of Amino Acids in the <Italic>Escherichia Coli</Italic

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Roles of Amino Acids in the <Italic>Escherichia Coli</Italic Article pubs.acs.org/biochemistry Roles of Amino Acids in the Escherichia coli Octaprenyl Diphosphate Synthase Active Site Probed by Structure-Guided Site-Directed Mutagenesis † ∥ † ‡ § † ‡ Keng-Ming Chang, Shih-Hsun Chen, Chih-Jung Kuo, , Chi-Kang Chang, Rey-Ting Guo, , ∥ † ‡ § Jinn-Moon Yang, and Po-Huang Liang*, , , † Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan ‡ Taiwan International Graduate Program, Academia Sinica, Taipei 115, Taiwan § Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan ∥ Department of Biological Science and Technology, National Chiao Tung University, Hsin-Chu 300, Taiwan *S Supporting Information ABSTRACT: Octaprenyl diphosphate synthase (OPPS) catalyzes consecutive condensation reactions of farnesyl diphosphate (FPP) with five molecules of isopentenyl diphosphates (IPP) to generate C40 octaprenyl diphosphate, which constitutes the side chain of ubiquinone or menaquinone. To understand the roles of active site amino acids in substrate binding and catalysis, we conducted site- directed mutagenesis studies with Escherichia coli OPPS. In conclusion, D85 is the most important residue in the first DDXXD motif for both FPP and IPP binding through an H-bond network involving R93 and R94, respectively, whereas R94, K45, R48, and H77 are responsible for IPP binding by providing H-bonds and ionic interactions. K170 and T171 may stabilize the farnesyl carbocation intermediate to facilitate the reaction, whereas R93 and K225 may stabilize the catalytic base (MgPPi) for HR proton abstraction after IPP condensation. K225 and K235 in a flexible loop may interact with FPP when the enzyme becomes a closed conformation, which is therefore crucial for catalysis. Q208 is near the hydrophobic part of IPP and is important for IPP binding and catalysis. group of prenyltransferases catalyze consecutive con- consecutive condensation reactions of FPP with five IPPs densation reactions of designated numbers of C iso- A 5 leading to a C40 product, two sulfate ions, S1 and S2, which pentenyl diphosphate (IPP) with C15 farnesyl diphosphate mimic the pyrophosphate group of the substrates, are bound in 1−3 (FPP) to generate linear isoprenoid natural products. On the active site; S1 is bound with a Mg2+ coordinated by the first the basis of the stereochemistry of double bonds formed in the DDXXD motif, but surprisingly, S2 is held by a positively IPP condensation reactions, these enzymes are classified as 4 charged pocket formed by Lys, His, and Arg residues, rather trans or cis types. FPP is synthesized by FPP synthase (FPPS), than the conserved DDXXD motifs.14 By aligning with the also a trans-prenyltransferase, through the condensation of two more recently published ternary complex structures of FPPS IPPs with its allylic isomer, dimethylallyl diphosphate with an inactive DMAPP thiol analogue (DMSPP) and IPP,15 (DMAPP).5,6 From FPP, the trans-prenyltransferases synthe- S1 and S2 sites are presumably for FPP and IPP binding, size C20 geranylgeranyl diphosphate as a precursor of many natural products (e.g., Taxol) and as used for protein post- respectively. Since then, a site-directed mutagenesis study has fi not been performed in FPPS, OPPS, or any other trans-prenyl- translational modi cation, C25 farnesylgeranyl diphosphate to − tranferase to verify the exact catalytic roles and the importance form ether-linked lipids in thermophilic archaea, and C30 C50 products that constitute side chains of ubiquinone and of the active site residues. There is no available structure for menaquinone.7,8 trans-prenyltransferase with bound FPP or IPP. A key feature for trans-prenyltransferases is that they all In the previous study, we utilized a bromo-substituted IPP possess two Asp-rich DDXXD motifs. Previous site-directed analogue (Br-IPP) to slow the IPP condensation step and mutagenesis studies reported that all Asp residues in these successfully trapped the farnesyl carbocation intermediate as motifs of FPPS except the last one in the second motif are farnesol (FOH) with a base in the Escherichia coli OPPS important for both substrate binding and catalysis before 9−13 detailed structural information was available. However, as Received: January 17, 2012 indicated by our crystal structures of Thermotoga maritima Revised: April 3, 2012 octaprenyl diphosphate synthase (OPPS) that catalyzes the Published: April 3, 2012 © 2012 American Chemical Society 3412 dx.doi.org/10.1021/bi300069j | Biochemistry 2012, 51, 3412−3419 Biochemistry Article reaction.16 This result further strengthened the ionization− results clarify or reveal the roles of previously proposed or condensation−elimination sequential mechanism previously unidentified important amino acids and enhance our under- proposed for FPPS reaction,17 where the allylic substrate standing of the catalytic mechanism of trans-prenyltransferases. (DMAPP in FPPS and FPP in OPPS) releases pyrophosphate to form the carbocation intermediate, which is attacked by IPP ■ EXPERIMENTAL PROCEDURES to form the second carbocation intermediate, followed by Materials. Radiolabeled [14C]IPP (55 μCi/mmol) and abstraction of the H proton on the IPP moiety to generate the R [3H]FPP (17 Ci/mmol) were purchased from Amersham C -extended product. On the basis of this reaction mechanism, 5 Pharmacia Biotech., and FPP was obtained from Sigma. we examined the possible catalytic roles of the potential active PfuTurbo DNA polymerase was obtained from Life Tech- site amino acids by performing site-directed mutagenesis on nologies, Inc. The plasmid mini-prep kit, the DNA gel E. coli OPPS using its structure derived by homology modeling extraction kit, and NiNTA resin were purchased from Qiagen. from the available T. maritima OPPS structure14 and comparing 15 The protein expression kit, including the pET16b and that structure with the E. coli FPPS complex structure. Our pET32Xa/LIC vectors, and competent JM109 and BL21(DE3) cells were obtained from Novagen. The QuikChange site- Table 1. Primers Used To Construct E. coli Mutant OPPS directed mutagenesis kit was obtained from Stratagene. All commercial buffers and reagents used are of the highest grade. mutant primer sequencea Homology Modeling of the E. coli OPPS Structure. D84A 5′-GCTACACGCCGACGTTGT-3′ The sequence homology between E. coli and T. maritima OPPS D85A 5′-ACGACGCCGTTGTGGAT-3′ was calculated with CLUSTAL W multiple-sequence alignment D88A 5′-GACGTTGTGGCAGAATCAGAT-3′ software.18 On the basis of the sequence homology, the three- R93A 5′-CAGATATGGCCAGGGGTAAA-3′ dimensional (3D) model of E. coli OPPS was generated from R94A 5′-ATATGCGCGCGGGTAAAG-3′ the crystal structure (Protein Data Bank entry 1V4E) of T. K45A 5′-CGGTGCACGTATTCGTCC-3′ maritima OPPS14 by using MODELER19 encoded in Insight II R48A 5′-GTAAACGTATTGCTCCGATGATT-3′ (Accelrys, Inc.), which uses a spatial restraint method to build H77A 5′-AGTTTATCGCCACGGCGA-3′ the 3D protein structure. The structure with the lowest viola- D211A 5′-TTGATCGCCGATTTACTCG-3′ tion score and lowest energy was chosen as the candidate. The D212A 5′-TGATCGACGCATTACTCGAT-3′ distribution of the backbone dihedral angles of the model was D215A 5′-GATTTACTCGCCTACAATGCC-3′ evaluated by a Ramachandran plot using PROCHECK.20 The K170L 5′-ATCTACAGCCTGACTGCGC-3′ Prostat module of Insight II was used to analyze the properties K225L 5′-AACAGTTAGGTCTAAACGTCGG-3′ of bonds, angles, and torsions. Profile-3D21 was used to check K235L 5′-AAGGTTTACCGACGCTGC-3′ structure and sequence compatibility. T171V 5′-ATCTACAGCAAGGTTGCGC-3′ Site-Directed Mutagenesis of E. coli OPPS. OPPS Q208A 5′-GCTTTCGCGTTGATCGAC-3′ mutants were prepared by using the QuikChange site-directed aThe mutagenic nucleotides are underlined. mutagenesis kit on the E. coli OPPS-encoding gene cloned in Figure 1. Alignment of amino acid sequences of E. coli OPPS (E-OPPs), T. maritima OPPS (T-OPPs), E. coli FPPS (E-FPPs), and avian FPPS (A-FPPs). The two conserved DDXXD motifs are top-lined. Stars denote the amino acid residues mutated for E. coli OPPS in this study. 3413 dx.doi.org/10.1021/bi300069j | Biochemistry 2012, 51, 3412−3419 Biochemistry Article the vector as previously described.22,23 The mutagenic oligo- nucleotides prepared by Biobasic Inc. for performing site- directed mutagenesis are listed in Table 1. Each specific amino acid mutation was confirmed by sequencing the entire gene of the cloning plasmid obtained from an overnight culture. The correct construct was subsequently transformed into E. coli BL21(DE3) cells for protein expression. The protein purification procedure was described previously.14 Briefly, the cells were disrupted and purified using NiNTA column chromatography followed by removal of the His tag using FXa. The purified OPPS mutants were checked by SDS−PAGE. Measurements of Kinetic Parameters for Mutant E. coli OPPS. For enzyme activity measurements, each mutant OPPS was used at 0.1 μM. The enzyme reaction was initiated in a 200 μLbuffer solution containing 100 mM Hepes (pH 7.5), 5 μM μ 14 ° FPP, 50 M[ C]IPP, 50 mM KCl, and 0.5 mM MgCl2 at 25 C. The reaction was terminated by addition of 10 mM (final concen- tration) EDTA, and the product was extracted with 1-butanol. The product was quantitated by counting the radioactivity in the butanol phase ([14C]IPP was in the aqueous phase) using a Beckman LS6500 scintillation counter. The steady-state kcat and Km values of the mutant enzymes were calculated on the basis of the the initial rates of IPP consumption as previously described.22,23 Circular Dichroism (CD) Experiments.
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