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Structure and Reactivity of Human Mitochondrial 2,4-Dienoyl-CoA Reductase

ENZYME-LIGAND INTERACTIONS IN A DISTINCTIVE SHORT-CHAIN REDUCTASE ACTIVE SITE*
  • Magnus S. Alphey
    Affiliations
    Division of Biological Chemistry and Molecular Microbiology, School of Life Sciences, University of Dundee, Dundee, DD1 5EH, United Kingdom
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  • Wenhua Yu
    Affiliations
    Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong SAR, P. R. China
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  • Emma Byres
    Affiliations
    Division of Biological Chemistry and Molecular Microbiology, School of Life Sciences, University of Dundee, Dundee, DD1 5EH, United Kingdom
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  • Ding Li
    Affiliations
    Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong SAR, P. R. China
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  • William N. Hunter
    Correspondence
    To whom correspondence should be addressed. Tel.: 44-1382-345745; Fax: 44-1382-345764;
    Affiliations
    Division of Biological Chemistry and Molecular Microbiology, School of Life Sciences, University of Dundee, Dundee, DD1 5EH, United Kingdom
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  • Author Footnotes
    * This work was supported by The Wellcome Trust and Research Grants Council of the Hong Kong Special Administrative Region, China (Project 9040660 (CityU 1110/01M)). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
      Fatty acid catabolism by β-oxidation mainly occurs in mitochondria and to a lesser degree in peroxisomes. Poly-unsaturated fatty acids are problematic for β-oxidation, because the enzymes directly involved are unable to process all the different double bond conformations and combinations that occur naturally. In mammals, three accessory proteins circumvent this problem by catalyzing specific isomerization and reduction reactions. Central to this process is the NADPH-dependent 2,4-dienoyl-CoA reductase. We present high resolution crystal structures of human mitochondrial 2,4-dienoyl-CoA reductase in binary complex with cofactor, and the ternary complex with NADP+ and substrate trans-2,trans-4-dienoyl-CoA at 2.1 and 1.75 Å resolution, respectively. The enzyme, a homotetramer, is a short-chain dehydrogenase/reductase with a distinctive catalytic center. Close structural similarity between the binary and ternary complexes suggests an absence of large conformational changes during binding and processing of substrate. The site of catalysis is relatively open and placed beside a flexible loop thereby allowing the enzyme to accommodate and process a wide range of fatty acids. Seven single mutants were constructed, by site-directed mutagenesis, to investigate the function of selected residues in the active site thought likely to either contribute to the architecture of the active site or to catalysis. The mutant proteins were overexpressed, purified to homogeneity, and then characterized. The structural and kinetic data are consistent and support a mechanism that derives one reducing equivalent from the cofactor, and one from solvent. Key to the acquisition of a solvent-derived proton is the orientation of substrate and stabilization of a dienolate intermediate by Tyr-199, Asn-148, and the oxidized nicotinamide.
      Essential fatty acids and derivatives that mammals acquire from exogenous sources or by endogenous biosynthesis fulfill critical functions in numerous metabolic pathways, in endocrine, and signaling processes (
      • Reddy J.K.
      • Hashimoto T.
      ,
      • Gurr M.I.
      • Harwood J.L.
      ). Fatty acids also provide much of the cells energy following degradation in a sequence of four enzyme-catalyzed reactions termed the β-oxidation cycle (
      • Reddy J.K.
      • Hashimoto T.
      ,
      • Kunau W.H.
      • Dommes V.
      • Schulz H.
      ,
      • Hiltunen J.K.
      • Qin Y.
      ), a highly exergonic metabolic process so named because oxidation occurs at Cβ of a fatty acyl-coenzyme A (CoA)
      The abbreviations used are: CoA, coenzyme A; DECR, 2,4-dienoyl-CoA reductase; mDECR, mitochondrial 2,4-dienoyl-CoA reductase; ESRF, European Synchrotron Radiation Facility; NCS, non-crystallographic symmetry; r.m.s.d., root mean square deviation; SDR, shortchain dehydrogenase/reductase; SeMet, selenomethionine.
      1The abbreviations used are: CoA, coenzyme A; DECR, 2,4-dienoyl-CoA reductase; mDECR, mitochondrial 2,4-dienoyl-CoA reductase; ESRF, European Synchrotron Radiation Facility; NCS, non-crystallographic symmetry; r.m.s.d., root mean square deviation; SDR, shortchain dehydrogenase/reductase; SeMet, selenomethionine.
      derivative preceding cleavage of the Cα–Cβ bond. The initial step leading into β-oxidation is fatty acid activation by formation of a thiolester bond with CoA in a reaction catalyzed by acyl-CoA synthetase. The oxidation of the Cα–Cβ bond produces an olefin, then hydration and oxidation produce a carbonyl group at Cβ. The fourth step is cleavage of the β-keto ester in a reverse Claisen condensation resulting in a new acyl-CoA derivative truncated by two C atoms, which are used to produce acetyl-CoA. These reactions proceed until eventually only acetyl-CoA is produced. Extensive studies have afforded a very clear picture of structure, mechanism, and specificity of the four enzymes (acyl-CoA dehydrogenase, enoyl-CoA hydratase, 3-l-hydroxyacyl-CoA dehydrogenase, and β-ketoacyl-CoA thiolase), which form this β-oxidation cycle (
      • Kim J.J.
      • Battaile K.P.
      ).
      Saturated fatty acids are simply processed in a continuous fashion by these four enzymes. However, most unsaturated fatty acids of biological origin contain cis-double bonds, and, if additional double bonds are present, they occur at three carbon intervals and so are unconjugated. Such molecules are problematic for the β-oxidation cycle, and in mammals three auxiliary enzymes are required to generate the conformation suitable for oxidation. The first such auxiliary enzyme is enoyl-CoA isomerase, the product of which can proceed to a further complete round of β-oxidation and then the initial oxidation step to produce a 2,4-dienoyl-CoA. This is a poor substrate for enoyl-CoA hydratase, and another auxiliary enzyme 2,4-dienoyl-CoA reductase (DECR) now comes into play. DECR has broad substrate specificity and can process all unsaturated fatty acids with double bonds originating at even-numbered positions and most of those with the double bonds originating at odd-numbered positions irrespective of the double bond stereochemistry (
      • Kunau W.H.
      • Dommes V.
      • Schulz H.
      ,
      • Hiltunen J.K.
      • Qin Y.
      ,
      • Kim J.J.
      • Battaile K.P.
      ,
      • Geisbrecht B.V.
      • Liang X.
      • Morrell J.C.
      • Schulz H.
      • Gould S.J.
      ). Two structurally unrelated forms of DECR are known, both of which exploit NADPH to catalyze the reduction of a 2,4-dienoyl-CoA to an enoyl-CoA. A monomeric Escherichia coli enzyme, typical of that found in bacteria, contains the prosthetic groups flavin mononucleotide and flavin adenine dinucleotide, and a 4Fe-4S cluster in a three-domain structure (
      • Hubbard P.A.
      • Liang X.
      • Schulz H.
      • Kim J.J.
      ) and produces a trans-2-enoyl-CoA, which directly enters the β-oxidation cycle.
      Our interest is in mammalian DECR, which produces a trans-3-enoyl-CoA (Fig. 1). This CoA derivative is subsequently isomerized by the third auxiliary enzyme dienoyl-CoA isomerase to trans-2-enoyl-CoA, a conformation suitable for β-oxidation. DECR activity is present in mitochondria and peroxisomes (
      • Cvetanovic M.
      • de la Garza Moreno M.
      • Dommes D.L.
      • Kunau W.H.
      ,
      • Hakkola E.H.
      • Hiltunen J.K.
      ,
      • Koivuranta K.T.
      • Hakkola E.H.
      • Hiltunen J.K.
      ,
      • Helander H.M.
      • Koivuranta K.T.
      • Horelli-Kuitunen N.
      • Palvimo J.J.
      • Palotie A.
      • Hiltunen J.K.
      ,
      • Gurvitz A.
      • Wabnegger L.
      • Yagi A.I.
      • Binders M.
      • Hartig A.
      • Ruis H.
      • Hamilton B.
      • Dawes I.W.
      • Hiltunen J.K.
      • Rottensteiner H.
      ,
      • Das A.K.
      • Uhler M.D.
      • Hajra A.K.
      ,
      • De Nys K.
      • Meyhi E.
      • Mannaerts G.P.
      • Fransen M.
      • Van Veldhoven P.P.
      ,
      • Chu X.
      • Yu W.
      • Chen G.
      • Li D.
      ). The presence of the mitochondrial reductase (mDECR) ensures that levels of CoA are not depleted to a level that could severely impair the oxidative function of that organelle. Peroxisomal DECR is more active toward longer chain substrates than mDECR (
      • De Nys K.
      • Meyhi E.
      • Mannaerts G.P.
      • Fransen M.
      • Van Veldhoven P.P.
      ); indeed a function of peroxisomal β-oxidation is to modify the longer chain fatty acids into forms readily processed by the mitochondrial enzymes. It is worth noting that, among the three auxiliary enzymes, DECR may catalyze the rate-limiting step. The Vmax of the two isomerases are in the range of several hundreds up to 1000 μmol/min/mg, whereas the Vmax of mDECR is 30 μmol/min/mg. Thus DECR may be the control site for unsaturated fatty acid oxidation. This would be consistent with the importance of the enzyme activity for human survival, because deficiency of DECR is lethal (
      • Roe C.R.
      • Miliington D.S.
      • Norwood D.L.
      • Kodo N.
      • Sprecher H.
      • Mohammed B.S.
      • Nada M.
      • Schulz H.
      • McVie R.
      ).
      Figure thumbnail gr5
      Fig. 5The catalytic center of human mDECR. A similar orientation to is employed. Atomic positions are colored pink for P, blue for N, red for O, gray for the protein C atoms, black for cofactor C, and pink for substrate C. The substrate S and a conserved water molecule are shown as black and marine spheres, respectively. Selected hydrogen bonds are depicted as black dashed lines, and the substrate Cα and Cγ atoms and nicotinamide hydride donor C4 are labeled.
      Figure thumbnail gr4
      Fig. 4The shape and size of the mDECR active site. A, the surface of subunit A is gray, that of subunit D is brown, and selected amino acids are labeled. The cofactor and substrate are depicted as stick models, colored black and pink, respectively. The cyan chicken wire is an omit difference density map calculated with coefficients (Fo - Fc) and αc and contoured at 2σ. Atoms of the aliphatic tail did not contribute to Fc or αc. B, residues involved in binding NADP+ and trans-2,trans-4-hexadienoyl-CoA in the mDECR active site. A similar view to the top panel has been selected. The substrate is colored pink, the S atom is depicted as a black sphere, and the cofactor is black. For the protein structure, C atomic positions are gray, N blue, and O red, and water molecules are marine colored spheres. Note that Phe-311′, Glu-326′, and Glu-310′ are from the adjacent D subunit. Hydrogen bonding interactions have been omitted for the purpose of clarity.
      Figure thumbnail gr1
      Fig. 1The type of reaction catalyzed by mammalian 2,4-dienoyl-CoA reductase.
      Eukaryotic DECR belongs to the Short-chain Dehydrogenase/Reductase (SDR) superfamily of which there are over 3000 members with sequence identities at the 15–30% level between distinct enzymes (
      • Oppermann U.
      • Filling C.
      • Hult M.
      • Shafqat N.
      • Wu X.
      • Lindh M.
      • Shafqat J.
      • Nordling E.
      • Kallberg Y.
      • Persson B.
      • Jörnvall H.
      ,
      • Kallberg Y.
      • Oppermann U.
      • Jörnvall H.
      • Persson B.
      ,
      • Duax W.L.
      • Pletnev V.
      • Addlagatta A.
      • Bruenn J.
      • Weeks C.M.
      ). Family members display an enormous spread of substrate specificity regulating diverse biological processes. Crystal structures of SDR family members have delineated seven motifs that are important for the protein structure, the recognition, binding, and orientation of the cofactor (NADH or NADPH) and catalysis (
      • Oppermann U.
      • Filling C.
      • Hult M.
      • Shafqat N.
      • Wu X.
      • Lindh M.
      • Shafqat J.
      • Nordling E.
      • Kallberg Y.
      • Persson B.
      • Jörnvall H.
      ,
      • Kallberg Y.
      • Oppermann U.
      • Jörnvall H.
      • Persson B.
      ,
      • Duax W.L.
      • Pletnev V.
      • Addlagatta A.
      • Bruenn J.
      • Weeks C.M.
      ,
      • Tanaka N.
      • Nonaka T.
      • Tanabe T.
      • Yoshimoto T.
      • Tsuru D.
      • Mitsui Y.
      ,
      • Tanaka N.
      • Nonaka T.
      • Nakanishi M.
      • Deyashiki Y.
      • Hara A.
      • Mitsui Y.
      ,
      • Filling C.
      • Berndt K.D.
      • Benach J.
      • Knapp S.
      • Prozorovski T.
      • Nordling E.
      • Ladenstein R.
      • Jörnvall H.
      • Oppermann U.
      ,
      • Shi R.
      • Lin S.X.
      ). Human mDECR carries five of these motifs that are implicated in aspects of structure stability and cofactor binding but lacks two that occur in the catalytic site. DECR, though similar, is distinct from other enoyl-thiolester reductases, because it catalyzes an unusual addition onto a conjugated diene, whereas the other enzymes are generally involved in fatty acid elongation in which they reduce a 2,3 (or α,β) double bond. A detailed, elegant biochemical and nuclear magnetic resonance study of rat liver mDECR by Fillgrove and Anderson (
      • Fillgrove K.L.
      • Anderson V.E.
      ) identified that the stereochemical course of the reduction is novel and that a specific lysine is essential for catalysis. In classic SDRs this lysine occurs in combination with a tyrosine in an active site YXXXK motif (where X is any amino acid) but in enoyl reductases the motif is YXXMXXXK. In the amino acid sequence of mDECR the nearest tyrosine is some 14 residues on the N-terminal side of the essential lysine. A serine is generally in direct association with the catalytic tyrosine, but analysis of mDECR sequences does not reveal a candidate for this residue. Structural information was sought to identify the “ubiquitous” tyrosine-serine pair or indeed to identify if different residue types contribute to the function of DECR.
      The gene for human mDECR encodes a polypeptide of 335 amino acids, but it is thought likely that the mature enzyme is truncated by removal of a mitochondrial targeting sequence (
      • Koivuranta K.T.
      • Hakkola E.H.
      • Hiltunen J.K.
      ). We have characterized a truncated version of recombinant human mDECR lacking the N-terminal 34 residues, which is active (
      • Chu X.
      • Yu W.
      • Chen G.
      • Li D.
      ) and provides highly ordered crystals. Molecular replacement calculations were unsuccessful, and so phases were first obtained from a selenomethionine (SeMet) derivative. Subsequently, we obtained a ternary complex structure and in conjunction with site-directed mutagenesis and kinetic analyses were able to test ideas concerning the contributions of specific residues to enzyme activity. We now detail the enzyme structure, the determinants of cofactor and substrate recognition, the identification of key residues, and their contributions to mDECR function.

      MATERIALS AND METHODS

      Sample Preparation and Crystallization—The plasmid carrying the gene encoding truncated mDECR with an N-terminal His6 tag was heat-shock transformed into a bacterial expression system (E. coli BL21(DE3), Novagen) and used according to established methods (
      • Chu X.
      • Yu W.
      • Chen G.
      • Li D.
      ). To prepare SeMet mDECR the plasmid was transformed into the methionine auxotrophic strain of E. coli, B834(DE3) (Novagen), and selected on Luria-Bertani agar plates containing 100 μg ml-1 ampicillin. Bacteria were cultured in M9 minimal media containing SeMet and expression induced at mid-log phase with 0.4 mm isopropyl-β-d-thiogalactopyranoside, and cell growth continued overnight at 25 °C. Cells were harvested by centrifugation (2,500 × g, 4 °C for 10 min), resuspended in binding buffer (50 mm potassium phosphate, pH 7.4, 500 mm NaCl, 5 mm imidazole), and, following the addition of deoxyribonuclease-I and lysozyme, were lysed using a One-shot Cell Disrupter (Constant Systems). Insoluble debris was separated by centrifugation (27,000 × g, 4 °C for 20 min), and the supernatant was passed through a 0.2-μm syringe filter and applied to a Ni2+-resin column (Amersham Biosciences HiTrap). Following a wash with 50 mm potassium phosphate and 10 mm imidazole, pH 7.4, the product was eluted with a linear imidazole gradient from 0 to 500 mm. Fractions were analyzed by SDS-PAGE, and those containing mDECR were pooled and dialyzed overnight against 50 mm potassium phosphate, pH 7.4, 100 mm NaCl, 5 mm β-mercaptoethanol, and 0.1 mm EDTA. The sample was then subjected to anion exchange chromatography on a 6-ml Resource Q column (Amersham Biosciences). Fractions containing mDECR were again pooled and dialyzed overnight in 50 mm potassium phosphate, pH 7.4, 5 mm β-mercaptoethanol, and 0.1 mm EDTA then concentrated to ∼10 mg ml-1 for crystallization. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry and SDS-PAGE confirmed that highly purified enzyme had been obtained, and SeMet was fully incorporated in the derivative.
      Isomorphous crystals of native and SeMet mDECR in complex with NADP+ and in ternary complex with substrate grew under similar conditions. Monoclinic blocks with space group P21, of dimensions ∼0.4 × 0.2 × 0.1 mm3, appeared overnight from a drop consisting of 1 μl of protein solution, 1 μl of reservoir (16% polyethylene glycol 4000 w/v, 180 mm ammonium sulfate, 80 mm sodium acetate (pH 4.6) to which had been added 20% glycerol, 4 mm NADP+), and 0.5 μl of 30% ethylene glycol. A ternary complex of mDECR with the oxidized cofactor and a substrate was prepared by incubating the protein solution with 4 mm NADP+ and 120 mm trans-2,trans-4-hexadienoyl-CoA (50-fold excess) for several hours prior to crystallization. All crystals were placed in a solution of artificial mother liquor containing 10% ethylene glycol for ∼10 s and flash-cooled in a stream of nitrogen gas at -170 °C, characterized using in-house facilities, and then transported to the European Synchrotron Radiation Facility (ESRF), Grenoble, France.
      Data Collection and Processing—Diffraction data were measured from SeMet mDECR in binary complex with NADP+ at a wavelength selected to maximize the anomalous dispersion from selenium, and the data (ESRF beam line ID29, Dataset 1, Table I) were recorded on an ADSC Quantum210 charge-coupled device detector. Another crystal of the SeMet protein, grown in a solution containing the reaction product (3-hexenoyl-CoA) and NADP+, provided Dataset 2 from beam line ID14 EH2 using an ADSC Quantum Q4 charge-coupled device detector. Subsequent analysis showed that the reaction product was not ordered, and this therefore represents a binary complex similar to that produced from Dataset 1, which, for completeness sake, is detailed in Table I. Data from a ternary complex of native mDECR (Dataset 3) were also measured on beam line ID14 EH2. The data were indexed, integrated, and scaled using either MOSFLM (
      • Leslie A.G.W.
      ) or DENZO and SCALEPACK (
      • Otwinowski Z.
      • Minor W.
      ). Data manipulation was achieved using the CCP4 suite of programs (
      Collaborative Computational, Project 4
      ).
      Table IProcessing and refinement statistics for human mDECR
      Dataset 1 SeMet binary complexDataset 2 SeMet binary complexDataset 3 native ternary complex
      PDB code1w731w8d1w6u
      Space group P21 with unit cell dimensions
         a (Å)62.9562.8763.28
         b (Å)131.74131.70131.66
         c (Å)71.3070.5970.85
      b (°)92.592.692.6
      Resolution range (Å)/wavelength (Å)70.0-2.1/0.979170.0-2.2/0.975630.0-1.75/0.9756
      No. reflections252,983212,544388,390
      No. unique reflections/redundancy67,331/3.856,224/3.8110,749/3.3
      Wilson B (Å2)32.339.026.7
      Completeness (%)99.7 (98.8)
      Numbers in parenthesis refer to the highest resolution shell
      96.9 (96.9)95.5 (90.8)
      I/σI8.0 (3.2)11.9 (1.9)23.1 (2.2)
      Rsym (%)6.3 (20.8)8.5 (50.9)5.1 (58.8)
      Ranom (%)7.2 (15.8)6.1 (30.5)
      Residues/atoms1,126/8,5661,126/8,5661,135/8,616
      Water molecules533219582
      Rwork (%)/no. observations18.8/61,87420.9/53,78122.7/105,117
      Rfree (%)/no. observations22.6/330225.5/2,93426.8/5,590
      Average isotropic thermal parameters (Å2)
         Overall/main chain/side chain28.7/27.4/30.145.1/44.2/46.233.2/33.4/34.2
         Waters/cofactor/substrate35.7/38.0/-41.9/51.3/-48.1/35.8/81.1
         r.m.s. bond lengths (Å)/angles (°)0.009/1.20.009/1.20.007/1.15
         r.m.s. planarity (Å)/G-factor0.003/0.10.003/0.10.013/0.1
      a Numbers in parenthesis refer to the highest resolution shell
      SAD Phasing, Structure Solution, Model Building, and Refinement—25 of the 28 selenium positions in the asymmetric unit were identified using the program SOLVE (
      • Terwilliger T.C.
      • Berendzen J.
      ) and used for phase calculation, giving a figure-of-merit of 0.35 to 2.1 Å resolution. Density modification and histogram matching with non-crystallographic symmetry (NCS) averaging using the program DM (
      • Cowtan K.
      ) increased the figure-of-merit to 0.76 resulting in an electron density map of excellent quality (Fig. 2A). The programs ARPwARP (
      • Perrakis A.
      • Morris R.
      • Lamzin V.S.
      ) and RESOLVE (
      • Terwilliger T.C.
      ) in combination produced a polyalanine model of mDECR onto which side chains were assigned using O (
      • Jones T.A.
      • Zou J.Y.
      • Cowan S.W.
      • Kjeldgaard M.
      ). A subset of data (5%) was set aside for the calculation of Rfree (
      • Brünger A.T.
      ) and used as a guide for refinement protocols. The Hendrickson-Lattman coefficients derived from the phasing calculations and the NCS were included as restraints in the early stages of refinement. Rounds of refinement with REFMAC5 (
      • Murshudov G.N.
      • Vagin A.A.
      • Dodson E.J.
      ) combined with map inspection and manual intervention, the addition of water molecules and cofactor completed the model of the binary complex. The structure of the ternary complex was solved by rigid body refinement of the SeMet protein model and completed with similar refinement procedures. Several residues were truncated to alanine on the basis that the side-chain density was ill defined, and multiple side-chain conformations were also modeled for several residues. Full details are provided in the deposited coordinates (Table I). The figures were prepared with Molscript (
      • Kraulis P.J.
      ), Raster3D (
      • Merrit E.A.
      • Bacon D.J.
      ), and PyMOL (
      • DeLano W.L.
      ).
      Figure thumbnail gr2
      Fig. 2A, the experimentally phased electron density map (light brown chicken wire) contoured at 1.5σ covering key residues in active site A together with the refined model (Dataset 1, binary complex). Note that the initial phases were sufficiently accurate to identify well ordered water molecules. B, an omit difference density map (blue chicken wire) for NADP+ (stick model) in the ternary complex, calculated with coefficients (Fo - Fc), αc, and contoured at the 2σ level. Fo and Fc represent the observed and calculated structure factors respectively, αc the calculated phases. The atoms shown here (gray represents C, blue N, red O, and yellow P) did not contribute to Fc or αc. The dual conformation of the α-phosphate is evident.
      Construction of Human mDECR Mutants—The Plasmid Mini kit and synthetic oligonucleotides were purchased from Tech Dragon Co. of Hong Kong. A QuikChange mutagenesis kit (Stratagene) in conjunction with the plasmid pLM1::HDER (
      • Chu X.
      • Yu W.
      • Chen G.
      • Li D.
      ) as template was used to construct the N148A, T197A, Y199A, S210A, K214A, E310A, and E310Q mutant expression plasmids. The following primers and their antisense primers (not shown) were used to introduce the mutated sequence: 1) g ata aac aat gca gca ggg gct ttt att tct cct act gaa ag (N148A); 2) gca gca ttt ctt tct att act gct atc tat gct gag act gg (T197A); 3) ctt tct att act act atc gct gct gag act ggt tca gg (Y199A); 4) ca ggt ttt gta gta cca gct gct tct gcc aaa gca gg (S210A); 5) cca agt gct tct gcc gca gca ggt gtg gaa gcc (K214A); 6) gta ctt att tca ggg gca ttc aac gac ctg ag (E310A); and 7) gta ctt att tca ggg caa ttc aac gac ctg ag (E310Q).
      PCR amplification was performed using Pfu DNA polymerase, and samples were subjected to 13 cycles of 0.5 min of denaturation at 95 °C, 1 min of annealing at 60–63 °C, and 12 min of elongation at 72 °C in a Mastercycler (Eppendorf). The mutant-carrying plasmid was transformed into E. coli HB101 competent cells (Novagen) by electroporation (Bio-Rad) for screening purposes. Positive clones were identified, and the DNA sequenced to verify the presence of the desired mutations and the absence of any PCR-generated random mutations. Plasmids were then transformed in E. coli strain BL21(DE3) cells for expression purposes.
      Expression and Purification of Human mDECR Wild-Type and Mutant Proteins—Established methods were again used to prepare the samples (
      • Chu X.
      • Yu W.
      • Chen G.
      • Li D.
      ). The proteins, all observed with >95% purity by SDS-PAGE, were stored at -80 °C in 50 mm potassium phosphate buffer, pH 7.5, 0.1 mm EDTA, 5% glycerol, and 5 mm β-mercaptoethanol.
      Activity Assay and Kinetic Studies—The trans-2,trans-4-hexadienoyl-CoA thiolester was synthesized from the corresponding free acid and CoA by the mixed anhydride method and purified by reverse-phase high-performance liquid chromatography. The assay mixture contained 50 mm potassium phosphate, pH 6.0, 100 μm EDTA, 125 μm NADPH, and 1 μg of enzyme in a final volume of 1.0 ml. The mixtures were preincubated for 20 min at room temperature, reactions were initiated by the addition of 40 μm trans-2,trans-4-hexadienoyl-CoA, and the decrease in absorbance at 340 nm was monitored for 60 s. One enzyme unit was defined as the activity that converts 1 μmol of trans-2,trans-4-hexadienoyl-CoA to trans-3-hexenoyl-CoA per minute. Determination of Km and the Vmax values was performed using the same assay buffer with substrate concentrations ranging from 1 to 100 μm (
      • Chu X.
      • Yu W.
      • Chen G.
      • Li D.
      ). Measurements were made at five substrate concentrations, and averages of two assays were used for each point. Results are given in Table II.
      Table IIKinetic data for human mDECR
      SampleKm for substrateVmaxActivityKm for NADPH
      μmμmol/min/mg%μm
      Wild-type14.3 ± 2.730.3 ± 2.51007.7 ± 1.1
      N148A4.2 ± 2.00.93 ± 0.14323.5 ± 2.1
      T197A11.6 ± 2.55.6 ± 0.51941.4 ± 1.2
      Y199A28.5 ± 22.60.3 ± 0.1145.0 ± 13
      S210A54.0 ± 32.00.16 ± 0.060.581.0 ± 39
      K214A8.1 ± 3.20.0096 ± 0.00120.035.7 ± 0.6
      E310A40.6 ± 12.414.5 ± 1.94821.4 ± 2.4
      E310Q40.0 ± 18.028.4 ± 5.2949.7 ± 1.5

      RESULTS AND DISCUSSION

      General Comments and Quality of the Structures—The binary structure of recombinant human mDECR in complex with NADP+ was solved by exploiting the anomalous dispersion signal from SeMet. An electron density map of excellent quality resulted from the experimentally derived phases (Fig. 2A). This structure provided the starting model for the analysis of the isomorphous DECR·NADP+·substrate ternary complex. The structures were refined satisfactorily using high resolution data, and the resulting models display good stereochemistry as indicated by analysis with PROCHECK (
      • Laskowski R.A.
      • MacArthur M.W.
      • Moss D.S.
      • Thornton J.M.
      ); details are presented in Table I. Over 90% of residues in each structure are in favorable regions with respect to φ/Ψ combinations in Ramachandran plots (not shown). There is a single outlier, residue 254 in subunit A of the ternary complex, that is near a break in the electron density maps. Several side chains were modeled and satisfactorily refined as dual conformers, and one phosphate of NADP+ was also modeled in two conformations, both of which are staggered with respect to the adjacent phosphate group (Fig. 2B). The aliphatic tail of the substrate trans-2,trans-4-dienoyl-CoA was only added at a late stage of the ternary complex analysis, and map interpretation was greatly aided by being able to position the sulfur atom and carbonyl group. However, the electron and difference density maps remain noisy in this area, and the isotropic thermal parameters (or B-factors, where B = 8π22, and is the mean displacement of atoms along the normal to the reflecting planes) are higher than the surrounding protein atoms suggesting a degree of flexibility and/or conformational disorder at the catalytic center. This will be discussed later. Several residues at the N and C termini, and a flexible loop in each subunit, near the active site, are disordered, and not included in the models. It is common for this particular loop to be disordered in SDR structures, although in rare circumstances the formation of a ternary complex leads to an ordering of the residues (
      • Tanaka N.
      • Nonaka T.
      • Tanabe T.
      • Yoshimoto T.
      • Tsuru D.
      • Mitsui Y.
      ). Secondary structure elements were assigned by automated methods, using PROMOTIF (
      • Hutchinson E.G.
      • Thornton J.M.
      ) and visual inspection, and are mapped onto the amino acid sequence in Fig. 3A. Although studying a truncated version of human mDECR, we used a numbering scheme consistent with that of the full-length protein.
      Figure thumbnail gr3
      Fig. 3A, the amino acid sequence of the truncated mDECR with the assignment of secondary structure. Strands are labeled β1 to β7, helices α1 to α11, and 310-helical segments θ1 to θ3. B, ribbon diagram of subunit A. The N and C termini are labeled; β-strands are represented as cyan arrows, α-helices and 310-helical segments as blue and purple ribbons, respectively. Elements of secondary structure are numbered according to the top panel. NADP+ and the substrate trans-2,trans-4-hexadienoyl-CoA are depicted as stick models and colored blue and green, respectively. C, the homotetramer with individual subunits colored separately; substrate and cofactor are shown as stick models. Subunit A is red and oriented in similar view as in the middle panel.
      The asymmetric unit comprises a functional tetramer with subunits labeled A, B, C, and D. The refined model of the ternary complex comprises residues 35–245 and 252–328 for subunit A, 34–245 and 256–329 for subunit B, 36–249 and 256–328 for subunit C, and 34–245 and 258–327 for subunit D. The root mean square deviation (r.m.s.d.) of 1133 Cα atoms common to the binary and ternary complex structures is ∼0.28 Å. Overlays of individual subunits produce r.m.s.d. values in the narrow range 0.35–0.44 Å for between 278 and 286 Cα atoms positions, respectively. Visual inspection of the superimposed molecules (not shown) confirms this high degree of structural conservation between binary and ternary complexes, between all subunits, the positions of side chains, the conformations of NADP+ and, with few exceptions, substrate and interactions with mDECR and even numerous well conserved water molecules. Such consistency between the different complexes suggests the enzyme does not undergo large conformational changes upon binding and processing substrate and that it is only necessary to describe the ternary complex and one enzyme active site. A few specific differences between subunits with respect to protein-substrate interactions will be mentioned later.
      Architecture of the mDECR Subunit and Functional Tetramer—The fold of the mDECR subunit is typical of the classic SDR family (Fig. 3B), a single α/β domain with a dinucleotide-binding “Rossmann” fold where a seven stranded β-sheet (strands β1–7) is sandwiched between six α-helices, three on each side (α4, α5, and α7; and α2, α3, and α9). A C-terminal extension from the Rossmann fold comprises α10 and α11 on either side of a 310-helix (θ3) to complete the structure. This extension contributes to subunit-subunit associations that form the functional tetramer and not a distinct sub-domain used to bind substrate as observed in the extended subgroup of the SDR family (
      • Kallberg Y.
      • Oppermann U.
      • Jörnvall H.
      • Persson B.
      ).
      Like many SDR family members, mDECR is a homotetramer (Fig. 3C) with 222-point group symmetry resulting in a rectangular block of approximate dimensions 75 × 75 × 80 Å. Each subunit interacts with the three partners using three types of inter subunit interface. Subunits A and B are related by an NCS 2-fold axis of symmetry near α9. This 2-fold axis also runs alongside β7 of each monomer, creating a twisted 14-stranded, anti-parallel β-sheet running the length of A and B. The AB association forms the largest area of interaction covering ∼2330 Å2. This AB (or the equivalent CD) interface, which is mainly non-polar, constitutes 15% of the total surface of a subunit and is stabilized by ten hydrogen bonds provided by residues from the loops between α8 and α9, and α9 and β7, along with interactions from residues in the secondary structure elements β7, θ2, and α9. An additional hydrogen bond is observed between subunit A Ser-45 O and subunit B Ser-290 Oγ (not shown).
      Subunits A and C are related by an NCS 2-fold axis of symmetry that runs between, and perpendicular to, α5 and α7 of each subunit. The interface surface area here is 1510 Å2, or 10% of a subunit surface area. A total of 20 hydrogen bonds in three regions, involving residues from α5, α6, α7, and θ1, stabilize this interface. Subunits A and D are related by a 2-fold NCS axis of symmetry between and perpendicular to α10. A total of eight hydrogen bonds involving residues from α6, α8, α10, and θ1 help to maintain this interface, which has an accessible surface area of about 1340 Å2, or 9% of a subunit surface area.
      The cofactor and substrate-binding sites from diagonally opposing subunits (A to D and B to C) are situated on the same side of the tetramer and separated by only about 30 Å (Fig. 3C). There is no evidence to implicate this quaternary structure in the mechanism of catalysis, rather, as will be described, oligomerization creates part of the CoA binding and active sites and serves to localize four catalytic centers.
      Cofactor Binding—NADP+ binds in an extended conformation in an L-shaped depression 28 Å long, 22 Å wide, and 15 Å deep, created by the C-terminal ends of the β-strands in the Rossmann fold (Figs. 3B and 4A). The pattern of hydrogen bonds formed between NADP+ and SDR family members is in general well conserved (
      • Tanaka N.
      • Nonaka T.
      • Tanabe T.
      • Yoshimoto T.
      • Tsuru D.
      • Mitsui Y.
      ). In Fig. 4B the residues that form the binding site for cofactor and substrate are shown, and potential hydrogen bonding interactions are listed in Table III. In mDECR the cofactor participates in 15 conventional hydrogen bonding interactions with residues that line the binding site (Table III); six involve main-chain and nine side-chain groups. In addition, there are hydrogen bonds with five ordered water molecules, which in turn interact with functional groups of the protein. One of these water molecules mediates a link from the cofactor adenine to the β-phosphate of substrate.
      Table IIIPotential hydrogen bonding contacts (≤3.5 Å) formed by the cofactor (NADP+) and substrate (trans-2,trans-4-dienoyl CoA) in enzyme (mDECR) active site A
      CofactorEnzyme/solventSubstrateEnzyme/solvent
      Adenine N6Oδ1 Asp-117Adenine N6Oϵ2 Glu-326 subunit D
      Adenine N6WaterAdenine N1Oϵ2 Glu-326 subunit D
      Adenine N7WaterAdenine N1Water
      Adenine N1N Val-118Adenine O2′Oγ Ser-157
      Adenine O2′WaterAdenine O1POγ Ser-157
      Adenine O1PNη2 Arg-91Adenine O3′Water
      Adenine O1PNξ Lys-92β-phosphate O3PWater
      Adenine O2POγ Ser-90CoA OHWater
      Adenine O2PN Lys-92CoA carbonyl 1Cα Gly-147
      C-H ... O interactions
      Adenine O3PN Arg-91CoA amide 1Water
      Adenine O3PNϵ Arg-91CoA carbonyl 2Water
      Adenine O3′WaterCoA carbonyl 2Water
      Adenine O3′Oγ1 Thr-69CoA S1O2′ nicotinamide ribose
      α-Phosphate O2POγ1 Thr-69CoA S1Water
      β-Phosphate O2PN Leu-71CoA carbonyl 3Oη Tyr-199
      Nicotinamide O3′O Asn-144
      Nicotinamide O3′Nξ Lys-214
      Nicotinamide O2′Nξ Lys-214
      Nicotinamide O2′S1 CoA
      Nicotinamide C4O Gly-241
      C-H ... O interactions
      Nicotinamide C5O Pro-240
      C-H ... O interactions
      Nicotinamide C5Water
      C-H ... O interactions
      Nicotinamide C6O Thr-196
      C-H ... O interactions
      Nicotinamide O7N Ile-243
      a C-H ... O interactions
      The adenine moiety, in anti conformation with respect to the ribose, is placed in a well defined cleft sandwiched between strands β3 and β4 lying on a hydrophobic bed created by the side chains of Val-118, Ala-145, and Ile-167 and covered by the side chains of Arg-91 and Lys-92. The side chain of Asp-117 is held in place to bind the adenine by hydrogen bonds donated from the side chain and amide group of Arg-119. Five hydrogen bond associations involve the adenine 2′-phosphate and contribute significantly to cofactor binding. Two basic residues, Arg-91 and Lys-92, bind this phosphate and are recognized as a principal factor in discrimination for NADPH utilization over NADH in the SDR family (
      • Tanaka N.
      • Nonaka T.
      • Nakanishi M.
      • Deyashiki Y.
      • Hara A.
      • Mitsui Y.
      ). Hydrogen bonds are formed between adenine N1 and the amide group of Val-118, between adenine N6 and the side chain of Asp-117, and the ribose O3 with Thr-69 Oγ (not shown).
      The pyridine nucleotide adopts a syn conformation with respect to the ribose with a single intramolecular hydrogen bond formed between the N7 amide and one of the phosphate oxygen atoms. Residues contributed from β4 and α5 line the back wall of the pocket where the nicotinamide binds, and residues from loop β1-α2 interact with the cofactor. The model is incomplete between β6 and α8, at one side of the catalytic center, due to flexibility of this loop. Interactions made by the nicotinamide moiety with the protein include hydrogen bonds from the ribose to the carbonyl group of Asn-144 and the side chain of Lys-214 (Table III). A hydrogen bond is accepted by the nicotinamide carboxamide from the amide of Ile-243. Approximately 25% of SDR family members have isoleucine at this position (
      • Duax W.L.
      • Pletnev V.
      • Addlagatta A.
      • Bruenn J.
      • Weeks C.M.
      ). Nearby, residues from the C-terminal extension of a partner subunit, in particular Glu-310 and Phe-311, create a wall on one side of the catalytic center near a key residue Tyr-199 (Figs. 4B and 5).
      Three C-H · · · O hydrogen bonds involve C4, C5, and C6 of the nicotinamide interacting with the carbonyl oxygen atoms of Gly-241, Pro-240, and Thr-196, respectively (Table III). Though weak (
      • Leonard G.A.
      • McAuley-Hecht K.
      • Brown T.
      • Hunter W.N.
      ), these interactions will contribute to the association of protein with cofactor, help to align the nicotinamide to facilitate hydride transfer from C4, and may even contribute to the formation of the transition state during catalysis. The presence of the C4 and C5 C-H · · · O hydrogen bonds in SDR cofactor complexes has been observed previously (
      • Duax W.L.
      • Pletnev V.
      • Addlagatta A.
      • Bruenn J.
      • Weeks C.M.
      ,
      • McLuskey K.
      • Gibellini F.
      • Carvalho P.
      • Avery M.A.
      • Hunter W.N.
      ), but the interaction involving C6 has not.
      Substrate Binding and Broad Substrate Specificity—The substrate, trans-2,trans-4-hexadienoyl-CoA, binds with the CoA moiety on the surface of the enzyme (Fig. 4) with the adenine positioned near the N-terminal region of α5 nestling down on Phe-311 and forming a hydrogen bond with the side chain of Glu-326, both from a partner subunit. The rest of the substrate trails over β4, running roughly parallel with the cofactor and positions the aliphatic tail down in the catalytic center. The β-phosphate of CoA is about 3.7 Å distant from Arg-119, which binds the cofactor adenine.
      The substrate participates in eight hydrogen bonding interactions with water molecules and five (in subunit A, Table III) that are direct with side-chain functional groups. Of note is a hydrogen bond formed between the substrate S and the 2′-ribose hydroxyl of the nicotinamide cofactor (Fig. 5). In subunits A, B, and C the carbonyl group of the thiolester tail accepts a hydrogen bond donated by Tyr-199 Oη and is between 3.7 and 3.9 Å distant from Asn-148 Nδ2. In subunit D, which has the least well defined substrate tail, the distance is nearly 5 Å. The substrate does not form a large number of strong interactions with the enzyme, and this suggests a relatively loose binding, which may contribute to the efficient release of product following catalysis.
      The enzyme reduces a wide range of substrates of varying lengths of aliphatic tail and with different stereochemical conformations. The active site pocket is large enough to accommodate substrates with chain lengths exceeding the six carbons of the derivative used in this analysis and with different conformations. At one side of the catalytic center there is the flexible active site loop, which presumably is able to facilitate binding of these longer substrates. The aliphatic tail of trans-2,trans-4-hexadienoyl-CoA displays B-factors of between 77 and 85 Å2, which, together with noisy difference density maps in these areas, suggests a high degree of conformational freedom of the substrate itself. Such flexibility is likely to also contribute to the broad range of CoA derivatives processed by mDECR.
      The Catalytic Center and Comparisons with SDRs—Catalysis occurs at one end of the NADPH binding site, above the nicotinamide C4 and near Tyr-199 (Figs. 3 and 5). A tetrad of spatially conserved active site residues (a serine, a tyrosine, lysine, and asparagines) occurs in many of the classic SDR family members suggestive of a conserved reaction mechanism (
      • Filling C.
      • Berndt K.D.
      • Benach J.
      • Knapp S.
      • Prozorovski T.
      • Nordling E.
      • Ladenstein R.
      • Jörnvall H.
      • Oppermann U.
      ). An architectural comparison, using DALI (
      • Holm L.
      • Sander C.
      ) was used to identify and align close SDR homologues. The most similar homologue was 3-hydroxyacyl-CoA dehydrogenase (Protein Data Bank (PDB) code 1E6W (
      • Berman H.M.
      • Westbrook J.
      • Feng Z.
      • Gilliland G.
      • Bhat T.N.
      • Weissig H.
      • Shindyalov I.N.
      • Bourne P.E.
      ,
      • Powell A.J.
      • Read J.A.
      • Banfield M.J.
      • Gunn-Moore F.
      • Yan S.D.
      • Lustbader J.
      • Stern A.R.
      • Stern D.M.
      • Brady R.L.
      )) with a Z-score of 30.8 and an r.m.s.d. of 1.6 Å for 251 Cα atoms. Two other enzymes of note are pteridine reductase (1E7W; z score of 29.1 (
      • Gourley D.G.
      • Schüttelkopf A.W.
      • Leonard G.A.
      • Luba J.
      • Hardy L.W.
      • Beverley S.M.
      • Hunter W.N.
      )) with an r.m.s.d. of 2.1 Å for 268 Cα atoms and enoyl-acyl carrier protein reductase (1ENY; Z-score of 25.3 (
      • Rozwarski D.A.
      • Vilchèze C.
      • Sugantino M.
      • Bittman R.
      • Sacchettini J.C.
      )) with an r.m.s.d. of 2.6 Å also for 268 Cα atoms. The Z-score is a measure of the statistical significance of the best domain-domain alignment and was determined by DALI (
      • Holm L.
      • Sander C.
      ). Typically, two dissimilar proteins will have a Z-score < 2.0. These alignments revealed that in mDECR the organization of key functional groups at the catalytic center is distinct from other SDR family members of known structure.
      Lys-214, strictly conserved in all SDR active sites, donates hydrogen bonds to O2 and O3 of the nicotinamide ribose (Fig. 5). This active site lysine is thought to lower the pKa of the catalytic tyrosine and stabilize the transition state. In human mDECR, the distance between Tyr-199 Oη and Lys-214 Nζ is almost 7.0 Å with the substrate carbonyl and nicotinamide 2′-hydroxyl groups between them. Nearby, however, Tyr-199 and Ser-210 occupy positions that differ from all other SDRs. Tyr-199 is more buried and in a different orientation compared with the catalytic tyrosine of other SDRs (Fig. 6). Ser-210 of mDECR occupies the position equivalent to the Cβ and Cγ atoms of the catalytic Tyr-168 of an example SDR, namely 3-hydroxyacyl-CoA dehydrogenase. The position normally occupied by the catalytic serine in other SDR structures (Ser-155, Fig. 6) is, replaced by mDECR Thr-197. Asn-148 Nδ2 is 4.3 and 3.4 Å away from Ser-210 Oγ and Tyr-199 Oη, respectively. Filling et al. (
      • Filling C.
      • Berndt K.D.
      • Benach J.
      • Knapp S.
      • Prozorovski T.
      • Nordling E.
      • Ladenstein R.
      • Jörnvall H.
      • Oppermann U.
      ) report that an asparagine is part of a “catalytic tetrad” used by the SDR family and that its main chain interacts with an active site water molecule serving to form a proton relay system. The residue they discuss is equivalent to Asn-143 not the important Asn-148 of mDECR (not shown).
      Figure thumbnail gr6
      Fig. 6Comparison of key active site residues of human mDECR with those of 3-hydroxyacyl-CoA dehydrogenase. A similar coloring scheme to is employed. The dehydrogenase model is shown as thin transparent lines and blue labels.
      Tyr-199 has one of the most ordered side chains in the structure exhibiting average B-factors of 19.5, 21.5, 22.3, and 23.0 Å2 in subunits A, B, C, and D, respectively. For comparison the average side-chain B-factor in the ternary complex is 33.6 Å2. The hydroxyl group of this residue accepts a hydrogen bond donated by Ser-210 and donates one to the carbonyl of the substrate. The benzyl group is cradled by Thr-197 and Val-207 (not shown) below and above, respectively, with a bulky Phe-311 from a partner subunit at the side. The result is that the phenolic group of Tyr-199 is precisely positioned ready to participate in the enzyme mechanism and unlikely to undergo any major conformational changes.
      Mechanistic Considerations—Knowledge of the ternary complex structure enabled us to make informed decisions when targeting specific residues by site-directed mutagenesis and kinetic analysis to investigate specific contributions to enzyme reactivity (Table II). This information facilitates a description of the enzyme mechanism and comments on the contributions of key residues.
      Short-chain reductases mainly catalyze the reduction of C=O, C=N, and less commonly, as in mDECR, C=C double bonds. The mechanism proposed for such a reduction can be described in two parts. Firstly, hydride transfer from a nicotinamide cofactor to a carbon atom of the substrate occurs and secondly the substrate acquires a solvent-derived proton (
      • Kallberg Y.
      • Oppermann U.
      • Jörnvall H.
      • Persson B.
      ,
      • Duax W.L.
      • Pletnev V.
      • Addlagatta A.
      • Bruenn J.
      • Weeks C.M.
      ,
      • Tanaka N.
      • Nonaka T.
      • Tanabe T.
      • Yoshimoto T.
      • Tsuru D.
      • Mitsui Y.
      ,
      • Fillgrove K.L.
      • Anderson V.E.
      ,
      • Gourley D.G.
      • Schüttelkopf A.W.
      • Leonard G.A.
      • Luba J.
      • Hardy L.W.
      • Beverley S.M.
      • Hunter W.N.
      ). The first part of the mechanism involves cofactor binding and orientation of the nicotinamide to create the floor of the catalytic site. Once the substrate is correctly positioned the pyridine nucleotide cofactor donates the C4 pro-4S hydride to Cδ, the electrophilic carbon of the conjugated thiolester (
      • Fillgrove K.L.
      • Anderson V.E.
      ). The ternary complex structure places the hydride donor C4 3.5 Å from the substrate Cδ, in the correct orientation to accept the pro-4S hydride (Fig. 5).
      Short-chain reductases depend on one reducing equivalent provided by a water molecule, and the second part of the DECR-catalyzed reduction is addition of such a proton to Cα (
      • Fillgrove K.L.
      • Anderson V.E.
      ). There is no obvious side chain in the active site to donate H+ directly to Cα, and we note that human mDECR has optimum activity near pH 6 (
      • Chu X.
      • Yu W.
      • Chen G.
      • Li D.
      ). In many cases, (e.g. 24), networks of water molecules and extensive proton relay systems, which are tucked under the conserved lysine, are invoked to try and explain how a proton is acquired in the second stage of the reduction. This theory does not seem necessary or applicable in the case of mDECR. Rather, we propose that the mDECR catalytic center generates a dienolate intermediate (Fig. 7). The intermediate can then acquire a proton from solvent, and, in the model of the ternary complex, Cα is indeed solvent-accessible.
      Figure thumbnail gr7
      Fig. 7The mechanism of the mDECR-catalyzed reaction.
      Stabilization of the dienolate intermediate is crucial for the function of the enzyme. The alignment of the thiolester and carbonyl moiety of substrate and the association with a well ordered section of the active site, in particular direct interactions with Tyr-199 and Asn-148, serves to align the aliphatic tail in the proximity of the reduced pyridine nucleotide and contributes to polarization and stabilization of the dienolate intermediate. A similar role has been assigned to the active site tyrosine in an enoyl-carrier protein reductase called InhA, and its association with a C16 fatty acyl substrate is noted (
      • Rozwarski D.A.
      • Vilchèze C.
      • Sugantino M.
      • Bittman R.
      • Sacchettini J.C.
      ,
      • Parikh S.
      • Moynihan D.P.
      • Xiao G.
      • Tonge P.J.
      ). The Cα of substrate is 4.5 Å from the nicotinamide, and, because hydride transfer is accompanied by the development of a positive charge on the pyridine moiety, this might serve to stabilize the dienolate intermediate.
      The presence of an acidic side chain, Glu-310, near the catalytic center of mDECR, is reminiscent of the Leishmania major pteridine reductase structure where an aspartate is directly involved in catalysis by providing a proton (abstracted from solvent) for the active site tyrosine (
      • Gourley D.G.
      • Schüttelkopf A.W.
      • Leonard G.A.
      • Luba J.
      • Hardy L.W.
      • Beverley S.M.
      • Hunter W.N.
      ). Mutation of mDECR Glu-310 to glutamine and alanine results in enzymes that retain 94 and 48% activity, respectively, compared with wild-type with increased Km values for substrate and, in the latter case, a significantly increased Km for the cofactor. In conjunction with the structural model this suggests that Glu-310 contributes to the conformation of the catalytic center by holding the nearby Phe-311 in place, which in turn helps to precisely position the side chain of Tyr-199. We can rule out a direct contribution to catalysis from this residue. In a similar fashion, Thr-197 when mutated to a smaller alanine gives a protein with almost 20% activity. Here, however, the major influence is on the binding of cofactor with a large increase in Km. This is logical, because the side chain of Thr-197 is near the nicotinamide.
      The mutation of Asn-148 to alanine is intriguing and results in low enzyme activity (3% of wild-type) with tighter binding of substrate but reduced affinity for cofactor. The disruption to the network of hydrogen bonds formed with the residues at this part of the catalytic center and removal of a functional group that stabilizes the dienolate intermediate will presumably influence enzyme activity. The alteration to the side chain may allow the tail of the substrate to participate in more van der Waals interactions with the protein and thereby improve binding though at a significant cost to catalytic efficiency due to misalignment and a reduced influence toward stabilizing the intermediate. It is unclear, on the basis of the structural model, why the Km for cofactor increases.
      Mutation of Lys-214 to alanine results in an inactive protein, yet it can still bind NADPH with similar affinity to the wild-type enzyme. This is in direct contrast to the behavior of a similarly mutated sample of enoyl-acyl carrier protein reductase (
      • Rozwarski D.A.
      • Vilchèze C.
      • Sugantino M.
      • Bittman R.
      • Sacchettini J.C.
      ). In the case of mDECR, it is likely that the alignment of the cofactor, when bound to the mutant protein, is simply not conducive to the catalytic reaction, but that water molecules fill the space vacated by removal of the lysine side chain and are able to compensate some of the cofactor binding interactions. The mutation of Tyr-199 to alanine reduces the affinity of the protein for both substrate and cofactor. A low activity is still retained, which would be compatible with the role of the tyrosine to bind substrate and to work in concert with other residues to stabilize the transition state. Although Tyr-199 could, in theory, provide the second proton used in the reduction, the orientation of the substrate Cα with respect to Tyr-199 Oη in the ternary complex is not compatible with such a function.
      Finally, mutation of Ser-210 to alanine was the most deleterious with respect to binding of both substrate and cofactor. Clearly, disruption to the hydrogen bonding interactions in the catalytic center will reduce efficiency and influence substrate binding. The hydroxyl group of Tyr-199 would not be confined to being a hydrogen bond donor in the direction of the substrate, if the Ser-210 hydroxyl group is removed. However, it is unclear why such a pronounced increase in Km for cofactor is observed. Removal of the serine hydroxyl group may provide enough space into which the side chain of the adjacent Lys-214 can rotate. Interaction with Tyr-199 Oη might then stabilize an active site conformation with much lower affinity for cofactor.

      Acknowledgments

      We thank the ESRF for synchrotron access and G. Leonard, N. Ramsden, and C. Bond for advice and help.

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