Journal of Biological Chemistry · 2004 · 12 citations · 24 references
Life SciencesBiochemistryMolecular BiologyEnzyme MechanismEnzymologyStructural BiologyCatalytic MechanismsEnzyme InhibitionEnzyme StructureEnzyme-substrate RecognitionProtein StructureStructural EnzymologyActive Site CharacterizationEnzyme Inhibition KineticsEnzymesNad+-dependent EnzymesNad-dependent EnzymesNovel OxidoreductasesSequence HomologySubstrate Binding Sites
Escherichia coli YiaK catalyzes the reduction of 2,3-diketo-l-gulonate in the presence of NADH. It belongs to a large family of oxidoreductases that is conserved in archaea, bacteria, and eukaryotes but shows no sequence homology to other proteins. We report here the crystal structures at up to 2.0-Å resolution of YiaK alone and in complex with NAD-tartrate. YiaK has a new polypeptide backbone fold and a novel mode of recognizing the NAD cofactor. In addition, NAD is bound in an unusual conformation, at the interface of a dimer of the enzyme. The crystallographic analysis unexpectedly revealed the binding of tartrate in the active site. Enzyme kinetics studies confirm that tartrate and the related d-malate are inhibitors of YiaK. In contrast to most other enzymes where substrate binding produces a more closed conformation, the binding of NAD-tartrate to YiaK produces a more open active site. The free enzyme conformation is incompatible with NAD binding. His44 is likely the catalytic residue of the enzyme. Escherichia coli YiaK catalyzes the reduction of 2,3-diketo-l-gulonate in the presence of NADH. It belongs to a large family of oxidoreductases that is conserved in archaea, bacteria, and eukaryotes but shows no sequence homology to other proteins. We report here the crystal structures at up to 2.0-Å resolution of YiaK alone and in complex with NAD-tartrate. YiaK has a new polypeptide backbone fold and a novel mode of recognizing the NAD cofactor. In addition, NAD is bound in an unusual conformation, at the interface of a dimer of the enzyme. The crystallographic analysis unexpectedly revealed the binding of tartrate in the active site. Enzyme kinetics studies confirm that tartrate and the related d-malate are inhibitors of YiaK. In contrast to most other enzymes where substrate binding produces a more closed conformation, the binding of NAD-tartrate to YiaK produces a more open active site. The free enzyme conformation is incompatible with NAD binding. His44 is likely the catalytic residue of the enzyme. In Escherichia coli, yiaK is the first gene in a nine-gene operon (1Sofia H.J. Burland V. Daniels D.L. Plunkett G. Blattner R. Nucleic Acids Res. 1994; 22: 2576-2586Crossref PubMed Scopus (215) Google Scholar), yiaKLMNOPQRS (yiaK-S), that is believed to be required for the utilization of rare sugars for growth. Mutational deactivation of the regulator of this operon, yiaJ, located just prior to yiaK but in the opposite orientation, leads to the constitutive expression of all the proteins of the operon, which enables bacteria carrying this mutation to utilize the rare pentose l-lyxose (2Badia J. Ibanez E. Sabate M. Baldoma L. Aguilar J. J. Biol. Chem. 1998; 273: 8376-8381Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). However, not all of the genes of the operon are needed for the metabolism of this sugar. It is believed that l-lyxose is first converted to l-xylulose. Then the YiaP, YiaR, and YiaS proteins, encoded in the second half of the operon, can convert l-xylulose to d-xylulose 5-phosphate, which can in turn enter the pentose phosphate pathway (3Ibanez E. Gimenez R. Pedraza T. Baldoma L. Aguilar J. Badia J. J. Bacteriol. 2000; 182: 4625-4627Crossref PubMed Scopus (10) Google Scholar). Therefore, it is likely that l-xylulose is only an intermediate from the action of the YiaK-S proteins on the genuine substrate(s) of this operon, which is currently still unknown (4Ibanez E. Campos E. Baldoma L. Aguilar J. Badia J. J. Bacteriol. 2000; 182: 4617-4624Crossref PubMed Scopus (21) Google Scholar). The YiaK protein catalyzes the reduction of 2,3-diketo-l-gulonate (DKG) 1The abbreviations used are: DKG, 2,3-diketo-l-gulonate; JK loop, loop linking helices αJ and αK. in the presence of NADH, and the product of the reduction is believed to be 3-keto-l-gulonate (see Fig. 1A) (5Yew W.S. Gerlt J.A. J. Bacteriol. 2002; 184: 302-306Crossref PubMed Scopus (100) Google Scholar, 6Volk W.A. Larsen J.L. J. Biol. Chem. 1962; 237: 2454-2457Abstract Full Text PDF PubMed Google Scholar). DKG can be obtained from the hydrolysis of l-dehydroascorbate. Another operon in E. coli, yif-sga (now known as ula), is involved in the fermentation of l-ascorbate (5Yew W.S. Gerlt J.A. J. Bacteriol. 2002; 184: 302-306Crossref PubMed Scopus (100) Google Scholar). The yiaK-S operon may be involved in the utilization of l-dehydroascorbate. YiaK belongs to a large family of putative oxidoreductases that have members from bacteria and archaea, as well as eukaryotes (see Fig. 1B). There are four additional homologs of YiaK in E. coli, two of which are shown in Fig. 1B. Many members of this family are annotated as type 2 malate/lactate dehydrogenase in the various sequence data bases, although there is only minimal biochemical evidence supporting this classification. Moreover, members of this family do not share any recognizable sequence homology to other NAD(P)-binding proteins. For example, the YiaK proteins do not contain the GXGXX(G/A) dinucleotide binding signature motif (7Wierenga R.K. Terpstra P. Hol W.G. J. Mol. Biol. 1986; 187: 101-107Crossref PubMed Scopus (996) Google Scholar). As a matter of fact, these proteins do not share any significant sequence homology with any other proteins that have known three-dimensional structures. To help understand the biochemical functions of these enzymes, we have determined the crystal structures of the free enzyme of YiaK and its complex with NAD-tartrate at up to a 2.0-Å resolution. The structures reveal a new polypeptide backbone fold for the enzyme as well as a novel mode of binding the NAD cofactor, establishing the YiaK enzymes as a new class of NAD(P)-dependent oxidoreductases. In addition, our crystallographic analysis unexpectedly showed the binding of tartrate in the active site. Enzyme kinetics studies confirm that tartrate and the related d-malate are competitive inhibitors of YiaK. Large conformational differences are observed between the free enzyme and the NAD-tartrate complex. In contrast to most other enzymes in which substrate binding produces a more closed conformation, the binding of NAD-tartrate to YiaK produces a more open active site. The conformation of the free enzyme of YiaK is incompatible with NAD binding. Expression and Purification of YiaK—The full-length yiaK gene from E. coli, Northeast Structural Genomics Consortium target ER82, was cloned into a pET21d (Novagen) derivative, generating plasmid pER82-21. The resulting recombinant protein contains eight non-native residues (LEHHHHHH) at the C terminus. The construct was sequence-verified by standard DNA sequence analysis. E. coli BL21 (DE3) pMGK cells, a rare codon-enhanced strain, were transformed with pER82-21. A single isolate was cultured in MJ minimal media (8Jansson M. Li Y.-C. Jendeberg L. Anderson S. Montelione G.T. Nilsson B. J. Biomol. NMR. 1996; 7: 131-141Crossref PubMed Scopus (151) Google Scholar) supplemented with selenomethionine, lysine, phenylalanine, threonine, isoleucine, leucine, and valine for the production of selenomethionine-labeled YiaK (9Doublie S. Kapp U. Aberg A. Brown K. Strub K. Cusack S. FEBS Lett. 1996; 384: 219-221Crossref PubMed Scopus (51) Google Scholar). Initial growth was carried out at 30 °C until the A600 of the culture reached 1.0 unit. The incubation temperature was then decreased to 17 °C, and protein expression was induced by the addition of isopropyl-β-d-thiogalactopyranoside at a final concentration of 1 mm. Following overnight incubation, the cells were harvested by centrifugation. Selenomethionyl YiaK was purified by standard methods. Cell pellets were resuspended in lysis buffer (50 mm NaH2PO4 (pH 8.0), 300 mm NaCl, 10 mm imidazole, and 5 mm β-mercaptoethanol) and disrupted by sonication. The resulting lysate was clarified by centrifugation at 26,000 × g for 45 min at 4 °C. The supernatant was loaded onto a nickel-nitrilotriacetic acid column (Qiagen) and eluted in lysis buffer containing 250 mm imidazole. Fractions containing partially purified YiaK were pooled and loaded onto a gel filtration column (Superdex 75, Amersham Biosciences), and the running buffer contained 10 mm Tris (pH 8.0) and 5 mm dithiothreitol. The purified YiaK protein was concentrated to 10 mg/ml. Sample purity (>97%) and molecular mass (37.6 kDa) were verified by SDS-PAGE and matrix-assisted laser desorption ionization time-of-flight mass spectrometry, respectively. The yield of purified protein was ∼16 mg/liter. Crystallization of YiaK—The free enzyme of YiaK was crystallized at 29 °C by the hanging-drop vapor diffusion method. The reservoir solution contained 19% (w/v) polyethylene glycol 3350, 200 mm lithium sulfate, and 10 mm dithiothreitol. The crystals were transferred to paratone for cryoprotection and then flash-frozen in liquid propane for data collection at 100 K. The crystals belong to the space group P21, with cell parameters of a = 58.0 Å, b = 51.2 Å, c = 108.9 Å, and β = 103.8°. There are two molecules in the asymmetric unit. Crystals of YiaK in complex with NAD were obtained with a reservoir solution containing 20% (w/v) polyethylene glycol 3350, 200 mm ammonium tartrate, and 20 mm NAD. The protein was pre-incubated with a 10 mm concentration of the substrate 2,3-diketo-l-gulonate (a kind gift from W. S. Yew and J. A. Gerlt). The crystals grew to full size in 3 days at 20 °C and were flash-frozen in liquid propane. They belong to the space group P1 with cell parameters of a = 75.5 Å, b = 81.3 Å, c = 113.2 Å, α = 79.6°, β = 77.2°, and γ = 82.0°. There are four dimers in the asymmetric unit. Structure Determination of YiaK-free Enzyme—Multiple wavelength anomalous diffraction data sets on a crystal of the free enzyme of YiaK were collected at 100 K at the peak, edge, and remote absorption wavelengths of selenium to 1.8-Å resolution at the X12C beamline of the National Synchrotron Light Source. The diffraction images were processed with the HKL package (10Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar). SOLVE was used for locating the selenium sites and for phasing and automated model building, which correctly placed 85% of the residues in the two monomers (11Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar). The remaining residues of the dimer were built with the program XtalView (12McRee D.E. J. Struct. Biol. 1999; 125: 156-165Crossref PubMed Scopus (2022) Google Scholar). A complete model, including the initiating selenomethionine of each monomer, was built during the refinement with crystallography NMR software (13Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.-S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar). A 2-fold non-crystallographic symmetry restraint was applied during the refinement. The electron density also showed the binding of two sulfate ions to each monomer. The data processing and refinement statistics are summarized in Table I.Table ISummary of crystallographic informationStructureFree enzymeComplex with NAD-tartrateMaximum resolution (Å)2.02.2Number of observations312,251234,793Rmerge (%)aRmerge=∑h∑i|Ihi-〈Ih〉|/∑h∑iIhi. The numbers in parentheses are for the highest resolution shell.10.8 (46.1)7.3 (16.3)Number of reflections66,809108,721Resolution range used in refinement29−2.024−2.2Completeness (%)99 (99)95 (89)R factor (%)bR=∑h|Fho-Fhc|/∑hFho18.2 (23.8)19.0 (22.0)Free R factor (%)22.2 (27.6)24.9 (28.8)r.m.s.d.cRoot mean square deviation. in bond lengths (Å)0.0050.007r.m.s.d.cRoot mean square deviation. in bond angles (°)1.11.1a Rmerge=∑h∑i|Ihi-〈Ih〉|/∑h∑iIhi. The numbers in parentheses are for the highest resolution shell.b R=∑h|Fho-Fhc|/∑hFhoc Root mean square deviation. Open table in a new tab Structure Determination of YiaK in Complex with NAD and Tartrate—An x-ray diffraction data set to 2.2 Å was collected on a crystal grown in the presence of NAD and DKG at the X4A beamline of the National Synchrotron Light Source. The structure was solved by the molecular replacement method with the program COMO (14Jogl G. Tao X. Xu Y. Tong L. Acta Crystallogr. Sect. D. PubMed Scopus Google Scholar) the free enzyme dimer structure as the of the monomers were into the electron density with conformational differences were in and for the other electron density for NAD was observed for four of these the of the substrate DKG, the electron showed the presence of tartrate in the active sites of the four The crystallographic is summarized in Table (5Yew W.S. Gerlt J.A. J. Bacteriol. 2002; 184: 302-306Crossref PubMed Scopus (100) Google Scholar). The buffer contained mm Tris (pH mm NADH, 2 mm DKG the of the and YiaK. The of the enzyme in the presence of of various was determined by the of at and structure of the YiaK-free enzyme and its complex with NAD-tartrate have in the and Structure crystal structure of the free enzyme of E. coli YiaK has determined at Å resolution by the wavelength anomalous diffraction method W.A. PubMed Scopus Google Scholar). The of the selenium and the of the were determined with the program SOLVE (11Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar). protein is with with residues of in each monomer. of these residues from each are located in the dimer interface to a of 10 residues are not conserved in other members of the family 1B). The model for the free enzyme contains residues for each of the four sulfate and The model has R and The crystal structure of YiaK in complex with NAD and tartrate has determined at resolution by the molecular replacement method (14Jogl G. Tao X. Xu Y. Tong L. Acta Crystallogr. Sect. D. PubMed Scopus Google Scholar). There are four dimers of the enzyme in this and the eight monomers are A resulting in and of these dimers and have NAD and tartrate in active For the other two the active of in each and has a electron density for NAD but density for tartrate, the active of the other in each and is The monomers with active sites have and the mean square between of any of these monomers is The two monomers and with active sites have that are to the free enzyme (see Structure of the YiaK a structure of the YiaK contains and and The structure can be into and this is by the that the as in the conformational that from binding (see is of residues from the and of the protein and contains a and at the and a that of this The residue is of and has electron is the of the It contains a which is on by two helices and In most of the other of this is in the structure of the with the two helices on this of the and located at the two of the The αJ has with the of the monomer, this conformation of the is to be on its of the is in and it the of the also the αJ (see the C of this a loop helices αJ and and this JK loop is involved in binding the NAD as well as (see in the with the program L. J. Mol. Biol. PubMed Scopus Google Scholar) not any other structures with a backbone fold to that of that this of YiaK may a new fold for a is an on the of in the between and A and residues with two helices and and a and A reveal that YiaK as dimers in the with an interface of the of each is at the dimer that the dimers be The are by studies not which that YiaK as dimers in solution as the that the conformation of the is to be and that the NAD binding is at the dimer interface (see it is likely that YiaK functions only as a The interface between the two monomers is by the of in and the two helices and of the is in the of the the of the in the other monomer. αJ to the two of this by with and The of the JK loop in is located to the loop of the other monomer. In and are located from the dimer interface in this interface are conserved the members of this family of enzymes that may all as A of NAD NAD in the and dimers has well electron density The is with monomer, and residues from of the other help the binding for the of the and The in a by the of the JK and residue numbers the other and However, these residues are conserved the YiaK family members 1B). The and the of the of the are at the of the in and The group is to the of residue and the of of the molecules on the a bond with the of residue The group also shows with the conserved residue of the other The group of the with the of and the of that YiaK may NAD as the (see The two on the to the are to the of which is conserved the YiaK family members 1B). The is placed the of the in this binding a can only on the of the The observed between NAD and YiaK to be to this family of Many NAD(P)-dependent contain a fold that is for binding the dinucleotide D. PubMed Scopus Google Scholar), and the group with the GXGXX(G/A) motif as well as the of the this motif (7Wierenga R.K. Terpstra P. Hol W.G. J. Mol. Biol. 1986; 187: 101-107Crossref PubMed Scopus (996) Google Scholar, J. 1996; PubMed Scopus Google Scholar). In the that NAD in YiaK has a novel backbone and the YiaK proteins do not contain the GXGXX(G/A) sequence the group with a as well as the ammonium of a residue from by with the of a is not observed in the complex The group on the to the is to in YiaK may be to that from an residue at the of the of the (7Wierenga R.K. Terpstra P. Hol W.G. J. Mol. Biol. 1986; 187: 101-107Crossref PubMed Scopus (996) Google Scholar, P. 1997; PubMed Scopus Google Scholar) and that the YiaK family members with a conserved residue at this may only be to NAD as the cofactor. However, members of the family have a residue at this as the cofactor. The conformation of NAD in the YiaK complex is and is from is observed for NAD in with enzymes containing a fold the of the in the are from each other by Å in the two conformational is of a the bond linking the and the as well as a of the bond of the this NAD conformation has observed in a complex with a of V. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The conformation of NAD in complex with the type of this enzyme A large of have observed for the in complex with various enzymes P. 1997; PubMed Scopus Google Scholar). of 2,3-diketo-l-gulonate 1A) in the solution at 20 mm concentration in an to the binding mode of this substrate to the enzyme. To our crystallographic on the x-ray diffraction data that a tartrate is bound in the active with electron density is in the reservoir solution at 200 mm and this was required for crystals of the enzyme in the presence of NAD. is the in the active and its and are to conserved residues of the enzyme of the of the two with the of and the other with a is conserved the YiaK family of enzymes, is conserved 1B). also a bond to the of tartrate The is to the of and at the of and it also have with the of this The group of tartrate is to the of which is the likely catalytic of the enzyme (see The that tartrate is likely an active of YiaK. confirm that tartrate is a of the enzyme. mm concentration this a of the of YiaK. which the for the group at the as tartrate can also the enzyme with the other with the opposite is a of YiaK as with of these can as a substrate of the enzyme in the reduction of NAD to NADH. a Open reveal a large conformational in YiaK the binding of NAD and as the and differences in and between the free enzyme and the NAD-tartrate complex The conformational is observed in which from a the active for the binding of NAD helices and αJ and the JK loop also significant differences between the free enzyme and the NAD-tartrate complex As these residues are involved in NAD binding as well as dimer there is also a in the of the dimer NAD-tartrate binding of the dimer in the a of is needed to the second into the conformational in YiaK in an unusual as the active of the enzyme is more open in the complex with NAD and tartrate and A and In most other enzymes a more closed conformation the binding of from the In of the tartrate is to the The of NAD is from the by tartrate, but the has significant to the a conformational of the enzyme is for the binding of NAD and tartrate, as the structure of the free enzyme is incompatible with the presence of these molecules in the active the of His44 and in the free enzyme are in with the of the cofactor, residues the binding of the group of In the crystal structure of the free each contains two sulfate ions in the active site. of these with the of the other sulfate is the of tartrate second sulfate is to the of and to the from the of It is that the observed free enzyme conformation is by the binding of sulfate, the two molecules in the crystal of the complex C and that have no tartrate, sulfate bound to the active that are the as that observed in the free enzyme of the the structure of the NAD-tartrate the of tartrate is located Å from the of the the the group on the is to the of the conserved His44 Therefore, this binding mode may that of the product of the It is likely that His44 functions as the of the enzyme for the It the from the group of the and this is to the from the to the NAD. For the reduction of DKG, this in the as a acid by the and the from to the However, the at the of tartrate and 3-keto-l-gulonate are opposite of each other It is not this can a binding mode as It may have to be bound in the opposite to with the enzyme. In this orientation, the of with the of and although a more closed conformation of may be needed for these It is that in addition to may also have a in the from the substrate for at crystals of the complex in the of tartrate have not In we have determined the crystal structures of the E. coli 2,3-diketo-l-gulonate alone and in complex with NAD and The NAD is bound to a with a novel polypeptide backbone establishing the YiaK family of enzymes as a new class of oxidoreductases. Structural and solution studies that YiaK functions as a in and of the enzyme are required for the binding of the NAD cofactor. In contrast to most other enzymes, these in YiaK a more open active site. and the related d-malate are inhibitors of the enzyme. His44 is likely the catalytic residue of the enzyme. We W. S. Yew and J. A. Gerlt for the gift of the 2,3-diketo-l-gulonate substrate of this enzyme and and for up the X4A
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