780 Biochemistry 2002, 41, 780-787 Pyruvate Site of Pyruvate Phosphate Dikinase: Crystal Structure of the Enzyme-Phosphonopyruvate Complex, and Mutant Analysis†,‡ Osnat Herzberg,*,§ Celia C. H. Chen,§ Sijiu Liu,§ Aleksandra Tempczyk,§ Andrew Howard,| Min Wei,⊥ Dongmei Ye,⊥ and Debra Dunaway-Mariano⊥ Center for AdVanced Research in Biotechnology, UniVersity of Maryland Biotechnology Institute, 9600 Gudelsky DriVe, RockVille, Maryland 20850, Department of Chemistry, UniVersity of New Mexico, Albuquerque, New Mexico 87131, Illinois Institute of Technology, Chicago, Illinois 60616, and AdVanced Photon Source, Argonne National Laboratory, 9700 South Cass AVenue, Argonne, Illinois 60439 ReceiVed September 17, 2001; ReVised Manuscript ReceiVed NoVember 5, 2001 ABSTRACT: Crystals of pyruvate phosphate dikinase in complex with a substrate analogue inhibitor, 2+ phosphonopyruvate (Ki ) 3 µM), have been obtained in the presence of Mg . The structure has been determined and refined at 2.2 Å resolution, revealing that the Mg2+-bound phosphonopyruvate binds in the R/â-barrel’s central channel, at the C-termini of the â-strands. The mode of binding resembles closely the previously proposed PEP substrate binding mode, inferred by the homology of the structure (but not sequence homology) to pyruvate kinase. Kinetic analysis of site-directed mutants, probing residues involved in inhibitor binding, showed that all mutations resulted in inactivation, confirming the key role that these residues play in catalysis. Comparison between the structure of the PPDK-phosphonopyruvate complex and the structures of two complexes of pyruvate kinase, one with Mg2+-bound phospholactate and the other with Mg2+-oxalate and ATP, revealed that the two enzymes share some key features that facilitate common modes of substrate binding. There are also important structural differences; most notably, the machinery for acid/base catalysis is different. 1 PPDK catalyzes the interconversion of ATP, Pi, and binding site is located within the N-terminal part of the 2+ pyruvate, with AMP, PPi, and PEP, in three Mg -dependent polypeptide chain, whereas the pyruvate/PEP binding site is partial reactions that involve phospho- and pyrophospho- located within the C-terminal part of the chain (4-7). enzyme intermediates (1, 2): The crystal structure of PPDK in the unbound state (8) revealed two remote substrate binding sites, one binding site E-His + ATP f E-His-PP‚AMP (I) for ATP/AMP and Pi/PPi and the second for pyruvate/PEP. - - ‚ + f - - ‚ ‚ E His PP AMP Pi E His P AMP PPi (II) The two binding sites are located 45 Å apart, and each is associated with a different structural domain. The N-terminal E-His-P + pyruvate f E-His + PEP (III) segment of the polypeptide chain contains a 240-residue The above reactions are reversible, and the outcome depends nucleotide binding domain that adopts the fold termed ATP- on the host organism (glycolytic ATP synthesis, or PEP grasp (9). The C-terminal 340-residue pyruvate/PEP binding R synthesis). Enzyme phosphorylation and pyrophosphorylation domain adopts an /â-barrel fold. A third domain contains occur on a histidine residue, His455 (3). The nucleotide the phosphoryl/pyrophosphate group acceptor/donor residue, His455. The locations of the nucleotide binding sites were inferred by analogy to other enzymes with the folds of the † Supported by NSF Grant MCB9813271 (O.H.) and NIH Grant GM36260 (D.D.-M.). Use of the National Synchrotron Light Source ATP-grasp, and the location of the PEP/pyruvate binding was supported by the U.S. Department of Energy, Basic Energy site by analogy to R/â-barrel enzymes. The structure implies Sciences, Office of Science, under Prime Contract DE-AC02- that the phosphohistidine domain undergoes a swivel motion 98CH110886, by the National Science Foundation, and by National Institutes of Health Grant 1P41 RR12408-01A1. Use of the Advanced to shuttle the phosphoryl group between the two remote Photon Source was supported by the U.S. Department of Energy, active sites. Basic Energy Sciences, Office of Science, under Contract W-31-109- The pyruvate/PEP binding site of PPDK may be compared Eng-38. ‡ The coordinates have been deposited in the Protein Data Bank with the active site of pyruvate kinase, an enzyme that (entries 1KBL and 1KC7). converts PEP to ATP directly, without the involvement of a * To whom correspondence should be addressed. Telephone: (301) phosphoenzyme intermediate. The pyruvate kinase active site 738-6245. Fax: (301) 738-6255. E-mail: [email protected]. R § University of Maryland Biotechnology Institute. also adopts an /â-barrel fold (10). The comparison revealed | Illinois Institute of Technology and Argonne National Laboratory. conservation of several key residues involved in substrate ⊥ University of New Mexico. binding, and similarity in their relative disposition in the 1 Abbreviations: PPDK, pyruvate phosphate dikinase; PEP, phos- active site. Thus, a model has been developed for the phoenolpyruvate; ATP, adenosine 5′-triphosphate; AMP, adenosine 5′- monophosphate; Pi, inorganic phosphate; PPi, inorganic pyrophosphate; pyruvate/PEP binding to PPDK based on the information Hepes, N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid. from pyruvate kinase site (8). Binding studies in the 10.1021/bi011799+ CCC: $22.00 © 2002 American Chemical Society Published on Web 12/19/2001 PPDK-Phosphonopyruvate Structure Biochemistry, Vol. 41, No. 3, 2002 781 enzyme-inhibitor complex were transferred to a cryogenic solution comprising 60% saturated ammonium sulfate, 20% glycerol, and 0.1 M Hepes buffer (pH 7.0). In addition, the cryogenic solution for the enzyme-inhibitor complex also contained 40 mM P-pyr and 40 mM MgCl2. After soaking in the cryogenic solution for 2 h, the crystal was flash-cooled in liquid propane cooled by liquid nitrogen. As with the FIGURE 1: Structures of PEP and P-pyr, the substrate and inhibitor crystal of the apoprotein, the space group of the enzyme- of PPDK, respectively. inhibitor crystal was P2, with one monomer in the asym- metric unit. The unit cell dimensions of the unbound protein crystalline state proved to be illusive, probably due to the crystal were as follows: a ) 89.8 Å, b ) 58.7 Å, c ) 102.9 ∼ relatively high Km value of the substrate ( 30 µM), and the Å, and â ) 94.8°. For the enzyme-inhibitor complex crystal, fact that, so far, crystals useful for X-ray work have been the cell dimensions were as follows: a ) 89.2 Å, b ) 58.4 obtained only from solutions containing a high concentration Å, c ) 102.6 Å, and â ) 95.0°. ∼ of ammonium sulfate ( 2 M). The high ionic strength of X-ray intensity data for both apoenzyme and enzyme- the crystallization solution is expected to lead to reduced 2+ inhibitor crystals were collected at 100 K. The native data substrate affinity, as the substrate and Mg cofactor are were collected to 1.9 Å resolution on the X12C beamline of charged. the National Synchrotron Light Source (NSLS, Brookhaven The current work became possible following the discovery National Laboratory, Upton, NY), and the enzyme-inhibitor that phosphonopyruvate (P-pyr), a compound that resembles complex data were collected to 1.73 Å resolution on the PEP (Figure 1), inhibits PPDK activity at micromolar IMCA-CAT 17-ID beamline at the Advanced Photon Source ) concentrations (Ki 3 µM). The crystal structure of the (Argonne National Laboratory). Diffraction data at a wave- - PPDK P-pyr complex, together with site-directed mutagen- length of 1.3 Å at NSLS beamline X12C were recorded on esis data, confirms the location of the pyruvate/PEP binding a 300 mm MAR research image plate, and at the IMCA- sites, and the identity of the residues that play key roles in CAT 17-ID beamline, diffraction data at a wavelength of catalysis. Also reported is the structure of the apoenzyme at 1.0 Å were recorded on a MAR 165 mm charge-coupled 1.94 Å resolution. This is a better resolution than that of device system. For the PPDK-inhibitor complex, several our previously reported structure (8), thus facilitating a more crystals were tested, and they all exhibited high mosaic reliable comparison between the bound and unbound enzyme. spread. For the best diffracting crystal, the estimated mosaic spread was close to 2.5°. This was the crystal used for data MATERIALS AND METHODS acquisition. Data collected on the X12C beamline were Expression, Protein Purification, and Site-Directed Mu- processed with HKL (12). Data collected on the IMCA-CAT tagenesis. Wild-type and mutant PPDK proteins from beamline were processed with the X-GEN package (13). The Clostridium symbiosum were expressed in Escherichia coli statistics of data processing are given in Table 1. Rmerge values and purified as described previously (11). Mutant proteins for the PPDK-inhibitor data indicate poor quality beyond were prepared from the plasmid pACYC184-D12 using a ∼1.9 Å resolution, which is attributed to the high mosaic PCR-based procedure analogous to that described in ref 5. spread of the crystal. Although refinement commenced using Primers for mutagenesis, 18-22 base pairs in length, were all data up to 1.8 Å resolution, it was ultimately completed synthesized by Gibco BRL Life Science. KpnI and BstXI at a truncated resolution of 2.2 Å. restriction sites were employed in the construction of the Structure Determination and Refinement. The starting E745Q, D769A, N768A, D620N, R617K, R561K, and model for refinement was that of the room-temperature wild- C831A mutants. The sequences of the isolated mutant genes type apo-PPDK structure refined at 2.3 Å resolution (PDB were determined by the Center for Agricultural Biotechnol- entry 1DIK), excluding solvent molecules. The structures ogy at the University of Maryland (College Park, MD). The were refined with the program CNS (14). Two cycles of mutant genes were expressed in E. coli JM101 cells as simulated annealing at 2500 K were followed by positional described in ref 5.