ABSTRACT
Energy converting NADH:ubiquinone oxidoreductase, complex I, is the first enzyme of respiratory chains in most eukaryotes and many bacteria. Mutations in genes encoding subunits of human complex I may lead to its dysfunction resulting in a diverse clinical pattern. The effect of mutations on the protein structure is not known. Here, we focus on mutations R88G, E246K, P252R and E377K that are found in subunit NDUFV1 comprising the NADH binding site of complex I. Homologous mutations were introduced into subunit NuoF of Aquifex aeolicus complex I and it was attempted to crystallize variants of the electron input module, NuoEF, with bound substrates in the oxidized and reduced state. The E377K variant did not form crystals most likely due to an improper protein assembly. The architecture of the NADH binding site is hardly affected by the other mutations indicating its unexpected structural robustness. The R88G, E246K and P252R mutations led to small local structural rearrangements that might be related to their pathogenicity. These minor structural changes involve substrate binding, product release and the putative formation of reactive oxygen species. The structural consequences of the mutations as obtained with the bacterial enzyme might thus help to contribute to the understanding of disease causing mutations.
ABSTRACT
Energy-converting NADH:ubiquinone oxidoreductase, respiratory complex I, is a major enzyme of energy metabolism that couples NADH oxidation and ubiquinone reduction with proton translocation. The NADH oxidation site features different enzymatic activities with various nucleotides. While the kinetics of these reactions are well described, only binding of NAD+ and NADH have been structurally characterized. Here, we report the structures of the electron input module of Aquifex aeolicus complex I with bound ADP-ribose and 3-acetylpyridine adenine dinucleotides at resolutions better than 2.0 Å. ADP-ribose acts as inhibitor by blocking the "ADP-handle" motif essential for nucleotide binding. The pyridine group of APADH is minimally offset from flavin, which could contribute to its poorer suitability as substrate. A comparison with other nucleotide co-structures surprisingly shows that the adenine ribose and the pyrophosphate moiety contribute most to nucleotide binding, thus all adenine dinucleotides share core binding modes to the unique Rossmann-fold in complex I.
Subject(s)
Adenosine Diphosphate Ribose , Electron Transport Complex I , Models, Molecular , Protein Binding , Electron Transport Complex I/metabolism , Electron Transport Complex I/chemistry , Adenosine Diphosphate Ribose/metabolism , Adenosine Diphosphate Ribose/chemistry , Binding Sites , NAD/metabolism , NAD/chemistry , Crystallography, X-Ray , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Oxidation-ReductionABSTRACT
NADH:ubiquinone oxidoreductase, respiratory complex I, plays a major role in cellular energy metabolism by coupling electron transfer with proton translocation. Electron transfer is catalyzed by a flavin mononucleotide and a series of iron-sulfur (Fe/S) clusters. As a by-product of the reaction, the reduced flavin generates reactive oxygen species (ROS). It was suggested that the ROS generated by the respiratory chain in general could damage the Fe/S clusters of the complex. Here, we show that the binuclear Fe/S cluster N1b is specifically damaged by H2O2, however, only at high concentrations. But under the same conditions, the activity of the complex is hardly affected, since N1b can be easily bypassed during electron transfer.
Subject(s)
Electron Transport Complex I , Iron-Sulfur Proteins , Electron Transport Complex I/metabolism , Iron-Sulfur Proteins/metabolism , Hydrogen Peroxide/metabolism , Iron/metabolism , Reactive Oxygen Species/metabolism , Electron Transport , Sulfur/metabolism , Oxidation-Reduction , Electron Spin Resonance SpectroscopyABSTRACT
Cytochrome bd-I catalyzes the reduction of oxygen to water with the aid of hemes b558, b595 and d. Here, effects of a mutation of E445, a ligand of heme b595 and of R448, hydrogen bonded to E445 are studied electrochemically in the E. coli enzyme. The equilibrium potential of the three hemes are shifted by up to 200 mV in these mutants. Strikingly the E445D and the R448N mutants show a turnover of 41 ± 2 % and 20 ± 4 %, respectively. Electrocatalytic studies confirm that the mutants react with oxygen and bind and release NO. These results point towards the ability of cytochrome bd to react even if the electron transfer is less favorable.
Subject(s)
Escherichia coli Proteins , Escherichia coli , Escherichia coli/metabolism , Oxidoreductases/genetics , Oxidoreductases/metabolism , Cytochromes/genetics , Cytochromes/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Electron Transport Chain Complex Proteins/genetics , Electron Transport Chain Complex Proteins/metabolism , Electron Transport , Oxygen/metabolism , Oxidation-ReductionABSTRACT
Escherichia coli contains two cytochrome bd oxidases, bd-I and bd-II. The structure of both enzymes is highly similar, but they exhibit subtle differences such as the accessibility of the active site through a putative proton channel. Here, we demonstrate that the duroquinol:dioxygen oxidoreductase activity of bd-I increased with alkaline pH, whereas bd-II showed a broad activity maximum around pH 7. Likewise, the pH dependence of NO release from the reduced active site, an essential property of bd oxidases, differed between the two oxidases as detected by UV/vis spectroscopy. Both findings may be attributed to differences in the proton channel leading to the active site heme d. The channel comprises a titratable residue (Asp58B in bd-I and Glu58B in bd-II). Conservative mutations at this position drastically altered NO release demonstrating its contribution to the process.
Subject(s)
Escherichia coli Proteins , Oxidoreductases , Oxidoreductases/metabolism , Escherichia coli , Cytochromes/chemistry , Protons , Escherichia coli Proteins/metabolism , Cytochrome b Group/genetics , Electron Transport Chain Complex Proteins/metabolism , Electron Transport Complex IV , Hydrogen-Ion ConcentrationABSTRACT
The reduction of oxygen to water is crucial to life and a central metabolic process. To fulfil this task, prokaryotes use among other enzymes cytochrome bd oxidases (Cyt bds) that also play an important role in bacterial virulence and antibiotic resistance. To fight microbial infections by pathogens, an in-depth understanding of the enzyme mechanism is required. Here, we combine bioinformatics, mutagenesis, enzyme kinetics and FTIR spectroscopy to demonstrate that proton delivery to the active site contributes to the rate limiting steps in Cyt bd-I and involves Asp58 of subunit CydB. Our findings reveal a previously unknown catalytic function of subunit CydB in the reaction of Cyt bd-I.
Subject(s)
Escherichia coli Proteins , Escherichia coli , Cytochrome b Group/genetics , Cytochrome b Group/metabolism , Cytochromes/chemistry , Electron Transport Chain Complex Proteins/metabolism , Electron Transport Complex IV/metabolism , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Oxidoreductases/metabolism , Oxygen/metabolism , Protons , Water/metabolismABSTRACT
Energy-converting NADH:ubiquinone oxidoreductase, respiratory complex I, is essential for cellular energy metabolism coupling NADH oxidation to proton translocation. The mechanism of proton translocation by complex I is still under debate. Its membrane arm contains an unusual central axis of polar and charged amino acid residues connecting the quinone binding site with the antiporter-type subunits NuoL, NuoM, and NuoN, proposed to catalyze proton translocation. Quinone chemistry probably causes conformational changes and electrostatic interactions that are propagated through these subunits by a conserved pattern of predominantly lysine, histidine, and glutamate residues. These conserved residues are thought to transfer protons along and across the membrane arm. The distinct charge distribution in the membrane arm is a prerequisite for proton translocation. Remarkably, the central subunit NuoM contains a conserved glutamate residue in a position that is taken by a lysine residue in the two other antiporter-type subunits. It was proposed that this charge asymmetry is essential for proton translocation, as it should enable NuoM to operate asynchronously with NuoL and NuoN. Accordingly, we exchanged the conserved glutamate in NuoM for a lysine residue, introducing charge symmetry in the membrane arm. The stably assembled variant pumps protons across the membrane, but with a diminished H+/e- stoichiometry of 1.5. Thus, charge asymmetry is not essential for proton translocation by complex I, casting doubts on the suggestion of an asynchronous operation of NuoL, NuoM, and NuoN. Furthermore, our data emphasize the importance of a balanced charge distribution in the protein for directional proton transfer.
Subject(s)
Cell Membrane , Electron Transport Complex I , Escherichia coli Proteins , NADH Dehydrogenase , Amino Acid Substitution , Cell Membrane/chemistry , Conserved Sequence , Electron Transport Complex I/chemistry , Electron Transport Complex I/genetics , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Glutamates/chemistry , Glutamates/genetics , Lysine/chemistry , Lysine/genetics , NADH Dehydrogenase/chemistry , NADH Dehydrogenase/genetics , Protons , Quinones/chemistryABSTRACT
Cytochrome bd is a triheme copper-free terminal oxidase in membrane respiratory chains of prokaryotes. This unique molecular machine couples electron transfer from quinol to O2 with the generation of a proton motive force without proton pumping. Apart from energy conservation, the bd enzyme plays an additional key role in the microbial cell, being involved in the response to different environmental stressors. Cytochrome bd promotes virulence in a number of pathogenic species that makes it a suitable molecular drug target candidate. This review focuses on recent advances in understanding the structure of cytochrome bd and the development of its selective inhibitors.
Subject(s)
Cytochromes , Escherichia coli Proteins , Cell Respiration , Cytochromes/metabolism , Electron Transport , Electron Transport Chain Complex Proteins/metabolism , Proton-Motive ForceABSTRACT
Respiratory complex I drives proton translocation across energy-transducing membranes by NADH oxidation coupled with (ubi)quinone reduction. In humans, its dysfunction is associated with neurodegenerative diseases. The Escherichia coli complex represents the structural minimal form of an energy-converting NADH:ubiquinone oxidoreductase. Here, we report the structure of the peripheral arm of the E. coli complex I consisting of six subunits, the FMN cofactor, and nine iron-sulfur clusters at 2.7 Å resolution obtained by cryo electron microscopy. While the cofactors are in equivalent positions as in the complex from other species, individual subunits are adapted to the absence of supernumerary proteins to guarantee structural stability. The catalytically important subunits NuoC and D are fused resulting in a specific architecture of functional importance. Striking features of the E. coli complex are scrutinized by mutagenesis and biochemical characterization of the variants. Moreover, the arrangement of the subunits sheds light on the unknown assembly of the complex.
Subject(s)
Electron Transport Complex I/chemistry , Electron Transport Complex I/metabolism , Escherichia coli/metabolism , Mutation , Binding Sites , Cryoelectron Microscopy , Electron Transport Complex I/genetics , Escherichia coli/chemistry , Escherichia coli/genetics , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Models, Molecular , Mutagenesis, Site-Directed , Protein Conformation , Protein Stability , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/metabolismABSTRACT
Cytochrome bd quinol:O2 oxidoreductases are respiratory terminal oxidases so far only identified in prokaryotes, including several pathogenic bacteria. Escherichia coli contains two bd oxidases of which only the bd-I type is structurally characterized. Here, we report the structure of the Escherichia coli cytochrome bd-II type oxidase with the bound inhibitor aurachin D as obtained by electron cryo-microscopy at 3 Å resolution. The oxidase consists of subunits AppB, C and X that show an architecture similar to that of bd-I. The three heme cofactors are found in AppC, while AppB is stabilized by a structural ubiquinone-8 at the homologous positions. A fourth subunit present in bd-I is lacking in bd-II. Accordingly, heme b595 is exposed to the membrane but heme d embedded within the protein and showing an unexpectedly high redox potential is the catalytically active centre. The structure of the Q-loop is fully resolved, revealing the specific aurachin binding.
Subject(s)
Cytochromes/metabolism , Escherichia coli/metabolism , Bacterial Outer Membrane Proteins/metabolism , Electron Transport Chain Complex Proteins/metabolism , Escherichia coli Proteins/metabolism , Oxidation-Reduction , Oxidoreductases/metabolism , Quinolones/metabolism , Ubiquinone/metabolismABSTRACT
NADH:ubiquinone oxidoreductase, respiratory complex I, plays a central role in cellular energy metabolism. As a major source of reactive oxygen species (ROS) it affects ageing and mitochondrial dysfunction. The novel inhibitor NADH-OH specifically blocks NADH oxidation and ROS production by complex I in nanomolar concentrations. Attempts to elucidate its structure by NMR spectroscopy have failed. Here, by using X-ray crystallographic analysis, we report the structure of NADH-OH bound in the active site of a soluble fragment of complex I at 2.0â Å resolution. We have identified key amino acid residues that are specific and essential for binding NADH-OH. Furthermore, the structure sheds light on the specificity of NADH-OH towards the unique Rossmann-fold of complex I and indicates a regulatory role in mitochondrial ROS generation. In addition, NADH-OH acts as a lead-structure for the synthesis of a novel class of ROS suppressors.
Subject(s)
Electron Transport Complex I/antagonists & inhibitors , Enzyme Inhibitors/chemistry , NAD/analogs & derivatives , Aquifex/enzymology , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Electron Transport Complex I/chemistry , Electron Transport Complex I/metabolism , Enzyme Inhibitors/pharmacology , Humans , Hydrogen Bonding , Models, Molecular , NAD/chemistry , NAD/metabolism , NAD/pharmacology , Protein BindingABSTRACT
Energy-converting NADH:ubiquinone oxidoreductase, respiratory complex I, plays a major role in cellular energy metabolism. It couples NADH oxidation and quinone reduction with the translocation of protons across the membrane, thus contributing to the protonmotive force. Complex I has an overall L-shaped structure with a peripheral arm catalyzing electron transfer and a membrane arm engaged in proton translocation. Although both reactions are arranged spatially separated, they are tightly coupled by a mechanism that is not fully understood. Using redox-difference UV-vis spectroscopy, an unknown redox component was identified in Escherichia coli complex I as reported earlier. A comparison of its spectrum with those obtained for different quinone species indicates features of a quinol anion. The re-oxidation kinetics of the quinol anion intermediate is significantly slower in the D213GH variant that was previously shown to operate with disturbed quinone chemistry. Addition of the quinone-site inhibitor piericidin A led to strongly decreased absorption peaks in the difference spectrum. A hypothesis for a mechanism of proton-coupled electron transfer with the quinol anion as catalytically important intermediate in complex I is discussed.
ABSTRACT
Cytochrome bd oxidase is a bacterial terminal oxygen reductase that was suggested to enable adaptation to different environments and to confer resistance to stress conditions. An electrocatalytic study of the cyt bd oxidases from Escherichia coli, Corynebacterium glutamicum and Geobacillus thermodenitrificans gives evidence for a different reactivity towards oxygen. An inversion of the redox potential values of the three hemes is found when comparing the enzymes from different bacteria. This inversion can be correlated with different protonated glutamic acids as evidenced by reaction induced FTIR spectroscopy. The influence of the microenvironment of the hemes on the reactivity towards oxygen is discussed.
Subject(s)
Corynebacterium glutamicum/enzymology , Cytochrome b Group/metabolism , Electrodes , Electron Transport Chain Complex Proteins/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Geobacillus/enzymology , Oxidoreductases/metabolism , Oxygen/metabolism , Catalysis , Oxygen/chemistryABSTRACT
Various malignant human diseases show disturbed signaling pathways due to increased activity of proteins within the epigenetic machinery. Recently, various novel inhibitors for epigenetic regulation have been introduced which promise a great therapeutic benefit. Inhibitors for the bromo- and extra-terminal domain (BET) family were of particular interest after inhibitors had shown a strong antiproliferative effect. More recently, the focus has increasingly shifted to bromodomains (BDs) outside the BET family. Based on previously developed inhibitors, we have optimized a small series of 4-acyl pyrroles, which we further analyzed by ITC, X-ray crystallography, selectivity studies, the NCI60 cell-panel, and GI50 determinations for several cancer cell lines. The inhibitors address both, BET and BRD7/9 BDs, with very high affinity and show a strong antiproliferative effect on various cancer cell lines that could not be observed for BD family selective inhibitors. Furthermore, a synergistic effect on breast cancer (MCF-7) and melanoma (SK-MEL-5) was proven.
Subject(s)
Antineoplastic Agents/chemistry , Chromosomal Proteins, Non-Histone/antagonists & inhibitors , Proteins/antagonists & inhibitors , Pyrroles/chemistry , Transcription Factors/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Binding Sites , Cell Line, Tumor , Cell Proliferation/drug effects , Chromosomal Proteins, Non-Histone/metabolism , Crystallography, X-Ray , Humans , Kinetics , Ligands , Molecular Dynamics Simulation , Protein Domains , Proteins/metabolism , Pyrroles/metabolism , Pyrroles/pharmacology , Structure-Activity Relationship , Transcription Factors/metabolismABSTRACT
Inositol pyrophosphates constitute a family of hyperphosphorylated signaling molecules involved in the regulation of glucose uptake and insulin sensitivity. While our understanding of the biological roles of inositol heptaphosphates (PP-InsP5) has greatly improved, the functions of the inositol octaphosphates ((PP)2-InsP4) have remained unclear. Here we present the synthesis of two enantiomeric cell-permeant and photocaged (PP)2-InsP4 derivatives and apply them to study the functions in living ß-cells. Photorelease of the naturally occurring isomer 1,5-(PP)2-InsP4 led to an immediate and concentration-dependent reduction of intracellular calcium oscillations, while other caged inositol pyrophosphates (3,5-(PP)2-InsP4, 5-PP-InsP5, 1-PP-InsP5, 3-PP-InsP5) showed no immediate effect. Furthermore, uncaging of 1,5-(PP)2-InsP4 but not 3,5-(PP)2-InsP4 induced translocation of the C2AB domain of granuphilin from the plasma membrane to the cytosol. Granuphilin is involved in membrane docking of secretory vesicles. This suggests that 1,5-(PP)2-InsP4 impacts ß-cell activity by regulating granule localization and/or priming and calcium signaling in concert.
Subject(s)
Calcium/metabolism , Inositol Phosphates/metabolism , Calcium/chemistry , Inositol Phosphates/chemical synthesis , Inositol Phosphates/chemistry , Molecular Conformation , PhotolysisABSTRACT
Cytochrome bd-I oxidase is a terminal reductase of bacterial respiratory chains produced under low oxygen concentrations, oxidative stress, and during pathogenicity. While the bulk of the protein forms transmembrane helices, a periplasmic domain, the Q-loop, is expected to be involved in binding and oxidation of (ubi)quinol. According to the length of the Q-loop, bd oxidases are classified into the S (short)- and the L (long)-subfamilies. Here, we show that either shortening the Q-loop of the Escherichia coli oxidase from the L-subfamily or replacing it by one from the S-subfamily leads to the production of labile and inactive variants, indicating a role for the extended Q-loop in the stability of the enzyme.
Subject(s)
Cytochrome b Group/chemistry , Cytochrome b Group/metabolism , Electron Transport Chain Complex Proteins/chemistry , Electron Transport Chain Complex Proteins/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Amino Acid Sequence , Cytochrome b Group/genetics , Electron Transport Chain Complex Proteins/genetics , Enzyme Stability/genetics , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Models, Molecular , Multienzyme Complexes/chemistry , Multienzyme Complexes/genetics , Multienzyme Complexes/metabolism , NADH, NADPH Oxidoreductases/chemistry , NADH, NADPH Oxidoreductases/genetics , NADH, NADPH Oxidoreductases/metabolism , Oxidation-Reduction , Oxidoreductases/genetics , Ubiquinone/analogs & derivatives , Ubiquinone/chemistry , Ubiquinone/metabolismABSTRACT
Cytochrome bd oxidases are terminal reductases of bacterial and archaeal respiratory chains. The enzyme couples the oxidation of ubiquinol or menaquinol with the reduction of dioxygen to water, thus contributing to the generation of the protonmotive force. Here, we determine the structure of the Escherichia coli bd oxidase treated with the specific inhibitor aurachin by cryo-electron microscopy (cryo-EM). The major subunits CydA and CydB are related by a pseudo two fold symmetry. The heme b and d cofactors are found in CydA, while ubiquinone-8 is bound at the homologous positions in CydB to stabilize its structure. The architecture of the E. coli enzyme is highly similar to that of Geobacillus thermodenitrificans, however, the positions of heme b595 and d are interchanged, and a common oxygen channel is blocked by a fourth subunit and substituted by a more narrow, alternative channel. Thus, with the same overall fold, the homologous enzymes exhibit a different mechanism.
Subject(s)
Cytochrome b Group/chemistry , Cytochrome b Group/metabolism , Electron Transport Chain Complex Proteins/chemistry , Electron Transport Chain Complex Proteins/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Sequence Homology, Amino Acid , Cytochrome b Group/ultrastructure , Electron Transport Chain Complex Proteins/ultrastructure , Escherichia coli Proteins/ultrastructure , Geobacillus/enzymology , Heme/chemistry , Heme/metabolism , Models, Molecular , Oxidoreductases/ultrastructure , Oxygen/metabolism , Protons , Substrate Specificity , Ubiquinone/chemistry , Ubiquinone/metabolism , WaterABSTRACT
Respiratory complex I plays a central role in cellular energy metabolism coupling NADH oxidation to proton translocation. In humans its dysfunction is associated with degenerative diseases. Here we report the structure of the electron input part of Aquifex aeolicus complex I at up to 1.8 Å resolution with bound substrates in the reduced and oxidized states. The redox states differ by the flip of a peptide bond close to the NADH binding site. The orientation of this peptide bond is determined by the reduction state of the nearby [Fe-S] cluster N1a. Fixation of the peptide bond by site-directed mutagenesis led to an inactivation of electron transfer and a decreased reactive oxygen species (ROS) production. We suggest the redox-gated peptide flip to represent a previously unrecognized molecular switch synchronizing NADH oxidation in response to the redox state of the complex as part of an intramolecular feed-back mechanism to prevent ROS production.
Subject(s)
Electron Transport Complex I/chemistry , Escherichia coli/metabolism , Reactive Oxygen Species/metabolism , Bacteria/chemistry , Bacteria/metabolism , Escherichia coli/chemistry , Escherichia coli Proteins/chemistry , Iron-Sulfur Proteins/chemistry , Mutagenesis, Site-Directed , NAD/chemistry , Oxidation-ReductionABSTRACT
The yeast ribosome-associated complex RAC and the Hsp70 homolog Ssb are anchored to the ribosome and together act as chaperones for the folding and co-translational assembly of nascent polypeptides. In addition, the RAC/Ssb system plays a crucial role in maintaining the fidelity of translation termination; however, the latter function is poorly understood. Here we show that the RAC/Ssb system promotes the fidelity of translation termination via two distinct mechanisms. First, via direct contacts with the ribosome and the nascent chain, RAC/Ssb facilitates the translation of stalling-prone poly-AAG/A sequences encoding for polylysine segments. Impairment of this function leads to enhanced ribosome stalling and to premature nascent polypeptide release at AAG/A codons. Second, RAC/Ssb is required for the assembly of fully functional ribosomes. When RAC/Ssb is absent, ribosome biogenesis is hampered such that core ribosomal particles are structurally altered at the decoding and peptidyl transferase centers. As a result, ribosomes assembled in the absence of RAC/Ssb bind to the aminoglycoside paromomycin with high affinity (KD = 76.6 nM) and display impaired discrimination between stop codons and sense codons. The combined data shed light on the multiple mechanisms by which the RAC/Ssb system promotes unimpeded biogenesis of newly synthesized polypeptides.
Subject(s)
Codon/genetics , Molecular Chaperones/physiology , Multiprotein Complexes/physiology , Peptide Chain Termination, Translational/physiology , Ribosomes/metabolism , Saccharomyces cerevisiae Proteins/physiology , Codon, Terminator/genetics , Nucleic Acid Conformation , Organelle Biogenesis , Paromomycin/metabolism , Polylysine/genetics , RNA, Ribosomal/chemistry , RNA, Ribosomal/genetics , Saccharomyces cerevisiae Proteins/biosynthesis , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Inorganic polyphosphate is a ubiquitous, linear biopolymer built of up to thousands of phosphate residues that are linked by energy-rich phosphoanhydride bonds. Polyphosphate kinases of the family 2 (PPK2) use polyphosphate to catalyze the reversible phosphorylation of nucleotide phosphates and are highly relevant as targets for new pharmaceutical compounds and as biocatalysts for cofactor regeneration. PPK2s can be classified based on their preference for nucleoside mono- or diphosphates or both. The detailed mechanism of PPK2s and the molecular basis for their substrate preference is unclear, which is mainly due to the lack of high-resolution structures with substrates or substrate analogs. Here, we report the structural analysis and comparison of a class I PPK2 (ADP-phosphorylating) and a class III PPK2 (AMP- and ADP-phosphorylating), both complexed with polyphosphate and/or nucleotide substrates. Together with complementary biochemical analyses, these define the molecular basis of nucleotide specificity and are consistent with a Mg2+ catalyzed in-line phosphoryl transfer mechanism. This mechanistic insight will guide the development of PPK2 inhibitors as potential antibacterials or genetically modified PPK2s that phosphorylate alternative substrates.