Chemical Antiquity in Metabolism.

ConspectusLife is an exergonic chemical reaction. The same was true when the very first cells emerged at life's origin. In order to live, all cells need a source of carbon, energy, and electrons to drive their overall reaction network (metabolism). In most cells, these are separate pathways. There is only one biochemical pathway that serves all three needs simultaneously: the acetyl-CoA pathway of CO2 fixation. In the acetyl-CoA pathway, electrons from H2 reduce CO2 to pyruvate for carbon supply, while methane or acetate synthesis are coupled to energy conservation as ATP. This simplicity and thermodynamic favorability prompted Georg Fuchs and Erhard Stupperich to propose in 1985 that the acetyl-CoA pathway might mark the origin of metabolism, at the same time that Steve Ragsdale and Harland Wood were uncovering catalytic roles for Fe, Co, and Ni in the enzymes of the pathway. Subsequent work has provided strong support for those proposals.In the presence of Fe, Co, and Ni in their native metallic state as catalysts, aqueous H2 and CO2 react specifically to formate, acetate, methane, and pyruvate overnight at 100 °C. These metals (and their alloys) thus replace the function of over 120 enzymes required for the conversion of H2 and CO2 to pyruvate via the pathway and its cofactors, an unprecedented set of findings in the study of biochemical evolution. The reactions require alkaline conditions, which promote hydrogen oxidation by proton removal and are naturally generated in serpentinizing (H2-producing) hydrothermal vents. Serpentinizing hydrothermal vents furthermore produce natural deposits of native Fe, Co, Ni, and their alloys. These are precisely the metals that reduce CO2 with H2 in the laboratory; they are also the metals found at the active sites of enzymes in the acetyl-CoA pathway. Iron, cobalt and nickel are relicts of the environments in which metabolism arose, environments that still harbor ancient methane- and acetate-producing autotrophs today. This convergence indicates bedrock-level antiquity for the acetyl-CoA pathway. In acetogens and methanogens growing on H2 as reductant, the acetyl-CoA pathway requires flavin-based electron bifurcation as a source of reduced ferredoxin (a 4Fe4S cluster-containing protein) in order to function. Recent findings show that H2 can reduce the 4Fe4S clusters of ferredoxin in the presence of native iron, uncovering an evolutionary precursor of flavin-based electron bifurcation and suggesting an origin of FeS-dependent electron transfer in proteins. Traditionally discussed as catalysts in early evolution, the most common function of FeS clusters in metabolism is one-electron transfer, also in radical SAM enzymes, a large and ancient enzyme family. The cofactors and active sites in enzymes of the acetyl-CoA pathway uncover chemical antiquity in metabolism involving metals, methyl groups, methyl transfer reactions, cobamides, pterins, GTP, S-adenosylmethionine, radical SAM enzymes, and carbon-metal bonds. The reaction sequence from H2 and CO2 to pyruvate on naturally deposited native metals is maximally simple. It requires neither nitrogen, sulfur, phosphorus, RNA, ion gradients, nor light. Solid-state metal catalysts tether the origin of metabolism to a H2-producing, serpentinizing hydrothermal vent.

Thiol-Mediated Enhancement of Nε-Acetyllysine Formation in Lens Proteins.

Lysine acetylation (AcK) is a prominent post-translational modification in eye lens crystallins. We have observed that AcK formation is preferred in some lysine residues over others in crystallins. In this study, we have investigated the role of thiols in such AcK formation. Upon incubation with acetyl-CoA (AcCoA), αA-Crystallin, which contains two cysteine residues, showed significantly higher levels of AcK than αB-Crystallin, which lacks cysteine residues. Incubation with thiol-rich γS-Crystallin resulted in higher AcK formation in αB-Crystallin from AcCoA. External free thiol (glutathione and N-acetyl cysteine) increased the AcK content in AcCoA-incubated αB-Crystallin. Reductive alkylation of cysteine residues significantly decreased (p < 0.001) the AcCoA-mediated AcK formation in αA-Crystallin. Introduction of cysteine residues within ∼5 Å of lysine residues (K92C, E99C, and V169C) in αB-Crystallin followed by incubation with AcCoA resulted in a 3.5-, 1.3- and 1.3-fold increase in the AcK levels when compared to wild-type αB-Crystallin, respectively. Together, these results suggested that AcK formation in α-Crystallin is promoted by the proximal cysteine residues and protein-free thiols through an S → N acetyl transfer mechanism.

Enhancement of essential cofactors for in vivo biocatalysis.

A scarcity of cofactors, necessary metabolites or substrates for in vivo enzymatic reactions, is among the major barriers for product synthesis in metabolically engineered cells. This work compares our recently developed cofactor-boosting strategy, which uses xylose reductase (XR) and lactose to increase the intracellular levels of reduced or oxidized nicotinamide adenine dinucleotide (phosphate) (NAD(P)H), adenosine triphosphate (ATP) and acetyl coenzymeA (acetyl-CoA), with other previously reported methods. We demonstrated that the XR/lactose approach enhances levels of sugar alcohols and sugar phosphates, which leads to elevated levels of crucial cofactors required by specific metabolic pathways. The patterns of cofactor enhancement are not uniform and depend upon the specific pathway components that are overexpressed. We term this model the "user-pool" model. Here, we investigated metabolite alteration in the fatty-alcohol-producing system in the presence of XR/lactose within an early time frame (5 min after the bioconversion started). All metabolite data were analyzed using untargeted metabolomics. We found that the XR/lactose system could improve fatty-alcohol production as early as 5 min after the bioconversion started. The enhancement of key cofactors and intermediates, such as hexitol, NAD(P)H, ATP, 3-phosphoglycerate, acetyl-CoA, 6-phosphogluconate (6-PG) and glutathione, was consistent with those previously reported on a longer time scale (after 1 h). However, measurements performed at the early time reported here showed detectable differences in metabolite enhancement patterns, such as those of ATP, NADPH, acetyl-CoA and glutathione. These data could serve as a basis for future analysis of metabolic flux alteration by the XR/lactose system. Comparative analysis of the cofactor enhancement by XR and other methods suggests that XR/lactose can serve as a simple tool to increase levels of various cofactors for microbial cell factories.

How to Build a Metalloenzyme: Lessons from a Protein-Based Model of Acetyl Coenzyme A Synthase.

"What I cannot create, I do not understand"─Richard Feynman. This sentiment motivates the entire field of artificial metalloenzymes. Naturally occurring enzymes catalyze reactions with efficiencies, rates, and selectivity that generally cannot be achieved in synthetic systems. Many of these processes represent vital building blocks for a sustainable society, including CO2 conversion, nitrogen fixation, water oxidation, and liquid fuel synthesis. Our inability as chemists to fully reproduce the functionality of naturally occurring enzymes implicates yet-unknown contributors to reactivity. To identify these properties, it is necessary to consider all of the components of naturally occurring metalloenzymes, from the active site metal(s) to large-scale dynamics. In this Account, we describe the holistic development of a metalloprotein-based model that functionally reproduces the acetyl coenzyme A synthase (ACS) enzyme.ACS catalyzes the synthesis of a thioester, acetyl coenzyme A, from gaseous carbon monoxide, a methyl group donated by a cobalt corrinoid protein, and coenzyme A. The active site of ACS contains a bimetallic nickel site coupled to a [4Fe-4S] cluster. This reaction mimics Monsanto's acetic acid synthesis and represents an ancient process for incorporating inorganic carbon into cellular biomass through the primordial Wood-Ljungdahl metabolic pathway. From a sustainability standpoint, the reversible conversion of C1 substrates into an acetyl group and selective downstream transfer to a thiolate nucleophile offer opportunities to expand this reactivity to the anthropogenic synthesis of liquid fuels. However, substantial gaps in our understanding of the ACS catalytic mechanism coupled with the enzyme's oxygen sensitivity and general instability have limited these applications. It is our hope that development of an artificial metalloenzyme that carries out ACS-like reactions will advance our mechanistic understanding and enable synthesis of robust compounds with the capacity for similar reactivity.To construct this model, we first focused on the catalytic proximal nickel (NiP) site, which has a single metal center bound by three bridging cysteine residues in a "Y"-shaped arrangement. With an initial emphasis on reproducing the general structure of a low-coordinate metal binding site, the type I cupredoxin, azurin, was selected as the protein scaffold, and a nickel center was incorporated into the mononuclear site. Using numerous spectroscopic and computational techniques, including electron paramagnetic resonance (EPR) spectroscopy, nickel-substituted azurin was shown to have similar electronic and geometric structures to the NiP center in ACS. A substrate access channel was installed, and both carbon monoxide and a methyl group were shown to bind individually to the reduced NiI center. The elusive EPR-active S = 1/2 Ni-CH3 species, which has never been detected in native ACS, was observed in the azurin-based model, establishing the capacity of a biological NiI species to support two-electron organometallic reactions. Pulsed EPR studies on the S = 1/2 Ni-CH3 species in azurin suggested a noncanonical electronic structure with an inverted ligand field, which was proposed to prevent irreversible site degradation. This model azurin protein was ultimately shown to perform carbon-carbon and carbon-sulfur bond formation using sequential, ordered substrate addition for selective, stoichiometric thioester synthesis. X-ray spectroscopic methods were used to provide characterization of the remaining catalytic intermediates, resolving some debate over key mechanistic details.The overall approach and strategies that we employed for the successful construction of a functional protein-based model of ACS are described in this Account. We anticipate that these principles can be adapted across diverse metalloenzyme classes, providing essential mechanistic details and guiding the development of next-generation, functional artificial metalloenzymes.

The role of the phosphate groups of trinitrophenyl adenosine 5'-triphosphate (TNP-ATP) in allosteric activation of pyruvate carboxylase and the inhibition of acetyl CoA-dependent activation.

A previous study showed that 2'-3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) was a weak allosteric activator of Rhizobium etli pyruvate carboxylase (RePC) in the absence of acetyl-CoA. On the other hand, TNP-ATP inhibited the allosteric activation of RePC by acetyl-CoA. Here, we aimed to study the role of triphosphate group of TNP-ATP on its allosteric activation of the enzyme and inhibition of acetyl-CoA-dependent activation of RePC using TNP-ATP and its derivatives, including TNP-ADP, TNP-AMP and TNP-adenosine. The pyruvate carboxylation activity was assayed to determine the effect of reducing the number of phosphate groups in TNP-ATP derivatives on allosteric activation and inhibition of acetyl-CoA activation of RePC and chicken liver pyruvate carboxylase (CLPC). Reducing the number of phosphate groups in TNP-ATP derivatives decreased the activation efficacy for both RePC and CLPC compared to TNP-ATP. The apparent binding affinity and inhibition of activation of the enzymes by acetyl-CoA were also diminished when the number of phosphate groups in the TNP-ATP derivatives was reduced. Whilst TNP-AMP activated RePC, it did not activate CLPC, but it did inhibit acetyl-CoA activation of both RePC and CLPC. Similarly, TNP-adenosine did not activate RePC; however, it did inhibit acetyl-CoA activation using a different mechanism compared to phosphorylated TNP-derivatives. These findings indicate that mechanisms of PC activation and inhibition of acetyl-CoA activation by TNP-ATP and its derivatives are different. This study provides the basis for possible drug development for treatment of metabolic diseases and cancers with aberrant expression of PC.

Gas channel rerouting in a primordial enzyme: Structural insights of the carbon-monoxide dehydrogenase/acetyl-CoA synthase complex from the acetogen Clostridium autoethanogenum.

Clostridium autoethanogenum, the bacterial model for biological conversion of waste gases into biofuels, grows under extreme carbon-monoxide (CO) concentrations. The strictly anaerobic bacterium derives its entire cellular energy and carbon from this poisonous gas, therefore requiring efficient molecular machineries for CO-conversion. Here, we structurally and biochemically characterized the key enzyme of the CO-converting metabolism: the CO-dehydrogenase/Acetyl-CoA synthase (CODH/ACS). We obtained crystal structures of natively isolated complexes from fructose-grown and CO-grown C. autoethanogenum cultures. Both contain the same isoforms and if the overall structure adopts the classic α2ß2 architecture, comparable to the model enzyme from Moorella thermoacetica, the ACS binds a different position on the CODH core. The structural characterization of a proteolyzed complex and the conservation of the binding interface in close homologs rejected the possibility of a crystallization artefact. Therefore, the internal CO-channeling system, critical to transfer CO generated at the C-cluster to the ACS active site, drastically differs in the complex from C. autoethanogenum. The 1.9-Å structure of the CODH alone provides an accurate picture of the new CO-routes, leading to the ACS core and reaching the surface. Increased gas accessibility would allow the simultaneous CO-oxidation and acetyl-CoA production. Biochemical experiments showed higher flexibility of the ACS subunit from C. autoethanogenum compared to M. thermoacetica, albeit monitoring similar CO-oxidation and formation rates. These results show a reshuffling of internal CO-tunnels during evolution of these Firmicutes, putatively leading to a bidirectional complex that ensure a high flux of CO-conversion toward energy conservation, acting as the main cellular powerplant.

Heteroexpression and biochemical characterization of thermostable citrate synthase from the cyanobacteria Anabaena sp. PCC7120.

The present study recombinantly expressed a citrate synthase from cyanobacteria Anabaena sp. PCC7120 (AnCS) in Escherichia coli and characterized its enzymatic activity. The molecular mass of native AnCS was 88,533.1 Da containing two 44,162.7 Da subunits. Recombinant AnCS revealed the highest activity at pH 9.0 and 25 °C. AnCS displayed high thermal stability with a half-life time (t1/2) of approximately 6.5 h at 60 °C, which was more thermostable than most CS from general organisms, but less than those from hyperthermophilic bacteria. The Km values of oxaloacetate and acetyl-CoA were 138.50 and 18.15 µM respectively, suggesting a higher affinity to acetyl-CoA than oxaloacetate. Our inhibition assays showed that AnCS activity was not severely affected by most metal ions, but was strongly inhibited by Cu2+ and Zn2+. Treatments with ATP, ADP, AMP, NADH, and DTT depressed the AnCS activity. Overall, our results provide information on the enzymatic properties of AnCS, which contributes to the basic knowledge on CS selection for industrial utilizations.

Human Acetyl-CoA Carboxylase 1 Is an Isomerase: Carboxyl Transfer Is Activated by Catalytic Effect of Isomerization.

Obesity and its related diseases such as cancer and diabetes are leading life-threatening issues in the modern world. Thus, new drugs toward obesity and obesity-caused diseases are highly desired. Human acetyl-CoA carboxylase 1 (hACC1) in charge of the rate-limiting step of the human fatty acid synthesis was recognized as an attractive target for rational drug design. The fundamental reaction mechanism and nature of the transition state of hACC1 remain unclear. In this study, combined quantum mechanics and molecular mechanics (QM/MM), molecular dynamics (MD), and free-energy simulations were performed to investigate the catalytic mechanism of the hACC1-catalyzed carboxyl-transfer reaction. Our computational results show a three-step mechanism for carboxyl transferase (CT)-catalyzed reaction, including isomerization of carboxybiotin, proton-transfer from acetyl-CoA to carboxybiotin, and carboxylation of acetyl-CoA enolate. Interestingly, isomerization of carboxybiotin is the rate-limiting step of the entire reaction pathway, indicating hACC1 has the catalytic effect of isomerization and thus might be an isomerase also. The activation free-energy barrier of carboxyl-transfer catalyzed by hACC1 was calculated to be 16.4 kcal/mol, in excellent agreement with the experimental result (16.7 kcal/mol). The obtained reaction mechanism together with the nature of the transition state provides helpful knowledge not only for future investigation of other ACCs but also for rational design of hACC1 inhibitors, such as TS analogue. The catalytic effect of hACC1 isomerization is discussed.

Metabolic Intermediates in Tumorigenesis and Progression.

Traditional antitumor drugs inhibit the proliferation and metastasis of tumour cells by restraining the replication and expression of DNA. These drugs are usually highly cytotoxic. They kill tumour cells while also cause damage to normal cells at the same time, especially the hematopoietic cells that divide vigorously. Patients are exposed to other serious situations such as a severe infection caused by a decrease in the number of white blood cells. Energy metabolism is an essential process for the survival of all cells, but differs greatly between normal cells and tumour cells in metabolic pathways and metabolic intermediates. Whether this difference could be used as new therapeutic target while reducing damage to normal tissues is the topic of this paper. In this paper, we introduce five major metabolic intermediates in detail, including acetyl-CoA, SAM, FAD, NAD+ and THF. Their contents and functions in tumour cells and normal cells are significantly different. And the possible regulatory mechanisms that lead to these differences are proposed carefully. It is hoped that the key enzymes in these regulatory pathways could be used as new targets for tumour therapy.

Characterization of acetyl-CoA synthetase kinetics and ATP-binding.

BACKGROUND: The superfamily of adenylating enzymes is a large family of enzymes broadly distributed from bacteria to humans. Acetyl-CoA synthetase (Acs), member of this family, is a metabolic enzyme with an essential role in Escherichia coli (E. coli) acetate metabolism, whose catalytic activity is regulated by acetylation/deacetylation in vivo. METHODS: In this study, the kinetics and thermodynamic parameters of deacetylated and acetylated E. coli Acs were studied for the adenylating step. Moreover, the role of the T264, K270, D500 and K609 residues in catalysis and ATP-binding was also determined by Isothermal titration calorimetry. RESULTS: The results showed that native Acs enzyme binds ATP in an endothermic way. The dissociation constant has been determined and ATP-binding showed no significant differences between acetylated and deacetylated enzyme, although kcat was much higher for the deacetylated enzyme. However, K609 lysine mutation resulted in an increase in ATP-Acs-affinity and in a total loss of enzymatic activity, while T264 and D500 mutant proteins showed a total loss of ATP-binding ability and a decrease in catalytic activity. K609 site-specified acetylation induced a change in Acs conformation which resulted in an exothermic and more energetic ATP-binding. CONCLUSIONS: The differences in ATP-binding could explain the broadly conserved inactivation of Acs when K609 is acetylated. GENERAL SIGNIFICANCE: The results presented in this study demonstrate the importance of the selected residues in Acs ATP-binding and represent an advance in our understanding of the adenylation step of the superfamily of adenylating enzymes and of their acetylation/deacetylation regulation.

A Biochemical Nickel(I) State Supports Nucleophilic Alkyl Addition: A Roadmap for Methyl Reactivity in Acetyl Coenzyme A Synthase.

Nickel-containing enzymes such as methyl coenzyme M reductase (MCR) and carbon monoxide dehydrogenase/acetyl coenzyme A synthase (CODH/ACS) play a critical role in global energy conversion reactions, with significant contributions to carbon-centered processes. These enzymes are implied to cycle through a series of nickel-based organometallic intermediates during catalysis, though identification of these intermediates remains challenging. In this work, we have developed and characterized a nickel-containing metalloprotein that models the methyl-bound organometallic intermediates proposed in the native enzymes. Using a nickel(I)-substituted azurin mutant, we demonstrate that alkyl binding occurs via nucleophilic addition of methyl iodide as a methyl donor. The paramagnetic NiIII-CH3 species initially generated can be rapidly reduced to a high-spin NiII-CH3 species in the presence of exogenous reducing agent, following a reaction sequence analogous to that proposed for ACS. These two distinct bioorganometallic species have been characterized by optical, EPR, XAS, and MCD spectroscopy, and the overall mechanism describing methyl reactivity with nickel azurin has been quantitatively modeled using global kinetic simulations. A comparison between the nickel azurin protein system and existing ACS model compounds is presented. NiIII-CH3 Az is only the second example of two-electron addition of methyl iodide to a NiI center to give an isolable species and the first to be formed in a biologically relevant system. These results highlight the divergent reactivity of nickel across the two intermediates, with implications for likely reaction mechanisms and catalytically relevant states in the native ACS enzyme.

New Insights into Behçet's Syndrome Metabolic Reprogramming: Citrate Pathway Dysregulation.

To date, a major research effort on Behçet's syndrome (BS) has been concentrated on immunological aspects. Little is known about the metabolic reprogramming in BS. Citrate is an intermediary metabolite synthesized in mitochondria, and when transported into the cytosol by the mitochondrial citrate carrier-SLC25A1-encoded protein-it is cleaved into acetyl-CoA and oxaloacetate by ATP citrate lyase (ACLY). In induced macrophages, mitochondrial citrate is necessary for the production of inflammatory mediators. The aim of our study was to evaluate SLC25A1 and ACLY expression levels in BS patients. Following a power analysis undertaken on few random samples, the number of enrolled patients was set. Thirty-nine consecutive BS patients fulfilling ISG criteria, and 21 healthy controls suitable for age and sex were recruited. BS patients were divided into two groups according to the presence (active) or absence (inactive) of clinical manifestations. Real-time PCR experiments were performed on PBMCs to quantify SLC25A1 and ACLY mRNA levels. Data processing through the Kruskal-Wallis test and Dunn's multiple comparison test as post hoc showed higher SLC25A1 and ACLY mRNA levels in BS patients compared to those in healthy controls. Therefore, SLC25A1 and ACLY upregulation suggests that metabolic reprogramming in BS involves the citrate pathway dysregulation.

Activity Essential Residue Analysis of Taxoid 10ß-O-Acetyl Transferase for Enzymatic Synthesis of Baccatin.

Taxoid 10ß-O-acetyl transferase (DBAT) is a key enzyme in the biosynthesis of the famous anticancer drug paclitaxel, which catalyses the formation of baccatin III from 10-deacetylbaccatin III (10-DAB). However, the activity essential residues of the enzyme are still unknown, and the acylation mechanism from its natural substrate 10-deacetylbaccatin III and acetyl CoA to baccatin III remains unclear. In this study, the homology modelling, molecular docking, site-directed mutagenesis, and kinetic parameter determination of the enzyme were carried out. The results showed that the enzyme mutant DBATH162A resulted in complete loss of enzymatic activity, suggesting that the residue histidine at 162 was essential to DBAT activity. Residues D166 and R363 which were located in the pocket of the enzyme by homology modelling and molecular docking were also important for DBAT activity through the site-directed mutations. Furthermore, four amino acid residues including S31 and D34 from motif SXXD, D372 and G376 from motif DFGWG also played important roles on acylation. This was the first report of the elucidation of the activity essential residues of DBAT, making it possible for the further structural-based re-design of the enzyme for efficient biotransformation of baccatin III and paclitaxel.

Native iron reduces CO2 to intermediates and end-products of the acetyl-CoA pathway.

Autotrophic theories for the origin of life propose that CO2 was the carbon source for primordial biosynthesis. Among the six known CO2 fixation pathways in nature, the acetyl-CoA (AcCoA; or Wood-Ljungdahl) pathway is the most ancient, and relies on transition metals for catalysis. Modern microbes that use the AcCoA pathway typically fix CO2 with electrons from H2, which requires complex flavin-based electron bifurcation. This presents a paradox: how could primitive metabolic systems have fixed CO2 before the origin of proteins? Here, we show that native transition metals (Fe0, Ni0 and Co0) selectively reduce CO2 to acetate and pyruvate-the intermediates and end-products of the AcCoA pathway-in near millimolar concentrations in water over hours to days using 1-40 bar CO2 and at temperatures from 30 to 100 °C. Geochemical CO2 fixation from native metals could have supplied critical C2 and C3 metabolites before the emergence of enzymes.

Structural and Functional Studies of the Daunorubicin Priming Ketosynthase DpsC.

Daunorubicin is a type II polyketide, one of a large class of polyaromatic natural products with anticancer, antibiotic, and antiviral activity. Type II polyketides are formed by the assembly of malonyl-CoA building blocks, though in rare cases, biosynthesis is initiated by the incorporation of a nonmalonyl derived starter unit, which adds molecular diversity to the poly-ß-ketone backbone. Priming mechanisms for the transfer of novel starter units onto polyketide synthases (PKS) are still poorly understood. Daunorubicin biosynthesis incorporates a unique propionyl starter unit thought to be selected for by a subclass ("DpsC type") of priming ketosynthases (KS III). To date, however, no structural information exists for this subclass of KS III enzymes. Although selectivity for self-acylation with propionyl-CoA has previously been implied, we demonstrate that DpsC shows no discrimination for self-acylation or acyl-transfer to the cognate acyl carrier protein, DpsG with short acyl-CoAs. We present five crystal structures of DpsC, including apo-DpsC, acetyl-DpsC, propionyl-DpsC, butyryl-DpsC, and a cocrystal of DpsC with a nonhydrolyzable phosphopantetheine (PPant) analogue. The DpsC crystal structures reveal the architecture of the active site, the molecular determinants for catalytic activity and homology to O-malonyl transferases, but also indicate distinct differences. These results provide a structural basis for rational engineering of starter unit selection in type II polyketide synthases.

iPSC-derived neuronal models of PANK2-associated neurodegeneration reveal mitochondrial dysfunction contributing to early disease.

Mutations in PANK2 lead to neurodegeneration with brain iron accumulation. PANK2 has a role in the biosynthesis of coenzyme A (CoA) from dietary vitamin B5, but the neuropathological mechanism and reasons for iron accumulation remain unknown. In this study, atypical patient-derived fibroblasts were reprogrammed into induced pluripotent stem cells (iPSCs) and subsequently differentiated into cortical neuronal cells for studying disease mechanisms in human neurons. We observed no changes in PANK2 expression between control and patient cells, but a reduction in protein levels was apparent in patient cells. CoA homeostasis and cellular iron handling were normal, mitochondrial function was affected; displaying activated NADH-related and inhibited FADH-related respiration, resulting in increased mitochondrial membrane potential. This led to increased reactive oxygen species generation and lipid peroxidation in patient-derived neurons. These data suggest that mitochondrial deficiency is an early feature of the disease process and can be explained by altered NADH/FADH substrate supply to oxidative phosphorylation. Intriguingly, iron chelation appeared to exacerbate the mitochondrial phenotype in both control and patient neuronal cells. This raises caution for the use iron chelation therapy in general when iron accumulation is absent.

Substrate-Induced Facilitated Dissociation of the Competitive Inhibitor from the Active Site of O-Acetyl Serine Sulfhydrylase Reveals a Competitive-Allostery Mechanism.

By classical competitive antagonism, a substrate and competitive inhibitor must bind mutually exclusively to the active site. The competitive inhibition of O-acetyl serine sulfhydrylase (OASS) by the C-terminus of serine acetyltransferase (SAT) presents a paradox, because the C-terminus of SAT binds to the active site of OASS with an affinity that is 4-6 log-fold (104-106) greater than that of the substrate. Therefore, we employed multiple approaches to understand how the substrate gains access to the OASS active site under physiological conditions. Single-molecule and ensemble approaches showed that the active site-bound high-affinity competitive inhibitor is actively dissociated by the substrate, which is not consistent with classical views of competitive antagonism. We employed fast-flow kinetic approaches to demonstrate that substrate-mediated dissociation of full length SAT-OASS (cysteine regulatory complex) follows a noncanonical "facilitated dissociation" mechanism. To understand the mechanism by which the substrate induces inhibitor dissociation, we resolved the crystal structures of enzyme·inhibitor·substrate ternary complexes. Crystal structures reveal a competitive allosteric binding mechanism in which the substrate intrudes into the inhibitor-bound active site and disengages the inhibitor before occupying the site vacated by the inhibitor. In summary, here we reveal a new type of competitive allosteric binding mechanism by which one of the competitive antagonists facilitates the dissociation of the other. Together, our results indicate that "competitive allostery" is the general feature of noncanonical "facilitated/accelerated dissociation" mechanisms. Further understanding of the mechanistic framework of "competitive allosteric" mechanism may allow us to design a new family of "competitive allosteric drugs/small molecules" that will have improved selectivity and specificity as compared to their competitive and allosteric counterparts.

Mechanism of host substrate acetylation by a YopJ family effector.

The Yersinia outer protein J (YopJ) family of bacterial effectors depends on a novel acetyltransferase domain to acetylate signalling proteins from plant and animal hosts. However, the underlying mechanism is unclear. Here, we report the crystal structures of PopP2, a YopJ effector produced by the plant pathogen Ralstonia solanacearum, in complex with inositol hexaphosphate (InsP6), acetyl-coenzyme A (AcCoA) and/or substrate Resistance to Ralstonia solanacearum 1 (RRS1-R)WRKY. PopP2 recognizes the WRKYGQK motif of RRS1-RWRKY to position a targeted lysine in the active site for acetylation. Importantly, the PopP2-RRS1-RWRKY association is allosterically regulated by InsP6 binding, suggesting a previously unidentified role of the eukaryote-specific cofactor in substrate interaction. Furthermore, we provide evidence for the reaction intermediate of PopP2-mediated acetylation, an acetyl-cysteine covalent adduct, lending direct support to the 'ping-pong'-like catalytic mechanism proposed for YopJ effectors. Our study provides critical mechanistic insights into the virulence activity of YopJ class of acetyltransferases.

Kinetic and Thermodynamic Analysis of Acetyl-CoA Activation of Staphylococcus aureus Pyruvate Carboxylase.

Allosteric regulation of pyruvate carboxylase (PC) activity is pivotal to maintaining metabolic homeostasis. In contrast, dysregulated PC activity contributes to the pathogenesis of numerous diseases, rendering PC a possible target for allosteric therapeutic development. Recent research efforts have focused on demarcating the role of acetyl-CoA, one of the most potent activators of PC, in coordinating catalytic events within the multifunctional enzyme. Herein, we report a kinetic and thermodynamic analysis of acetyl-CoA activation of the Staphylococcus aureus PC (SaPC)-catalyzed carboxylation of pyruvate to identify novel means by which acetyl-CoA synchronizes catalytic events within the PC tetramer. Kinetic and linked-function analysis, or thermodynamic linkage analysis, indicates that the substrates of the biotin carboxylase and carboxyl transferase domain are energetically coupled in the presence of acetyl-CoA. In contrast, both kinetic and energetic coupling between the two domains is lost in the absence of acetyl-CoA, suggesting a functional role for acetyl-CoA in facilitating the long-range transmission of substrate-induced conformational changes within the PC tetramer. Interestingly, thermodynamic activation parameters for the SaPC-catalyzed carboxylation of pyruvate are largely independent of acetyl-CoA. Our results also reveal the possibility that global conformational changes give rise to observed species-specific thermodynamic activation parameters. Taken together, our kinetic and thermodynamic results provide a possible allosteric mechanism by which acetyl-CoA coordinates catalysis within the PC tetramer.

Iron catalysis at the origin of life.

Iron-sulphur proteins are ancient and drive fundamental processes in cells, notably electron transfer and CO2 fixation. Iron-sulphur minerals with equivalent structures could have played a key role in the origin of life. However, the 'iron-sulphur world' hypothesis has had a mixed reception, with questions raised especially about the feasibility of a pyrites-pulled reverse Krebs cycle. Phylogenetics suggests that the earliest cells drove carbon and energy metabolism via the acetyl CoA pathway, which is also replete in Fe(Ni)S proteins. Deep differences between bacteria and archaea in this pathway obscure the ancestral state. These differences make sense if early cells depended on natural proton gradients in alkaline hydrothermal vents. If so, the acetyl CoA pathway diverged with the origins of active ion pumping, and ancestral CO2 fixation might have been equivalent to methanogens, which depend on a membrane-bound NiFe hydrogenase, energy converting hydrogenase. This uses the proton-motive force to reduce ferredoxin, thence CO2 . The mechanism suggests that pH could modulate reduction potential at the active site of the enzyme, facilitating the difficult reduction of CO2 by H2 . This mechanism could be generalised under abiotic conditions so that steep pH differences across semi-conducting Fe(Ni)S barriers drives not just the first steps of CO2 fixation to C1 and C2 organics such as CO, CH3 SH and CH3 COSH, but a series of similar carbonylation and hydrogenation reactions to form longer chain carboxylic acids such as pyruvate, oxaloacetate and α-ketoglutarate, as in the incomplete reverse Krebs cycle found in methanogens. We suggest that the closure of a complete reverse Krebs cycle, by regenerating acetyl CoA directly, displaced the acetyl CoA pathway from many modern groups. A later reliance on acetyl CoA and ATP eliminated the need for the proton-motive force to drive most steps of the reverse Krebs cycle. © 2017 IUBMB Life, 69(6):373-381, 2017.