Kinetic characterization of the C-terminal domain of Malonyl-CoA reductase.

Malonyl-CoA reductase utilizes two equivalents of NADPH to catalyze the reduction of malonyl-CoA to 3-hydroxypropionic acid (3HP). This reaction is part of the carbon fixation pathway in the phototrophic bacterium Chloroflexus aurantiacus. The enzyme is composed of two domains. The C-terminal domain catalyzes the reduction of malonyl-CoA to malonic semialdehyde, while the N-terminal domain catalyzes the reduction of the aldehyde to 3HP. The two domains can be produced independently and retain their enzymatic activity. This report focuses on the kinetic characterization of the C-terminal domain. Initial velocity patterns and inhibition studies showed the kinetic mechanism is ordered with NADPH binding first followed by malonyl-CoA. Malonic semialdehyde is released first, while CoA and NADP+ are released randomly. Analogs of malonyl-CoA showed that the thioester carbon is reduced, while the carboxyl group is needed for proper positioning. The enzyme transfers the pro-S hydrogen of NADPH to malonyl-CoA and pH rate profiles revealed that a residue with a pKa value of about 8.8 must be protonated for activity. Kinetic isotope effects indicated that NADPH is not sticky (that is, NADPH dissociates from the enzyme faster than the rate of product formation) and product release is partially rate-limiting. Moreover, the mechanism is stepwise with the pH dependent step occurring before or after hydride transfer. The findings from this study will aid in the development of an eco-friendly biosynthesis of 3HP which is an industrial chemical used in the production of plastics and adhesives.

Kinetic characterization of the N-terminal domain of Malonyl-CoA reductase.

Climate change is driving a search for environmentally safe methods to produce chemicals used in ordinary life. One such molecule is 3-hydroxypropionic acid, which is a platform industrial chemical used as a precursor for a variety of other chemical end products. The biosynthesis of 3-hydroxypropionic acid can be achieved in recombinant microorganisms via malonyl-CoA reductase in two separate reactions. The reduction of malonyl-CoA by NADPH to form malonic semialdehyde is catalyzed in the C-terminal domain of malonyl-CoA reductase, while the subsequent reduction of malonic semialdehyde to 3-hydroxypropionic acid is accomplished in the N-terminal domain of the enzyme. A new assay for the reverse reaction of the N-terminal domain of malonyl-CoA reductase from Chloroflexus aurantiacus activity has been developed. This assay was used to determine the kinetic mechanism and for isotope effect studies. Kinetic characterization using initial velocity patterns revealed random binding of the substrates NADP+ and 3-hydroxypropionic acid. Isotope effects showed substrates react to give products faster than they dissociate and that the products of the reverse reaction, NADPH and malonic semialdehyde, have a low affinity for the enzyme. Multiple isotope effects suggest proton and hydride transfer occur in a concerted fashion. This detailed kinetic characterization of the reaction catalyzed by the N-terminal domain of malonyl-CoA reductase could aid in engineering of the enzyme to make the biosynthesis of 3-hydroxypropionic acid commercially competitive with its production from fossil fuels.

Mechanism of biotin carboxylase inhibition by ethyl 4-[[2-chloro-5-(phenylcarbamoyl)phenyl]sulphonylamino]benzoate.

The rise of antibacterial-resistant bacteria is a major problem in the United States of America and around the world. Millions of patients are infected with antimicrobial resistant bacteria each year. Novel antibacterial agents are needed to combat the growing and present crisis. Acetyl-CoA carboxylase (ACC), the multi-subunit complex which catalyses the first committed step in fatty acid synthesis, is a validated target for antibacterial agents. However, there are at present, no commercially available antibiotics that target ACC. Ethyl 4-[[2-chloro-5-(phenylcarbamoyl)phenyl]sulfonylamino]benzoate (SABA1) is a compound that has been shown to have antibacterial properties against Pseudomonas aeruginosa and Escherichia coli. SABA1 inhibits biotin carboxylase (BC), the enzyme that catalyses the first half reaction of ACC. SABA1 inhibits BC via an atypical mechanism. It binds in the biotin binding site in the presence of ADP. SABA1 represents a potentially new class of antibiotics that can be used to combat the antibacterial resistance crisis.

Pyrrolocin C and equisetin inhibit bacterial acetyl-CoA carboxylase.

Antimicrobial resistance is a growing global health and economic concern. Current antimicrobial agents are becoming less effective against common bacterial infections. We previously identified pyrrolocins A and C, which showed activity against a variety of Gram-positive bacteria. Structurally similar compounds, known as pyrrolidinediones (e.g., TA-289, equisetin), also display antibacterial activity. However, the mechanism of action of these compounds against bacteria was undetermined. Here, we show that pyrrolocin C and equisetin inhibit bacterial acetyl-CoA carboxylase (ACC), the first step in fatty acid synthesis. We used transcriptomic data, metabolomic analysis, fatty acid rescue and acetate incorporation experiments to show that a major mechanism of action of the pyrrolidinediones is inhibition of fatty acid biosynthesis, identifying ACC as the probable molecular target. This hypothesis was further supported using purified proteins, demonstrating that biotin carboxylase is the inhibited component of ACC. There are few known antibiotics that target this pathway and, therefore, we believe that these compounds may provide the basis for alternatives to current antimicrobial therapy.

Optimization and Mechanistic Characterization of Pyridopyrimidine Inhibitors of Bacterial Biotin Carboxylase.

A major challenge for new antibiotic discovery is predicting the physicochemical properties that enable small molecules to permeate Gram-negative bacterial membranes. We have applied physicochemical lessons from previous work to redesign and improve the antibacterial potency of pyridopyrimidine inhibitors of biotin carboxylase (BC) by up to 64-fold and 16-fold against Escherichia coli and Pseudomonas aeruginosa, respectively. Antibacterial and enzyme potency assessments in the presence of an outer membrane-permeabilizing agent or in efflux-compromised strains indicate that penetration and efflux properties of many redesigned BC inhibitors could be improved to various extents. Spontaneous resistance to the improved pyridopyrimidine inhibitors in P. aeruginosa occurs at very low frequencies between 10-8 and 10-9. However, resistant isolates had alarmingly high minimum inhibitory concentration shifts (16- to >128-fold) compared to the parent strain. Whole-genome sequencing of resistant isolates revealed that either BC target mutations or efflux pump overexpression can lead to the development of high-level resistance.

Capillary electrophoretic assay of human acetyl-coenzyme A carboxylase 2.

Human acetyl-coenzyme A carboxylase 2 catalyzes the carboxylation of acetyl coenzyme A to form malonyl coenzyme A, along with the conversion of magnesium-adenosine triphosphate complex to magnesium-adenosine diphosphate complex. A simple off-column capillary electrophoresis assay for human acetyl-coenzyme A carboxylase 2 was developed based on the separation of magnesium-adenosine triphosphate complex, magnesium-adenosine diphosphate complex, acetyl coenzyme A and malonyl coenzyme A with detection by ultraviolet absorption at 256 nm. When Mg2+ was absent from the separation buffer, the zones due to magnesium-adenosine triphosphate complex and magnesium-adenosine diphosphate complex both split and migrated as two separate peaks. With Mg2+ added to the separation buffer, magnesium-adenosine triphosphate complex and magnesium-adenosine diphosphate complex produced single peaks, and the reproducibility of peak shape and area improved for human acetyl-coenzyme A carboxylase 2 assay components. The final separation buffer used was 30.0 mM HEPES, 3.0 mM MgCl2 , 2.5 mM KHCO3 , and 2.5 mM potassium citrate at pH 7.50. The same buffer was used for the enzyme-catalyzed reaction (off-column). Inhibition of human acetyl-coenzyme A carboxylase 2 by CP-640186, a known inhibitor, was detected using the capillary electrophoresis assay.

The many types of carbonic anhydrases in photosynthetic organisms.

Carbonic anhydrases (CAs) are enzymes that catalyze the interconversion of CO2 and HCO3-. In nature, there are multiple families of CA, designated with the Greek letters α through θ. CAs are ubiquitous in plants, algae and photosynthetic bacteria, often playing essential roles in the CO2 concentrating mechanisms (CCMs) which enhance the delivery of CO2 to Rubisco. As algal CCMs become better characterized, it is clear that different types of CAs are playing the same role in different algae. For example, an α-CA catalyzes the conversion of accumulated HCO3- to CO2 in the green alga Chlamydomonas reinhardtii, while a θ-CA performs the same function in the diatom Phaeodactylum tricornutum. In this review we argue that, in addition to its role of delivering CO2 for photosynthesis, other metabolic roles of CA have likely changed as the Earth's atmospheric CO2 level decreased. Since the algal and plant lineages diverged well before the decrease in atmospheric CO2, it is likely that plant, algae and photosynthetic bacteria all adapted independently to the drop in atmospheric CO2. In light of this, we will discuss how the roles of CAs may have changed over time, focusing on the role of CA in pH regulation, how CAs affect CO2 supply for photosynthesis and how CAs may help in the delivery of HCO3- for other metabolic reactions.

Acetyl-CoA carboxylase from Escherichia coli exhibits a pronounced hysteresis when inhibited by palmitoyl-acyl carrier protein.

Acetyl-CoA carboxylase (ACC) in bacteria is composed of three components: biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase. ACC catalyzes the first committed step in fatty acid synthesis: the carboxylation of acetyl-CoA to form malonyl-CoA via a two-step reaction. In the first half-reaction, biotin carboxylase catalyzes the ATP-dependent carboxylation of the vitamin biotin covalently linked to biotin carboxyl carrier protein. In the second half-reaction, the carboxyl group is transferred from biotin to acetyl-CoA by the enzyme carboxyltransferase, to form malonyl-CoA. In most Gram-negative and Gram-positive bacteria, the three components of ACC form a complex that requires communication for catalysis, and is subject to feedback inhibition by acylated-acyl carrier proteins. This study investigated the mechanism of inhibition of palmitoyl-acyl carrier protein (PACP) on ACC. Unexpectedly, ACC was found to exhibit a significant hysteresis, meaning ACC was subject to inhibition by PACP in a time dependent manner. Pull-down assays demonstrated PACP does not prevent formation of the multiprotein complex, while steady-state kinetic analyses showed PACP inhibited ACC activity allosterically. Structure-activity analyses revealed that the pantothenic acid moiety of PACP is responsible for the inhibition of ACC. This study provides the first evidence of the hysteretic nature of ACC.

Crystal Structure of Carboxyltransferase from Staphylococcus aureus Bound to the Antibacterial Agent Moiramide B.

The dramatic increase in the prevalence of antibiotic-resistant bacteria has necessitated a search for new antibacterial agents against novel targets. Moiramide B is a natural product, broad-spectrum antibiotic that inhibits the carboxyltransferase component of acetyl-CoA carboxylase, which catalyzes the first committed step in fatty acid synthesis. Herein, we report the 2.6 Å resolution crystal structure of moiramide B bound to carboxyltransferase. An unanticipated but significant finding was that moiramide B bound as the enol/enolate. Crystallographic studies demonstrate that the (4S)-methyl succinimide moiety interacts with the oxyanion holes of the enzyme, supporting the notion that an anionic enolate is the active form of the antibacterial agent. Structure-activity studies demonstrate that the unsaturated fatty acid tail of moiramide B is needed only for entry into the bacterial cell. These results will allow the design of new antibacterial agents against the bacterial form of carboxyltransferase.

Carbon Fixation Driven by Molecular Hydrogen Results in Chemolithoautotrophically Enhanced Growth of Helicobacter pylori.

UNLABELLED: A molecular hydrogen (H2)-stimulated, chemolithoautotrophic growth mode for the gastric pathogen Helicobacter pylori is reported. In a culture medium containing peptides and amino acids, H2-supplied cells consistently achieved 40 to 60% greater growth yield in 16 h and accumulated 3-fold more carbon from [(14)C]bicarbonate (on a per cell basis) in a 10-h period than cells without H2 Global proteomic comparisons of cells supplied with different atmospheric conditions revealed that addition of H2 led to increased amounts of hydrogenase and the biotin carboxylase subunit of acetyl coenzyme A (acetyl-CoA) carboxylase (ACC), as well as other proteins involved in various cellular functions, including amino acid metabolism, heme synthesis, or protein degradation. In agreement with this result, H2-supplied cells contained 3-fold more ACC activity than cells without H2 Other possible carbon dioxide (CO2) fixation enzymes were not up-expressed under the H2-containing atmosphere. As the gastric mucus is limited in carbon and energy sources and the bacterium lacks mucinase, this new growth mode may contribute to the persistence of the pathogen in vivo This is the first time that chemolithoautotrophic growth is described for a pathogen. IMPORTANCE: Many pathogens must survive within host areas that are poorly supplied with carbon and energy sources, and the gastric pathogen Helicobacter pylori resides almost exclusively in the nutritionally stringent mucus barrier of its host. Although this bacterium is already known to be highly adaptable to gastric niches, a new aspect of its metabolic flexibility, whereby molecular hydrogen use (energy) is coupled to carbon dioxide fixation (carbon acquisition) via a described carbon fixation enzyme, is shown here. This growth mode, which supplements heterotrophy, is termed chemolithoautotrophy and has not been previously reported for a pathogen.

Structural Analysis of Substrate, Reaction Intermediate, and Product Binding in Haemophilus influenzae Biotin Carboxylase.

Acetyl-CoA carboxylase catalyzes the first and regulated step in fatty acid synthesis. In most Gram-negative and Gram-positive bacteria, the enzyme is composed of three proteins: biotin carboxylase, a biotin carboxyl carrier protein (BCCP), and carboxyltransferase. The reaction mechanism involves two half-reactions with biotin carboxylase catalyzing the ATP-dependent carboxylation of biotin-BCCP in the first reaction. In the second reaction, carboxyltransferase catalyzes the transfer of the carboxyl group from biotin-BCCP to acetyl-CoA to form malonyl-CoA. In this report, high-resolution crystal structures of biotin carboxylase from Haemophilus influenzae were determined with bicarbonate, the ATP analogue AMPPCP; the carboxyphosphate intermediate analogues, phosphonoacetamide and phosphonoformate; the products ADP and phosphate; and the carboxybiotin analogue N1'-methoxycarbonyl biotin methyl ester. The structures have a common theme in that bicarbonate, phosphate, and the methyl ester of the carboxyl group of N1'-methoxycarbonyl biotin methyl ester all bound in the same pocket in the active site of biotin carboxylase and as such utilize the same set of amino acids for binding. This finding suggests a catalytic mechanism for biotin carboxylase in which the binding pocket that binds tetrahedral phosphate also accommodates and stabilizes a tetrahedral dianionic transition state resulting from direct transfer of CO2 from the carboxyphosphate intermediate to biotin.

Design, synthesis, and antibacterial properties of dual-ligand inhibitors of acetyl-CoA carboxylase.

There is an urgent demand for the development of new antibiotics due to the increase in drug-resistant pathogenic bacteria. A novel target is the multifunctional enzyme acetyl-CoA carboxylase (ACC), which catalyzes the first committed step in fatty acid synthesis and consists of two enzymes: biotin carboxylase and carboxyltransferase. Covalently attaching known inhibitors against these enzymes with saturated hydrocarbon linkers of different lengths generated dual-ligand inhibitors. Kinetic results revealed that the dual-ligands inhibited the ACC complex in the nanomolar range. Microbiology assays showed that the dual-ligand with a 15-carbon linker did not exhibit any antibacterial activity, while the dual-ligand with a 7-carbon linker displayed broad-spectrum antibacterial activity as well as a decreased susceptibility in the development of bacterial resistance. These results suggest that the properties of the linker are vital for antibacterial activity and show how inhibiting two different enzymes with the same compound increases the overall potency while also impeding the development of resistance.

Computational redesign of bacterial biotin carboxylase inhibitors using structure-based virtual screening of combinatorial libraries.

As the spread of antibiotic resistant bacteria steadily increases, there is an urgent need for new antibacterial agents. Because fatty acid synthesis is only used for membrane biogenesis in bacteria, the enzymes in this pathway are attractive targets for antibacterial agent development. Acetyl-CoA carboxylase catalyzes the committed and regulated step in fatty acid synthesis. In bacteria, the enzyme is composed of three distinct protein components: biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase. Fragment-based screening revealed that amino-oxazole inhibits biotin carboxylase activity and also exhibits antibacterial activity against Gram-negative organisms. In this report, we redesigned previously identified lead inhibitors to expand the spectrum of bacteria sensitive to the amino-oxazole derivatives by including Gram-positive species. Using 9,411 small organic building blocks, we constructed a diverse combinatorial library of 1.2×108 amino-oxazole derivatives. A subset of 9×106 of these compounds were subjected to structure-based virtual screening against seven biotin carboxylase isoforms using similarity-based docking by eSimDock. Potentially broad-spectrum antibiotic candidates were selected based on the consensus ranking by several scoring functions including non-linear statistical models implemented in eSimDock and traditional molecular mechanics force fields. The analysis of binding poses of the top-ranked compounds docked to biotin carboxylase isoforms suggests that: (1) binding of the amino-oxazole anchor is stabilized by a network of hydrogen bonds to residues 201, 202 and 204; (2) halogenated aromatic moieties attached to the amino-oxazole scaffold enhance interactions with a hydrophobic pocket formed by residues 157, 169, 171 and 203; and (3) larger substituents reach deeper into the binding pocket to form additional hydrogen bonds with the side chains of residues 209 and 233. These structural insights into drug-biotin carboxylase interactions will be tested experimentally in in vitro and in vivo systems to increase the potency of amino-oxazole inhibitors towards both Gram-negative as well as Gram-positive species.

Capillary electrophoresis-based assay of phosphofructokinase-1.

An assay was developed for phosphofructokinase-1 (PFK-1) using capillary electrophoresis (CE). In the glycolytic pathway, this enzyme catalyzes the rate-limiting step from fructose-6-phosphate and magnesium-bound adenosine triphosphate (Mg-ATP) to fructose-1,6-bisphosphate and magnesium-bound adenosine diphosphate (Mg-ADP). This enzyme has recently become a research target because of the importance of glycolysis in cancer and obesity. The CE assay for PFK-1 is based on the separation and detection by ultraviolet (UV) absorbance at 260 nm of Mg-ATP and Mg-ADP. The separation was enhanced by the addition of Mg²âº to the separation buffer. Inhibition studies of PFK-1 by aurintricarboxylic acid and palmitoyl coenzyme A were also performed. An IC50 value was determined for aurintricarboxylic acid, and this value matched values in the literature obtained using coupled spectrophotometric assays. This assay for PFK-1 directly monitors the enzyme-catalyzed reaction, and the CE separation reduces the potential of spectral interference by inhibitors.

Complex formation and regulation of Escherichia coli acetyl-CoA carboxylase.

Acetyl-CoA carboxylase is a biotin-dependent enzyme that catalyzes the regulated step in fatty acid synthesis. The bacterial form has three separate components: biotin carboxylase, biotin carboxyl carrier protein (BCCP), and carboxyltransferase. Catalysis by acetyl-CoA carboxylase proceeds via two half-reactions. In the first half-reaction, biotin carboxylase catalyzes the ATP-dependent carboxylation of biotin, which is covalently attached to BCCP, to form carboxybiotin. In the second half-reaction, carboxyltransferase transfers the carboxyl group from carboxybiotin to acetyl-CoA to form malonyl-CoA. All biotin-dependent carboxylases are proposed to have a two-site ping-pong mechanism in which the carboxylase and transferase activities are separate and do not interact. This posits two hypotheses: either biotin carboxylase and BCCP undergo the first half-reaction, BCCP dissociates, and then BCCP binds to carboxyltransferase, or all three constituents form an enzyme complex. To determine which hypothesis is correct, a steady-state enzyme kinetic analysis of Escherichia coli acetyl-CoA carboxylase was conducted. The results indicated the two active sites of acetyl-CoA carboxylase interact. Both in vitro and in vivo pull-down assays demonstrated that the three components of E. coli acetyl-CoA carboxylase form a multimeric complex and that complex formation is unaffected by acetyl-CoA, AMPPNP, and mRNA encoding carboxyltransferase. The implications of these findings for the regulation of acetyl-CoA carboxylase and fatty acid biosynthesis are discussed.

The three-dimensional structure of the biotin carboxylase-biotin carboxyl carrier protein complex of E. coli acetyl-CoA carboxylase.

Acetyl-coenzyme A (acetyl-CoA) carboxylase is a biotin-dependent, multifunctional enzyme that catalyzes the regulated step in fatty acid synthesis. The Escherichia coli enzyme is composed of a homodimeric biotin carboxylase (BC), biotinylated biotin carboxyl carrier protein (BCCP), and an α2ß2 heterotetrameric carboxyltransferase. This enzyme complex catalyzes two half-reactions to form malonyl-coenzyme A. BC and BCCP participate in the first half-reaction, whereas carboxyltransferase and BCCP are involved in the second. Three-dimensional structures have been reported for the individual subunits; however, the structural basis for how BCCP reacts with the carboxylase or transferase is unknown. Therefore, we report here the crystal structure of E. coli BCCP complexed with BC to a resolution of 2.49 Å. The protein-protein complex shows a unique quaternary structure and two distinct interfaces for each BCCP monomer. These BCCP binding sites are unique compared to phylogenetically related biotin-dependent carboxylases and therefore provide novel targets for developing antibiotics against bacterial acetyl-CoA carboxylase.

A capillary electrophoretic assay for acetyl coenzyme A carboxylase.

A simple off-column capillary electrophoretic (CE) assay for measuring acetyl coenzyme A carboxylase holoenzyme (holo-ACC) activity and inhibition was developed. The two reactions catalyzed by the holo-ACC components, biotin carboxylase (BC) and carboxyltransferase (CT), were simultaneously monitored in this assay. Acetyl coenzyme A (CoA), malonyl-CoA, adenosine triphosphate (ATP), and adenosine diphosphate (ADP) were separated by capillary electrophoresis, and the depletion of ATP and acetyl-CoA as well as the production of ADP and malonyl-CoA were monitored. Inhibition of holo-ACC by the BC inhibitor, 2-amino-N,N-dibenzyloxazole-5-carboxamide, and the carboxyltransferase inhibitor, andrimid, was confirmed using this assay. A previously reported off-column CE assay for only the CT component of ACC was optimized, and an off-column CE assay for the BC component of ACC also was developed.

The enzymes of biotin dependent CO2 metabolism: what structures reveal about their reaction mechanisms.

Biotin is the major cofactor involved in carbon dioxide metabolism. Indeed, biotin-dependent enzymes are ubiquitous in nature and are involved in a myriad of metabolic processes including fatty acid synthesis and gluconeogenesis. The cofactor, itself, is composed of a ureido ring, a tetrahydrothiophene ring, and a valeric acid side chain. It is the ureido ring that functions as the CO2 carrier. A complete understanding of biotin-dependent enzymes is critically important for translational research in light of the fact that some of these enzymes serve as targets for anti-obesity agents, antibiotics, and herbicides. Prior to 1990, however, there was a dearth of information regarding the molecular architectures of biotin-dependent enzymes. In recent years there has been an explosion in the number of three-dimensional structures reported for these proteins. Here we review our current understanding of the structures and functions of biotin-dependent enzymes. In addition, we provide a critical analysis of what these structures have and have not revealed about biotin-dependent catalysis.

Behavior of the ATP grasp domain of biotin carboxylase monomers and dimers studied using molecular dynamics simulations.

The enzyme biotin carboxylase (BC) uses adenosine triphosphate (ATP) to carboxylate biotin and is involved in fatty acid synthesis. Structural evidence suggests that the B domain of BC undergoes a large hinge motion of ∼45° when binding and releasing substrates. Escherichia coli BC can function as a natural homodimer and as a mutant monomer. Using molecular dynamics simulations, we evaluate the free energy profile along a closure angle of the B domain of E. coli BC for three cases: a monomer without bound Mg(2)ATP, a monomer with bound Mg(2)ATP, and a homodimer with bound Mg(2)ATP in one subunit. The simulation results show that a closed state is the most probable for the monomer with or without bound Mg(2)ATP. For the dimer with Mg(2)ATP in one of its subunits, communication between the two subunits was observed. Specifically, in the dimer, the opening of the subunit without Mg(2)ATP caused the other subunit to open, and hysteresis was observed upon reclosing it. The most stable state of the dimer is one in which the B domain of both subunits is closed; however, the open state for the B domain without Mg(2)ATP is only approximately 2k(B)T higher in free energy than the closed state. A simple diffusion model indicates that the mean times for opening and closing of the B domain in the monomer with and without Mg(2)ATP are much smaller than the overall reaction time, which is on the order of seconds.