Grover L. Waldrop

Frontiers, Opportunities, and Challenges in Biochemical and Chemical Catalysis of CO2 Fixation

Two major energy-related problems confront the world in the next 50 years. First, increased worldwide competition for gradually depleting fossil fuel reserves (derived from past photosynthesis) will lead to higher costs, both monetarily and politically. Second, atmospheric CO_2 levels are at their highest recorded level since records began. Further increases are predicted to produce large and uncontrollable impacts on the world climate. These projected impacts extend beyond climate to ocean acidification, because the ocean is a major sink for atmospheric CO2.1 Providing a future energy supply that is secure and CO_2-neutral will require switching to nonfossil energy sources such as wind, solar, nuclear, and geothermal energy and developing methods for transforming the energy produced by these new sources into forms that can be stored, transported, and used upon demand.

Multi-subunit acetyl-CoA carboxylases.

Acetyl-CoA carboxylase (ACC) catalyses the first committed step of fatty acid synthesis, the carboxylation of acetyl-CoA to malonyl-CoA. Two physically distinct types of enzymes are found in nature. Bacterial and most plant chloroplasts contain a multi-subunit ACC (MS-ACC) enzyme that is readily dissociated into its component proteins. Mammals, fungi, and plant cytosols contain the second type of ACC, a single large multifunctional polypeptide. This review will focus on the structures, regulation, and enzymatic mechanisms of the bacterial and plant MS-ACCs.

Movement of the Biotin Carboxylase B-domain as a Result of ATP Binding

Acetyl-CoA carboxylase catalyzes the first committed step in fatty acid synthesis. In Escherichia coli, the enzyme is composed of three distinct protein components: biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase. The biotin carboxylase component has served for many years as a paradigm for mechanistic studies devoted toward understanding more complicated biotin-dependent carboxylases. The three-dimensional x-ray structure of an unliganded form of E. coli biotin carboxylase was originally solved in 1994 to 2.4-Å resolution. This study revealed the architecture of the enzyme and demonstrated that the protein belongs to the ATP-grasp superfamily. Here we describe the three-dimensional structure of theE. coli biotin carboxylase complexed with ATP and determined to 2.5-Å resolution. The major conformational change that occurs upon nucleotide binding is a rotation of approximately 45o of one domain relative to the other domains thereby closing off the active site pocket. Key residues involved in binding the nucleotide to the protein include Lys-116, His-236, and Glu-201. The backbone amide groups of Gly-165 and Gly-166 participate in hydrogen bonding interactions with the phosphoryl oxygens of the nucleotide. A comparison of this closed form of biotin carboxylase with carbamoyl-phosphate synthetase is presented.

A Biotin Analog Inhibits Acetyl-CoA Carboxylase Activity and Adipogenesis

Acetyl-CoA carboxylase catalyzes the first committed step in the synthesis of long chain fatty acids. In this study, we observed that treatment of 3T3-L1 cells with biotin chloroacetylated at the 1′ nitrogen reduced the enzymatic activity of cytosolic acetyl-CoA carboxylase and concomitantly inhibited the differentiation of 3T3-L1 cells in a dose-dependent manner. Treatment with chloroacetylated biotin blocked the induction of PPARγ, STAT1, and STAT5A expression that normally occurs with adipogenesis. Moreover, addition of chloroacetylated biotin inhibited lipid accumulation, as judged by Oil Red O staining. Our results support recent studies that indicate that acetyl-CoA carboxylase may be a suitable target for an anti-obesity therapeutic.

OVEREXPRESSION AND KINETIC CHARACTERIZATION OF THE CARBOXYLTRANSFERASE COMPONENT OF ACETYL-COA CARBOXYLASE

Acetyl-CoA carboxylase catalyzes the first committed step in the biosynthesis of fatty acids. TheEscherichia coli form of the enzyme consists of a biotin carboxylase protein, a biotin carboxyl carrier protein, and a carboxyltransferase protein. In this report the overexpression of the genes for the carboxyltransferase component is described. The steady-state kinetics of the recombinant carboxyltransferase are characterized in the reverse direction, in which malonyl-CoA reacts with biocytin to form acetyl-CoA and carboxybiocytin. The initial velocity patterns indicated that the kinetic mechanism is equilibrium-ordered with malonyl-CoA binding before biocytin and the binding of malonyl-CoA to carboxyltransferase at equilibrium. The biotin analogs, desthiobiotin and 2-imidazolidone, inhibited carboxyltransferase. Both analogs exhibited parabolic noncompetitive inhibition, which means that two molecules of inhibitor bind to the enzyme. The pH dependence for both the maximum velocity (V) and the (V/K)biocytinparameters decreased at low pH. A single ionizing group on the enzyme with a pK of 6.2 or lower in the (V/K)biocytin profile and 7.5 in theV profile must be unprotonated for catalysis. Carboxyltransferase was inactivated by N-ethylmaleimide, whereas malonyl-CoA protected against inactivation. This suggests that a thiol in or near the active site is needed for catalysis. The rate of inactivation of carboxyltransferase byN-ethylmaleimide decreased with decreasing pH and indicated that the pK of the sulfhydryl group had a pK value of 7.3. It is proposed that the thiolate ion of a cysteine acts as a catalytic base to remove the N1′ proton of biocytin.

Structural evidence for substrate-induced synergism and half-sites reactivity in biotin carboxylase

Bacterial acetyl‐CoA carboxylase is a multifunctional biotin‐dependent enzyme that consists of three separate proteins: biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyltransferase (CT). Acetyl‐CoA carboxylase is a potentially attractive target for novel antibiotics because it catalyzes the first committed step in fatty acid biosynthesis. In the first half‐reaction, BC catalyzes the ATP‐dependent carboxylation of BCCP. In the second half‐reaction, the carboxyl group is transferred from carboxybiotinylated BCCP to acetyl‐CoA to produce malonyl‐CoA. A series of structures of BC from several bacteria crystallized in the presence of various ATP analogs is described that addresses three major questions concerning the catalytic mechanism. The structure of BC bound to AMPPNP and the two catalytically essential magnesium ions resolves inconsistencies between the kinetics of active‐site BC mutants and previously reported BC structures. Another structure of AMPPNP bound to BC shows the polyphosphate chain folded back on itself, and not in the correct (i.e., extended) conformation for catalysis. This provides the first structural evidence for the hypothesis of substrate‐induced synergism, which posits that ATP binds nonproductively to BC in the absence of biotin. The BC homodimer has been proposed to exhibit half‐sites reactivity where the active sites alternate or “flip‐flop” their catalytic cycles. A crystal structure of BC showed the ATP analog AMPPCF2P bound to one subunit while the other subunit was unliganded. The liganded subunit was in the closed or catalytic conformation while the unliganded subunit was in the open conformation. This provides the first structural evidence for half‐sites reactivity in BC.

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.

A tale of two functions: enzymatic activity and translational repression by carboxyltransferase

Acetyl-CoA Carboxylase catalyzes the first committed step in fatty acid synthesis. Escherichia coli acetyl-CoA carboxylase is composed of biotin carboxylase, carboxyltransferase and biotin carboxyl carrier protein functions. The accA and accD genes that code for the α- and β-subunits, respectively, are not in an operon, yet yield an α2β2 carboxyltransferase. Here, we report that carboxyltransferase regulates its own translation by binding the mRNA encoding its subunits. This interaction is mediated by a zinc finger on the β-subunit; mutation of the four cysteines to alanine diminished nucleic acid binding and catalytic activity. Carboxyltransferase binds the coding regions of both subunit mRNAs and inhibits translation, an inhibition that is relieved by the substrate acetyl-CoA. mRNA binding reciprocally inhibits catalytic activity. Preferential binding of carboxyltransferase to RNA in situ was shown using fluorescence resonance energy transfer. We propose an unusual regulatory mechanism by which carboxyltransferase acts as a ‘dimmer switch’ to regulate protein production and catalytic activity, while sensing the metabolic state of the cell through acetyl-CoA concentration.

Site-directed mutagenesis of ATP binding residues of biotin carboxylase: insight into the mechanism of catalysis

Acetyl-CoA carboxylase catalyzes the first committed step in fatty acid synthesis in all plants, animals, and bacteria. The Escherichia coli form is a multimeric protein complex consisting of three distinct and separate components: biotin carboxylase, carboxyltransferase, and the biotin carboxyl carrier protein. The biotin carboxylase component catalyzes the ATP-dependent carboxylation of biotin using bicarbonate as the carboxylate source and has a distinct architecture that is characteristic of the ATP-grasp superfamily of enzymes. Included in this superfamily are d-Ala d-Ala ligase, glutathione synthetase, carbamyl phosphate synthetase,N 5-carboxyaminoimidazole ribonucleotide synthetase, and glycinamide ribonucleotide transformylase, all of which have known three-dimensional structures and contain a number of highly conserved residues between them. Four of these residues of biotin carboxylase, Lys-116, Lys-159, His-209, and Glu-276, were selected for site-directed mutagenesis studies based on their structural homology with conserved residues of other ATP-grasp enzymes. These mutants were subjected to kinetic analysis to characterize their roles in substrate binding and catalysis. In all four mutants, theK m value for ATP was significantly increased, implicating these residues in the binding of ATP. This result is consistent with the crystal structures of several other ATP-grasp enzymes, which have shown specific interactions between the corresponding homologous residues and cocrystallized ADP or nucleotide analogs. In addition, the maximal velocity of the reaction was significantly reduced (between 30- and 260-fold) in the 4 mutants relative to wild type. The data suggest that the mutations have misaligned the reactants for optimal catalysis.

Discovery of novel antibacterials

Importance of the field: Antibiotics have existed in the environment for millennia, but it has only been in the past 80 years that humans have used them systematically to treat infections. This battle between humans and bacteria has led to an alarming increase in resistance to all clinically useful antibacterial agents. Thus, there is an imperative need for new agents to combat these resistant strains of bacteria. Areas covered in this review: The topics covered include natural product screening, identification and validation of new antibacterial targets and approaches for the discovery and optimization of antibacterial compounds. Last, an assessment of the major challenges facing antibacterial discovery is presented. What the reader will gain: The current strategies and methodologies for discovering and designing new antibacterial agents are evaluated as to their potential for generating the next round of therapeutics. Each topic is presented in a general, basic manner and will hopefully be a useful resource for students and newcomers to the field. Take home message: New antibacterial agents are desperately needed to fight the increasing number of antibiotic resistant pathogenic bacteria. New methodologies as well as traditional approaches should both be used for discovering antibiotics to meet this serious medical need.