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ABSTRACTAcetyl-CoA carboxylases (ACCs) convert acetyl-CoA to malonyl-CoA, a key step in fatty acid biosynthesis and autotrophic carbon fixation pathways. Three functionally distinct components, biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyltransferase (CT), are either separated or partially fused in different combinations, forming heteromeric ACCs. However, an ACC with fused BC-BCCP and separate CT has not been identified, leaving its catalytic mechanism unclear. Here, we identify two BC isoforms (BC1 and BC2) from Chloroflexus aurantiacus, a filamentous anoxygenic phototroph that employs 3-hydroxypropionate (3-HP) bi-cycle rather than Calvin cycle for autotrophic carbon fixation. We reveal that BC1 possesses fused BC and BCCP domains, where BCCP could be biotinylated by E. coli or C. aurantiacus BirA on Lys553 residue. Crystal structures of BC1 and BC2 at 3.2 Å and 3.0 Å resolutions, respectively, further reveal a tetramer of two BC1-BC homodimers, and a BC2 homodimer, all exhibiting similar BC architectures. The two BC1-BC homodimers are connected by an eight-stranded β-barrel of the partially resolved BCCP domain. Disruption of β-barrel results in dissociation of the tetramer into dimers in solution and decreased biotin carboxylase activity. Biotinylation of the BCCP domain further promotes BC1 and CTβ-CTα interactions to form an enzymatically active ACC, which converts acetyl-CoA to malonyl-CoA in vitro and produces 3-HP via co-expression with a recombinant malonyl-CoA reductase in E. coli cells. This study revealed a heteromeric ACC that evolves fused BC-BCCP but separate CTα and CTβ to complete ACC activity.IMPORTANCEAcetyl-CoA carboxylase (ACC) catalyzes the rate-limiting step in fatty acid biosynthesis and autotrophic carbon fixation pathways across a wide range of organisms, making them attractive targets for drug discovery against various infections and diseases. Although structural studies on homomeric ACCs, which consist of a single protein with three subunits, have revealed the “swing domain model” where the biotin carboxyl carrier protein (BCCP) domain translocates between biotin carboxylase (BC) and carboxyltransferase (CT) active sites to facilitate the reaction, our understanding of the subunit composition and catalytic mechanism in heteromeric ACCs remains limited. Here, we identify a novel ACC from an ancient anoxygenic photosynthetic bacterium Chloroflexus aurantiacus, it evolves fused BC and BCCP domain, but separate CT components to form an enzymatically active ACC, which converts acetyl-CoA to malonyl-CoA in vitro and produces 3-hydroxypropionate (3-HP) via co-expression with recombinant malonyl-CoA reductase in E. coli cells. These findings expand the diversity and molecular evolution of heteromeric ACCs and provide a structural basis for potential applications in 3-HP biosynthesis.

Acetyl-CoA carboxylases (ACCs) convert acetyl-CoA to malonyl-CoA, a key step in fatty acid biosynthesis and autotrophic carbon fixation pathways. Three functionally distinct components, biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyltransferase (CT), are either separated or partially fused in different combinations, forming heteromeric ACCs. However, an ACC with fused BC-BCCP and separate CT has not been identified, leaving its catalytic mechanism unclear. Here, we identify two BC isoforms (BC1 and BC2) from Chloroflexus aurantiacus , a filamentous anoxygenic phototroph that employs 3-hydroxypropionate (3-HP) bi-cycle rather than Calvin cycle for autotrophic carbon fixation. We reveal that BC1 possesses fused BC and BCCP domains, where BCCP could be biotinylated by E. coli or C. aurantiacus BirA on Lys553 residue. Crystal structures of BC1 and BC2 at 3.2 Å and 3.0 Å resolutions, respectively, further reveal a tetramer of two BC1-BC homodimers, and a BC2 homodimer, all exhibiting similar BC architectures. The two BC1-BC homodimers are connected by an eight-stranded β-barrel of the partially resolved BCCP domain. Disruption of β-barrel results in dissociation of the tetramer into dimers in solution and decreased biotin carboxylase activity. Biotinylation of the BCCP domain further promotes BC1 and CTβ-CTα interactions to form an enzymatically active ACC, which converts acetyl-CoA to malonyl-CoA in vitro and produces 3-HP via co-expression with a recombinant malonyl-CoA reductase in E. coli cells. This study revealed a heteromeric ACC that evolves fused BC-BCCP but separate CTα and CTβ to complete ACC activity.IMPORTANCEAcetyl-CoA carboxylase (ACC) catalyzes the rate-limiting step in fatty acid biosynthesis and autotrophic carbon fixation pathways across a wide range of organisms, making them attractive targets for drug discovery against various infections and diseases. Although structural studies on homomeric ACCs, which consist of a single protein with three subunits, have revealed the "swing domain model" where the biotin carboxyl carrier protein (BCCP) domain translocates between biotin carboxylase (BC) and carboxyltransferase (CT) active sites to facilitate the reaction, our understanding of the subunit composition and catalytic mechanism in heteromeric ACCs remains limited. Here, we identify a novel ACC from an ancient anoxygenic photosynthetic bacterium Chloroflexus aurantiacus , it evolves fused BC and BCCP domain, but separate CT components to form an enzymatically active ACC, which converts acetyl-CoA to malonyl-CoA in vitro and produces 3-hydroxypropionate (3-HP) via co-expression with recombinant malonyl-CoA reductase in E. coli cells. These findings expand the diversity and molecular evolution of heteromeric ACCs and provide a structural basis for potential applications in 3-HP biosynthesis., Competing Interests: The authors declare no conflict of interest.

Propionyl-CoA carboxylase (PCC) is a promising enzyme in the fields of biological CO 2 utilization, synthesis of natrual products, and so on. The activity and substrate specificity of PCC are dependent on its key subunit carboxyltransferase (CT). To obtain PCC with high enzyme activity, seven pccB genes encoding CT subunit from diverse microorganisms were expressed in recombinant E. coli , and PccB from Bacillus subtilis showed the highest activity in vitro. To further optimize this protein using directed evolution, a genetic screening system based on oxaloacetate availability was designed to enrich the active variants of PccB Bs. Four amino acid substitutions (D46G, L97Q, N220I and I391T) proved of great assistance in PccB Bs activity improvement, and a double mutant of PccB Bs (N220I/I391T) showed a 94-fold increase of overall catalytic efficiency indicated by k cat / K m. Moreover, this PccB Bs double mutant was applied in construction of new succinate biosynthetic pathway. This new pathway produces succinate from acetyl-CoA with fixation of two CO 2 molecules, which was confirmed by isotope labeling experiment with NaH13CO 3. Compared with previous succinate production based on carboxylation of phosphoenolpyruvate or pyruvate, this new pathway showed some advantages including higher CO 2 fixation potentiality and availability under aerobic conditions. In summary, this study developed a PCC with high enzyme activity which can be widely used in biotechnology field, and also demonstrated the feasibility of new succinate biosynthetic pathway with two CO 2 fixation reactions. • Several propionyl-CoA carboxylases (PCCs) were cloned and assayed in vitro. • Activity of B. subtilis PCC were notable improved by directed evolution. • The improved PCC was applied in a novel succinate biosynthetic pathway. • Succinate is produced from acetyl-CoA with fixation of two CO 2 molecules. [ABSTRACT FROM AUTHOR]

Through a structure-based drug design project (SBDD), potent small molecule inhibitors of pyruvate carboxylase (PC) have been discovered. A series of α-keto acids (7) and α-hydroxycinnamic acids (8) were prepared and evaluated for inhibition of PC in two assays. The two most potent inhibitors were 3,3′-(1,4-phenylene)bis[2-hydroxy-2-propenoic acid] (8u) and 2-hydroxy-3-(quinoline-2-yl)propenoic acid (8v) with IC 50 values of 3.0 ± 1.0 μM and 4.3 ± 1.5 μM respectively. Compound 8v is a competitive inhibitor with respect to pyruvate (K i = 0.74 μM) and a mixed-type inhibitor with respect to ATP, indicating that it targets the unique carboxyltransferase (CT) domain of PC. Furthermore, compound 8v does not significantly inhibit human carbonic anhydrase II, matrix metalloproteinase-2, malate dehydrogenase or lactate dehydrogenase. [ABSTRACT FROM AUTHOR]

Carboxyltransferase is one component of the multifunctional enzyme acetyl-CoA carboxylase which catalyzes the first committed step in fatty acid biosynthesis. Carboxyltransferase is an α2β2 heterotetramer and possesses two distinct but integrated functions. One function catalyzes the transfer of carbon dioxide from biotin to acetyl-CoA, whereas the other involves binding to the mRNA encoding both subunits. When carboxyltransferase binds to the mRNA both enzymatic activity and translation of the mRNA are inhibited. However, the substrate acetyl-CoA competes with mRNA for binding. Thus, mRNA binding by carboxyltransferase provides an effective mechanism for regulating enzymatic activity and gene expression. This conceptual review takes the position that regulation of enzymatic activity and gene expression of carboxyltransferase by binding to its own mRNA is at its most fundamental level the result of the symmetry in the chemical reaction catalyzed by the enzyme. The chemical reaction is symmetrical in that both substrates generate enolate anions during the course of catalysis. The chemical symmetry led to a structural symmetry in the enzyme where both the α and β subunits contain oxyanion holes that stabilize the enolate anions. Then the region of the mRNA that codes for the oxyanion holes provided the binding sites for carboxyltransferase. Thus, the symmetry of the chemical reaction formed the foundation for the evolution of the mechanism for regulation of carboxyltransferase.

Acetyl-CoA carboxylase (ACC) catalyzes the committed and rate-limiting step in fatty acid biosynthesis. The two partial reactions, carboxylation of biotin followed by carboxyl transfer to the acceptor acetyl-CoA, are performed by two separate domains in animal ACCs. The cyclic keto-enol insecticides and acaricides have been proposed to inhibit insect ACCs. In this communication, we show that the enol derivative of the cylic keto-enol insecticide spirotetramat inhibited ACCs partially purified from the insect species Myzus persicae and Spodoptera frugiperda , as well as the spider mite ( Tetranychus urticae ) ACC which was expressed in insect cells using a recombinant baculovirus. Steady-state kinetic analysis revealed competitive inhibition with respect to the carboxyl acceptor, acetyl-CoA, indicating that spirotetramat-enol bound to the carboxyltransferase domain of ACC. Interestingly, inhibition with respect to the biotin carboxylase substrate ATP was uncompetitive. Amino acid residues in the carboxyltransferase domains of plant ACCs are important for binding of established herbicidal inhibitors. Mutating the spider mite ACC at the homologous positions, for example L1736 to either isoleucine or alanine, and A1739 to either valine or serine, did not affect the inhibition of the spider mite ACC by spirotetramat-enol. These results indicated different binding modes of the keto-enols and the herbicidal chemical families. [ABSTRACT FROM AUTHOR]

Highlights: [•] Structures of pyruvate carboxylase were determined in the presence of three ligands. [•] Unexpectedly, none of the ligands directly coordinate the active site metal ion. [•] Structures suggest that substrate orientation remains fixed throughout catalysis. [•] 3-Bromopyruvate binds in the active site of pyruvate carboxylase. [Copyright &y& Elsevier]

The gene locus for acetyl-CoA carboxylase (ACC) involved in the primary metabolism was identified from the genomic library of Streptomyces toxytricini which produces a lipase inhibitor lipstatin. The 7.4 kb cloned gene was comprised of 5 ORFs including accD1, accA1, hmgL, fadST1, and stsF. In order to confirm the biochemical characteristics of AccA1, the gene was overexpressed in Escherichia coli cells, and the recombinant protein was purified through Ni2+ affinity chromatography. Because most of the expressed AccAl was biotinylated by host E. coli BirA in the presence of D-biotin, the non-biotinylated apo-AccA1 was purified after gene induction without D-biotin, followed by exclusion of holo-AccA1 using streptavidin beads. The separated apo-AccA1 was post-translationally biotinylated by S. toxytricini biotin apo-protein ligase (BPL) in a time- and enzyme-dependent manner. This result supports that this gene cluster of S. toxytricini encodes the functional ACC enzyme subunits to be biotinylated. [ABSTRACT FROM AUTHOR]

Acetyl-CoA carboxylase (ACC) catalyzes the first committed step in the synthesis of long-chain fatty acids. The crystal structure of the Escherichia coli carboxyltransferase component of ACC revealed an α2β2 subunit composition with two active sites and, most importantly, a unique zinc domain in each αβ pair that is absent in the eukaryotic enzyme. We show here that carboxyltransferase binds DNA. Half-maximal saturation of different single-stranded or double-stranded DNA constructs is seen at 0.5-1.0 μM, and binding is cooperative and nonspecific. The substrates (malonyl-CoA and biocytin) inhibit DNA:carboxyltransferase complex formation. More significantly, single-stranded DNA, double-stranded DNA, and heparin inhibit the reaction catalyzed by carboxyltransferase, with single-stranded DNA and heparin acting as competitive inhibitors. However, double-inhibition experiments revealed that both DNA and heparin can bind the enzyme in the presence of a bisubstrate analog (BiSA), and the binding of BiSA has a very weak synergistic effect on the binding of the second inhibitor (DNA or heparin) and vice versa. In contrast, DNA and heparin can also bind to the enzyme simultaneously, but the binding of either molecule has a strong synergistic effect on binding of the other. An important mechanistic implication of these observations is that the dual active sites of ACC are functionally connected. [ABSTRACT FROM AUTHOR]

Abstract: One consequence of the dramatic rise of antibiotic-resistant pathogenic bacteria is the need for new targets for antibiotics. Because membrane lipid biogenesis is essential for bacterial growth, enzymes of the fatty acid biosynthetic pathway offer attractive possibilities for the development of new antibiotics. Acetyl-coenzyme A carboxylase (ACC) catalyzes the first committed and regulated step in fatty acid biosynthesis in bacteria and thus is a prime target for development of antibiotics. ACC is a multifunctional enzyme composed of three separate proteins. The biotin carboxylase component catalyzes the ATP-dependent carboxylation of biotin. The biotin carboxyl carrier protein features a biotin molecule covalently attached at Lys122 of the Escherichia coli enzyme. The carboxyltransferase subunit catalyzes the transfer of a carboxyl group from biotin to acetyl-coenzyme A (acetyl-CoA) to form malonyl-CoA. The objective of this study was to develop an assay for high-throughput screening for inhibitors of the carboxyltransferase subunit. The carboxyltransferase reaction was assayed in the reverse direction in which malonyl-CoA reacts with biocytin (an analog of the biotin carboxyl carrier protein) to form acetyl-CoA and carboxybiotin. The production of acetyl-CoA was coupled to citrate synthase, which produced citrate and coenzyme A. The amount of coenzyme A formed was detected using 5,5′-dithiobis(2-nitrobenzoic acid) (Ellman’s reagent). The assay has been developed for use in both 96- and 384-well microplate formats and was validated using a known bisubstrate analog inhibitor of carboxyltransferase. The spectrophotometric readout in the visible absorbance range used in this assay does not generate the number of false negatives associated with frequently used NAD/NADH assay systems that rely on detection of NADH using UV absorbance. [Copyright &y& Elsevier]

Transcarboxylase is a 1.2 million Dalton (Da) multienzyme complex from Propionibacterium shermanii that couples two carboxylation reactions, transferring CO2- from methylmalonyl-CoA to pyruvate to yield propionyl-CoA and oxaloacetate. Crystal structures of the 5S metalloenzyme subunit, which catalyzes the second carboxylation reaction, have been solved in free form and bound to its substrate pyruvate, product oxaloacetate, or inhibitor 2-ketobutyrate. The structure reveals a dimer ofß8a8 barrels with an active site cobalt ion coordinated by a carbamylated lysine, except in the oxaloacetate complex in which the product's carboxylate group serves as a ligand instead. 5S and human pyruvate carboxylase (PC), an enzyme crucial to gluconeogenesis, catalyze similar reactions. A 5S-based homology model of the PC carboxyltransferase domain indicates a conserved mechanism and explains the molecular basis of mutations in lactic acidemia. PC disease mutations reproduced in 5S result in a similar decrease in carboxyltransferase activity and crystal structures with altered active sites. [ABSTRACT FROM AUTHOR]

The plastid accD gene encoding the carboxyltransferase E subunit of acetyl-coenzyme A carboxylase (ACCase) was cloned from potato. Potato accD (saccD) is 2487 bp in length with a 614 bp 5' upstream promoter region and an ORF of 1524 bp, corresponding to a polypeptide of 507 amino acids. The N-terminal region lacks recognizable motifs, while the C-terminal regions contains five motifs. Among these is motif II, PLIIVCASGGARMQE, the sole motif present in all available accD sequences of plants and animals, and of (. coli, suggesting that this motif may correspond to the catalytic site. saccD has the typical prokaryotic promoter signatures, TTGACA and TATCAA, which are -35 and -10-like sequences for plastid-encoded RNA polymerase (PEP), at positions -184 and -160, respectively. However, it seems to be transcribed by the nucleus-encoded RNA polymerase because it is expressed in tuber and root, and in the dark (under crippled PEP conditions) and its transcription initiation sites do not correspond to those of PEP. saccD is expressed in all potato tissues, i.e., leaf, stem, root, and tuber, and its transcript is produced at a similar rate in the light and dark, at different developmental stages, and during growth in the presence of different sugars and carbon sources. Taken together, our results suggest that potato accD is a housekeeping gene constitu-tively expressed in both chloroplast and amyloplast. [ABSTRACT FROM AUTHOR]

Acetyl-CoA carboxylase catalyzes the first committed step in the synthesis of long-chain fatty acids. The Escherichia coli form of the enzyme consists of a biotin carboxylase protein, a biotin carboxyl carrier protein, and a carboxyltransferase protein. In this report, the synthesis of a bisubstrate analog inhibitor of carboxyltransferase is described. The inhibitor was synthesized by covalently linking biotin to coenzyme A via an acyl bridge between the sulfur of coenzyme A and the 1′-N of biotin. The steady-state kinetics of carboxyltransferase are characterized in the reverse direction, in which malonyl-CoA reacts with biocytin to form acetyl-CoA and carboxybiocytin. The inhibitor exhibited competitive inhibition versus malonyl-CoA and noncompetitive inhibition versus biocytin, with a slope inhibition constant (Kis) of 23 ± 2 μM. The bisubstrate analog has an affinity for carboxyltransferase 350 times higher than biotin. This suggests the inhibitor will be useful in structural studies, as well as aid in the search for chemotherapeutic agents that target acetyl-CoA carboxylase. [Copyright &y& Elsevier]

Biotin-dependent carboxylases are widely distributed in nature and have central roles in the metabolism of fatty acids, amino acids, carbohydrates, and other compounds. The last decade has seen the accumulation of structural information on most of these large holoenzymes, including the 500-kDa dimeric yeast acetyl-CoA carboxylase, the 750-kDa α 6 β 6 dodecameric bacterial propionyl-CoA carboxylase, 3-methylcrotonyl-CoA carboxylase, and geranyl-CoA carboxylase, the 720-kDa hexameric bacterial long-chain acyl-CoA carboxylase, the 500-kDa tetrameric bacterial single-chain pyruvate carboxylase, the 370-kDa α 2 β 4 bacterial two-subunit pyruvate carboxylase, and the 130-kDa monomeric eukaryotic urea carboxylase. A common theme that has emerged from these studies is the dramatic structural flexibility of these holoenzymes despite their strong overall sequence conservation, evidenced both by the extensive diversity in the architectures of the holoenzymes and by the extensive conformational variability of their domains and subunits. This structural flexibility is crucial for the function and regulation of these enzymes and identifying compounds that can interfere with it represents an attractive approach for developing novel modulators and drugs. The extensive diversity observed in the structures so far and its biochemical and functional implications will be the focus of this review., (© 2017 Elsevier Inc. All rights reserved.)