Genetic analysis of acyl-CoA carboxylases involved in lipid accumulation in Rhodococcus jostii RHA1.

In actinomycetes, the acyl-CoA carboxylases, including the so-called acetyl-CoA carboxylases (ACCs), are biotin-dependent enzymes that exhibit broad substrate specificity and diverse domain and subunit arrangements. Bioinformatic analyses of the Rhodococcus jostii RHA1 genome found that this microorganism contains a vast arrange of putative acyl-CoA carboxylases domains and subunits. From the thirteen putative carboxyltransferase domains, only the carboxyltransferase subunit RO01202 and the carboxyltransferase domain present in the multidomain protein RO04222 are highly similar to well-known essential ACC subunits from other actinobacteria. Mutant strains in each of these genes showed that none of these enzymes is essential for R. jostii growth in rich or in minimal media with high nitrogen concentration, presumably because of their partial overlapping activities. A mutant strain in the ro04222 gene showed a decrease in triacylglycerol and mycolic acids accumulation in rich and minimal medium, highlighting the relevance of this multidomain ACC in the biosynthesis of these lipids. On the other hand, RO01202, a carboxyltransferase domain of a putative ACC complex, whose biotin carboxylase and biotin carboxyl carrier protein domain were not yet identified, was found to be essential for R. jostii growth only in minimal medium with low nitrogen concentration. The results of this study have identified a new component of the TAG-accumulating machinery in the oleaginous R. jostii RHA1. While non-essential for growth and TAG biosynthesis in RHA1, the activity of RO04222 significantly contributes to lipogenesis during single-cell oil production. Furthermore, this study highlights the high functional diversity of ACCs in actinobacteria, particularly regarding their essentiality under different environmental conditions. KEY POINTS: • R. jostii possess a remarkable heterogeneity in their acyl-carboxylase complexes. • RO04222 is a multidomain acetyl-CoA carboxylase involved in lipid accumulation. • RO01202 is an essential carboxyltransferase only at low nitrogen conditions.

Conformational Selection Governs Carrier Domain Positioning in Staphylococcus aureus Pyruvate Carboxylase.

Biotin-dependent enzymes employ a carrier domain to efficiently transport reaction intermediates between distant active sites. The translocation of this carrier domain is critical to the interpretation of kinetic and structural studies, but there have been few direct attempts to investigate the dynamic interplay between ligand binding and carrier domain positioning in biotin-dependent enzymes. Pyruvate carboxylase (PC) catalyzes the MgATP-dependent carboxylation of pyruvate where the biotinylated carrier domain must translocate ∼70 Šfrom the biotin carboxylase domain to the carboxyltransferase domain. Many prior studies have assumed that carrier domain movement is governed by ligand-induced conformational changes, but the mechanism underlying this movement has not been confirmed. Here, we have developed a system to directly observe PC carrier domain positioning in both the presence and absence of ligands, independent of catalytic turnover. We have incorporated a cross-linking trap that reports on the interdomain conformation of the carrier domain when it is positioned in proximity to a neighboring carboxyltransferase domain. Cross-linking was monitored by gel electrophoresis, inactivation kinetics, and intrinsic tryptophan fluorescence. We demonstrate that the carrier domain positioning equilibrium is sensitive to substrate analogues and the allosteric activator acetyl-CoA. Notably, saturating concentrations of biotin carboxylase ligands do not prevent carrier domain trapping proximal to the neighboring carboxyltransferase domain, demonstrating that carrier domain positioning is governed by conformational selection. This model of carrier domain translocation in PC can be applied to other multi-domain enzymes that employ large-scale domain motions to transfer intermediates during catalysis.

Translational Detection of Nonproteinogenic Amino Acids Using an Engineered Complementary Cell-Free Protein Synthesis Assay.

We developed a simple and rapid method for analyzing nonproteinogenic amino acids that does not require conventional chromatographic equipment. In this technique, nonproteinogenic amino acids were first converted to a proteinogenic amino acid through in vitro metabolism in a cell extract. The proteinogenic amino acid generated from the nonproteinogenic precursors were then incorporated into a reporter protein using a cell-free protein synthesis system. The titers of the nonproteinogenic amino acids could be readily quantified by measuring the activity of reporter proteins. This method, which combines the enzymatic conversion of target amino acids with translational analysis, makes amino acid analysis more accessible while minimizing the cost and time requirements. We anticipate that the same strategy could be extended to the detection of diverse biochemical molecules with clinical and industrial implications.

A revised model for the control of fatty acid synthesis by master regulator Spo0A in Bacillus subtilis.

In starving Bacillus subtilis cells, the accDA operon encoding two subunits of the essential acetyl-CoA carboxylase (ACC) has been proposed to be tightly regulated by direct binding of the master regulator Spo0A to a cis element (0A box) in the promoter region. When the 0A box is mutated, biofilm formation and sporulation have been reported to be impaired. Here, we present evidence that two 0A boxes, one previously known (0A-1) and another newly discovered (0A-2) in the accDA promoter region are positively and negatively regulated by Spo0A∼P respectively. Cells with mutated 0A boxes experience slight delays in sporulation, but eventually sporulate with high efficiency. In contrast, cells harboring a single mutated 0A-2 box are deficient for biofilm formation, while cells harboring either a mutated 0A-1 box or both mutated 0A boxes form biofilms. We further show that the essential ACC enzyme localizes on or near the cell membrane by directly observing a functional GFP fusion to one of the enzyme's subunits. Collectively, we propose a revised model in which accDA is primarily transcribed by a major σA -RNA polymerase, while Spo0A∼P plays an additional role in the fine-tuning of accDA expression upon starvation to support proper biofilm formation and sporulation.

Discovery and characterization of a novel C-terminal peptide carboxyl methyltransferase in a lassomycin-like lasso peptide biosynthetic pathway.

Lasso peptides belong to a peculiar family of ribosomally synthesized and post-translationally modified peptides (RiPPs)-natural products with an unusual isopeptide-bonded slipknot structure. Except for assembling of this unusual lasso fold, several further post-translational modifications of lasso peptides, including C-terminal methylation, phosphorylation/poly-phosphorylation, citrullination, and acetylation, have been reported recently. However, most of their biosynthetic logic have not been elucidated except the phosphorylated paeninodin lasso peptide. Herein, we identified two novel lassomycin-like lasso peptide biosynthetic pathways and, for the first time, characterized a novel C-terminal peptide carboxyl methyltransferase involved in these pathways. Our investigations revealed that this new family of methyltransferase could specifically methylate the C terminus of precursor peptide substrates, eventually leading to lassomycin-like C-terminal methylated lasso peptides. Our studies offer another rare insight into the extraordinary strategies of chemical diversification adopted by lasso peptide biosynthetic machinery and predicated two valuable sources for methylated lasso peptide discovery.

Proteolytic cleavage orchestrates cofactor insertion and protein assembly in [NiFe]-hydrogenase biosynthesis.

Metalloenzymes catalyze complex and essential processes, such as photosynthesis, respiration, and nitrogen fixation. For example, bacteria and archaea use [NiFe]-hydrogenases to catalyze the uptake and release of molecular hydrogen (H2). [NiFe]-hydrogenases are redox enzymes composed of a large subunit that harbors a NiFe(CN)2CO metallo-center and a small subunit with three iron-sulfur clusters. The large subunit is synthesized with a C-terminal extension, cleaved off by a specific endopeptidase during maturation. The exact role of the C-terminal extension has remained elusive; however, cleavage takes place exclusively after assembly of the [NiFe]-cofactor and before large and small subunits form the catalytically active heterodimer. To unravel the functional role of the C-terminal extension, we used an enzymatic in vitro maturation assay that allows synthesizing functional [NiFe]-hydrogenase-2 of Escherichia coli from purified components. The maturation process included formation and insertion of the NiFe(CN)2CO cofactor into the large subunit, endoproteolytic cleavage of the C-terminal extension, and dimerization with the small subunit. Biochemical and spectroscopic analysis indicated that the C-terminal extension of the large subunit is essential for recognition by the maturation machinery. Only upon completion of cofactor insertion was removal of the C-terminal extension observed. Our results indicate that endoproteolytic cleavage is a central checkpoint in the maturation process. Here, cleavage temporally orchestrates cofactor insertion and protein assembly and ensures that only cofactor-containing protein can continue along the assembly line toward functional [NiFe]-hydrogenase.

Preliminary studies on the antibacterial mechanism of Yanglingmycin.

The antibacterial mechanism of Yanglingmycin, a new dihydrooxazole antibiotic, was preliminarily investigated by symptomatology observation and physical and biochemical analysis. The electron microscopy observation exhibited that the bacterial cell became elongated, appeared breakage or even cavities on the cell surface after treated with Yanglingmycin. The content of reducing sugar and the activity levels of alanine transaminase and aspartate transaminase in treated group had a significant increase compared to control group. These results indicated that the integrity of bacteria cell membrane was damaged by the antibiotic. Furthermore, the activity of Accase and carboxyltransferase could be effectively inhibited by Yanglingmycin. Meanwhile, the addition of exogenous fatty acid resulted in the decrease or even loss of the antibacterial activity of Yanglingmycin. These findings implied that Yanglingmycin might take effect by inhibiting the activity of Accase, which resulted in the blockade of fatty acids and lipids biosynthesis.

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.

Pyruvate Occupancy in the Carboxyl Transferase Domain of Pyruvate Carboxylase Facilitates Product Release from the Biotin Carboxylase Domain through an Intermolecular Mechanism.

Protein structure, ligand binding, and catalytic turnover contributes to the governance of catalytic events occurring at spatially distinct domains in multifunctional enzymes. Coordination of these catalytic events partially rests on the ability of spatially discrete active sites to communicate with other allosteric and active sites on the same polypeptide chain (intramolecular) or on different polypeptide chains (intermolecular) within the holoenzyme. Often, communication results in long-range effects on substrate binding or product release. For example, pyruvate binding to the carboxyl transferase (CT) domain of pyruvate carboxylase (PC) increases the rate of product release in the biotin carboxylase (BC) domain. In order to address how CT domain ligand occupancy is "sensed" by other domains, we generated functional, mixed hybrid tetramers using the E218A (inactive BC domain) and T882S (low pyruvate binding, low activity) mutant forms of PC. The apparent Ka pyruvate for the pyruvate-stimulated release of Pi catalyzed by the T882S:E218A[1:1] hybrid tetramer was comparable to the wild-type enzyme and nearly 10-fold lower than that for the T882S homotetramer. In addition, the ratio of the rates of oxaloacetate formation to Pi release for the WT:T882S[1:1] and E218A:T882S[1:1] hybrid tetramer-catalyzed reactions was 0.5 and 0.6, respectively, while the T882S homotetramer exhibited a near 1:1 coupling of the two domains, suggesting that the mechanisms coordinating catalytic events is more complicated that we initially assumed. The results presented here are consistent with an intermolecular communication mechanism, where pyruvate binding to the CT domain is "sensed" by domains on a different polypeptide chain within the tetramer.

The bacterial signal transduction protein GlnB regulates the committed step in fatty acid biosynthesis by acting as a dissociable regulatory subunit of acetyl-CoA carboxylase.

Biosynthesis of fatty acids is one of the most fundamental biochemical pathways in nature. In bacteria and plant chloroplasts, the committed and rate-limiting step in fatty acid biosynthesis is catalyzed by a multi-subunit form of the acetyl-CoA carboxylase enzyme (ACC). This enzyme carboxylates acetyl-CoA to produce malonyl-CoA, which in turn acts as the building block for fatty acid elongation. In Escherichia coli, ACC is comprised of three functional modules: the biotin carboxylase (BC), the biotin carboxyl carrier protein (BCCP) and the carboxyl transferase (CT). Previous data showed that both bacterial and plant BCCP interact with signal transduction proteins belonging to the PII family. Here we show that the GlnB paralogues of the PII proteins from E. coli and Azospirillum brasiliense, but not the GlnK paralogues, can specifically form a ternary complex with the BC-BCCP components of ACC. This interaction results in ACC inhibition by decreasing the enzyme turnover number. Both the BC-BCCP-GlnB interaction and ACC inhibition were relieved by 2-oxoglutarate and by GlnB uridylylation. We propose that the GlnB protein acts as a 2-oxoglutarate-sensitive dissociable regulatory subunit of ACC in Bacteria.

Effect of molecular chaperones on aberrant protein oligomers in vitro: super-versus sub-stoichiometric chaperone concentrations.

Living systems protect themselves from aberrant proteins by a network of chaperones. We have tested in vitro the effects of different concentrations, ranging from 0 to 16 µm, of two molecular chaperones, namely αB-crystallin and clusterin, and an engineered monomeric variant of transthyretin (M-TTR), on the morphology and cytotoxicity of preformed toxic oligomers of HypF-N, which represent a useful model of misfolded protein aggregates. Using atomic force microscopy imaging and static light scattering analysis, all were found to bind HypF-N oligomers and increase the size of the aggregates, to an extent that correlates with chaperone concentration. SDS-PAGE profiles have shown that the large aggregates were predominantly composed of the HypF-N protein. ANS fluorescence measurements show that the chaperone-induced clustering of HypF-N oligomers does not change the overall solvent exposure of hydrophobic residues on the surface of the oligomers. αB-crystallin, clusterin and M-TTR can diminish the cytotoxic effects of the HypF-N oligomers at all chaperone concentration, as demonstrated by MTT reduction and Ca2+ influx measurements. The observation that the protective effect is primarily at all concentrations of chaperones, both when the increase in HypF-N aggregate size is minimal and large, emphasizes the efficiency and versatility of these protein molecules.

From Genome to Structure and Back Again: A Family Portrait of the Transcarbamylases.

Enzymes in the transcarbamylase family catalyze the transfer of a carbamyl group from carbamyl phosphate (CP) to an amino group of a second substrate. The two best-characterized members, aspartate transcarbamylase (ATCase) and ornithine transcarbamylase (OTCase), are present in most organisms from bacteria to humans. Recently, structures of four new transcarbamylase members, N-acetyl-L-ornithine transcarbamylase (AOTCase), N-succinyl-L-ornithine transcarbamylase (SOTCase), ygeW encoded transcarbamylase (YTCase) and putrescine transcarbamylase (PTCase) have also been determined. Crystal structures of these enzymes have shown that they have a common overall fold with a trimer as their basic biological unit. The monomer structures share a common CP binding site in their N-terminal domain, but have different second substrate binding sites in their C-terminal domain. The discovery of three new transcarbamylases, l-2,3-diaminopropionate transcarbamylase (DPTCase), l-2,4-diaminobutyrate transcarbamylase (DBTCase) and ureidoglycine transcarbamylase (UGTCase), demonstrates that our knowledge and understanding of the spectrum of the transcarbamylase family is still incomplete. In this review, we summarize studies on the structures and function of transcarbamylases demonstrating how structural information helps to define biological function and how small structural differences govern enzyme specificity. Such information is important for correctly annotating transcarbamylase sequences in the genome databases and for identifying new members of the transcarbamylase family.

Biosynthesis of the carbamoylated D-gulosamine moiety of streptothricins: involvement of a guanidino-N-glycosyltransferase and an N-acetyl-D-gulosamine deacetylase.

Streptothricins (STNs) are atypical aminoglycosides containing a rare carbamoylated D-gulosamine (D-GulN) moiety, and the antimicrobial activity of STNs has been exploited for crop protection. Herein, the biosynthetic pathway of the carbamoylated D-GulN moiety was delineated. An N-acetyl-D-galactosamine is first attached to the streptolidine lactam by the glycosyltransferse StnG and then epimerized to N-acetyl-D-gulosamine by the putative epimerase StnJ. After carbamoylation by the carbamoyltransferase StnQ, N-acetyl-D-GulN is deacetylated by StnI to furnish the carbamoylated D-GulN moiety. In vitro studies characterized two novel enzymes: StnG is an unprecedented GT-A fold N-glycosyltransferase that glycosylates the imine nitrogen atom of guanidine, and StnI is the first reported N-acetyl-D-GulN deacetylase.

A substrate-induced biotin binding pocket in the carboxyltransferase domain of pyruvate carboxylase.

Biotin-dependent enzymes catalyze carboxyl transfer reactions by efficiently coordinating multiple reactions between spatially distinct active sites. Pyruvate carboxylase (PC), a multifunctional biotin-dependent enzyme, catalyzes the bicarbonate- and MgATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in mammalian tissues. To complete the overall reaction, the tethered biotin prosthetic group must first gain access to the biotin carboxylase domain and become carboxylated and then translocate to the carboxyltransferase domain, where the carboxyl group is transferred from biotin to pyruvate. Here, we report structural and kinetic evidence for the formation of a substrate-induced biotin binding pocket in the carboxyltransferase domain of PC from Rhizobium etli. Structures of the carboxyltransferase domain reveal that R. etli PC occupies a symmetrical conformation in the absence of the biotin carboxylase domain and that the carboxyltransferase domain active site is conformationally rearranged upon pyruvate binding. This conformational change is stabilized by the interaction of the conserved residues Asp(590) and Tyr(628) and results in the formation of the biotin binding pocket. Site-directed mutations at these residues reduce the rate of biotin-dependent reactions but have no effect on the rate of biotin-independent oxaloacetate decarboxylation. Given the conservation with carboxyltransferase domains in oxaloacetate decarboxylase and transcarboxylase, the structure-based mechanism described for PC may be applicable to the larger family of biotin-dependent enzymes.

Structure, activity, and inhibition of the Carboxyltransferase ß-subunit of acetyl coenzyme A carboxylase (AccD6) from Mycobacterium tuberculosis.

In Mycobacterium tuberculosis, the carboxylation of acetyl coenzyme A (acetyl-CoA) to produce malonyl-CoA, a building block in long-chain fatty acid biosynthesis, is catalyzed by two enzymes working sequentially: a biotin carboxylase (AccA) and a carboxyltransferase (AccD). While the exact roles of the three different biotin carboxylases (AccA1 to -3) and the six carboxyltransferases (AccD1 to -6) in M. tuberculosis are still not clear, AccD6 in complex with AccA3 can synthesize malonyl-CoA from acetyl-CoA. A series of 10 herbicides that target plant acetyl-CoA carboxylases (ACC) were tested for inhibition of AccD6 and for whole-cell activity against M. tuberculosis. From the tested herbicides, haloxyfop, an arylophenoxypropionate, showed in vitro inhibition of M. tuberculosis AccD6, with a 50% inhibitory concentration (IC50) of 21.4 ± 1 µM. Here, we report the crystal structures of M. tuberculosis AccD6 in the apo form (3.0 Å) and in complex with haloxyfop-R (2.3 Å). The structure of M. tuberculosis AccD6 in complex with haloxyfop-R shows two molecules of the inhibitor bound on each AccD6 subunit. These results indicate the potential for developing novel therapeutics for tuberculosis based on herbicides with low human toxicity.

Functionally diverse biotin-dependent enzymes with oxaloacetate decarboxylase activity.

Biotin-dependent enzymes catalyze carboxylation, decarboxylation and transcarboxylation reactions that participate in the primary metabolism of a wide range of organisms. In all cases, the overall reaction proceeds via two half reactions that take place in physically distinct active sites. In the first half-reaction, a carboxyl group is transferred to the 1-N' of a covalently tethered biotin cofactor. The tethered carboxybiotin intermediate subsequently translocates to a second active site where the carboxyl group is either transferred to an acceptor substrate or, in some bacteria and archaea, is decarboxylated to biotin and CO2 in order to power the export of sodium ions from the cytoplasm. A homologous carboxyltransferase domain is found in three enzymes that catalyze diverse overall reactions: carbon fixation by pyruvate carboxylase, decarboxylation and sodium transport by the biotin-dependent oxaloacetate decarboxylase complex, and transcarboxylation by transcarboxylase from Propionibacterium shermanii. Over the past several years, structural data have emerged which have greatly advanced the mechanistic description of these enzymes. This review assembles a uniform description of the carboxyltransferase domain structure and catalytic mechanism from recent studies of pyruvate carboxylase, oxaloacetate decarboxylase and transcarboxylase, three enzymes that utilize an analogous carboxyltransferase domain to catalyze the biotin-dependent decarboxylation of oxaloacetate.

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.

Hydrogenase activity and proton-motive force generation by Escherichia coli during glycerol fermentation.

Proton motive force (Δp) generation by Escherichia coli wild type cells during glycerol fermentation was first studied. Its two components, electrical-the membrane potential (∆φ) and chemical-the pH transmembrane gradient (ΔpH), were established and the effects of external pH (pHex) were determined. Intracellular pH was 7.0 and 6.0 and lower than pHex at pH 7.5 and 6.5, respectively; and it was higher than pHex at pH 5.5. At high pHex, the increase of ∆φ (-130 mV) was only partially compensated by a reversed ΔpH, resulting in a low Δp. At low pHex ∆φ and consequently Δp were decreased. The generation of Δp during glycerol fermentation was compared with glucose fermentation, and the difference in Δp might be due to distinguished mechanisms for H(+) transport through the membrane, especially to hydrogenase (Hyd) enzymes besides the F0F1-ATPase. H(+) efflux was determined to depend on pHex; overall and N,N'-dicyclohexylcarbodiimide (DCCD)-inhibitory H(+) efflux was maximal at pH 6.5. Moreover, ΔpH was changed at pH 6.5 and Δp was different at pH 6.5 and 5.5 with the hypF mutant lacking all Hyd enzymes. DCCD-inhibited ATPase activity of membrane vesicles was maximal at pH 7.5 and decreased with the hypF mutant. Thus, Δp generation by E. coli during glycerol fermentation is different than that during glucose fermentation. Δp is dependent on pHex, and a role of Hyd enzymes in its generation is suggested.

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.

Crystal structure and biochemical properties of putrescine carbamoyltransferase from Enterococcus faecalis: Assembly, active site, and allosteric regulation.

Putrescine carbamoyltransferase (PTCase) catalyzes the conversion of carbamoylputrescine to putrescine and carbamoyl phosphate (CP), a substrate of carbamate kinase (CK). The crystal structure of PTCase has been determined and refined at 3.2 Å resolution. The trimeric molecular structure of PTCase is similar to other carbamoyltransferases, including the catalytic subunit of aspartate carbamoyltransferase (ATCase) and ornithine carbamoyltransferase (OTCase). However, in contrast to other trimeric carbamoyltransferases, PTCase binds both CP and putrescine with Hill coefficients at saturating concentrations of the other substrate of 1.53 ± 0.03 and 1.80 ± 0.06, respectively. PTCase also has a unique structural feature: a long C-terminal helix that interacts with the adjacent subunit to enhance intersubunit interactions in the molecular trimer. The C-terminal helix appears to be essential for both formation of the functional trimer and catalytic activity, since truncated PTCase without the C-terminal helix aggregates and has only 3% of native catalytic activity. The active sites of PTCase and OTCase are similar, with the exception of the 240's loop. PTCase lacks the proline-rich sequence found in knotted carbamoyltransferases and is unknotted. A Blast search of all available genomes indicates that 35 bacteria, most of which are Gram-positive, have an agcB gene encoding PTCase located near the genes that encode agmatine deiminase and CK, consistent with the catabolic role of PTCase in the agmatine degradation pathway. Sequence comparisons indicate that the C-terminal helix identified in this PTCase structure will be found in all other PTCases identified, suggesting that it is the signature feature of the PTCase family of enzymes.