Peeling back the many layers of competitive exclusion.

Baby chicks administered a fecal transplant from adult chickens are resistant to Salmonella colonization by competitive exclusion. A two-pronged approach was used to investigate the mechanism of this process. First, Salmonella response to an exclusive (Salmonella competitive exclusion product, Aviguard®) or permissive microbial community (chicken cecal contents from colonized birds containing 7.85 Log10Salmonella genomes/gram) was assessed ex vivo using a S. typhimurium reporter strain with fluorescent YFP and CFP gene fusions to rrn and hilA operon, respectively. Second, cecal transcriptome analysis was used to assess the cecal communities' response to Salmonella in chickens with low (≤5.85 Log10 genomes/g) or high (≥6.00 Log10 genomes/g) Salmonella colonization. The ex vivo experiment revealed a reduction in Salmonella growth and hilA expression following co-culture with the exclusive community. The exclusive community also repressed Salmonella's SPI-1 virulence genes and LPS modification, while the anti-virulence/inflammatory gene avrA was upregulated. Salmonella transcriptome analysis revealed significant metabolic disparities in Salmonella grown with the two different communities. Propanediol utilization and vitamin B12 synthesis were central to Salmonella metabolism co-cultured with either community, and mutations in propanediol and vitamin B12 metabolism altered Salmonella growth in the exclusive community. There were significant differences in the cecal community's stress response to Salmonella colonization. Cecal community transcripts indicated that antimicrobials were central to the type of stress response detected in the low Salmonella abundance community, suggesting antagonism involved in Salmonella exclusion. This study indicates complex community interactions that modulate Salmonella metabolism and pathogenic behavior and reduce growth through antagonism may be key to exclusion.

Campylobacter jejuni uses energy taxis and a dehydrogenase enzyme for l-fucose chemotaxis.

IMPORTANCE: In this study, we identify a separate role for the Campylobacter jejuni l-fucose dehydrogenase in l-fucose chemotaxis and demonstrate that this mechanism is not only limited to C. jejuni but is also present in Burkholderia multivorans. We now hypothesize that l-fucose energy taxis may contribute to the reduction of l-fucose-metabolizing strains of C. jejuni from the gastrointestinal tract of breastfed infants, selecting for isolates with increased colonization potential.

The coenzyme B12 precursor 5,6-dimethylbenzimidazole is a flavin antagonist in Salmonella.

Salmonella enterica subsp. enterica sv. Typhimurium str. LT2 (hereafter S. Typhimurium) synthesizes adenosylcobalamin (AdoCbl, CoB12) de novo only under anoxic conditions, but it can assemble the lower ligand loop (a.k.a. the nucleotide loop) and can form the unique C-Co bond present in CoB12 in the presence or absence of molecular oxygen. During studies of nucleotide loop assembly in S. Typhimurium, we noticed that the growth of this bacterium could be arrested by the lower ligand nucleobase, namely 5,6-dimethylbenzimidazole (DMB). Here we report in vitro and in vivo evidence that shows that the structural similarity of DMB to the isoalloxazine moiety of flavin cofactors causes its deleterious effect on cell growth. We studied DMB inhibition of the housekeeping flavin dehydrogenase (Fre) and three flavoenzymes that initiate the catabolism of tricarballylate, succinate or D-alanine in S. Typhimurium. Notably, while growth with tricarballylate was inhibited by 5-methyl-benzimidazole (5-Me-Bza) and DMB, growth with succinate or glycerol was arrested by DMB but not by 5-Me-Bza. Neither unsubstituted benzimidazole nor adenine inhibited growth of S. Typhimurium at DMB inhibitory concentrations. Whole genome sequencing analysis of spontaneous mutant strains that grew in the presence of inhibitory concentrations of DMB identified mutations effecting the cycA (encodes D-Ala/D-Ser transporter) and dctA (encodes dicarboxylate transporter) genes and in the coding sequence of the tricarballylate transporter (TcuC), suggesting that increased uptake of substrates relieved DMB inhibition. We discuss two possible mechanisms of inhibition by DMB.

In Salmonella enterica, the pathogenicity island 2 (SPI-2) regulator PagR regulates its own expression and the expression of a five-gene operon that encodes transketolase C.

The enteropathogen Salmonella enterica subsp. enterica sv. Typhimurium str. LT2 (hereafter S. Typhimurium) utilizes a cluster of genes encoded within the pathogenicity island 2 (SPI-2) of its genome to proliferate inside macrophages. The expression of SPI-2 is controlled by a complex network of transcriptional regulators and environmental cues, which now include a recently characterized DNA-binding protein named PagR. Growth of S. Typhimurium in low-phosphate, low-magnesium medium mimics conditions inside macrophages. Under such conditions, PagR ensures SPI-2 induction by upregulating the transcription of slyA, which encodes a known activator of SPI-2. Here, we report that PagR represses the expression of a divergently transcribed polycistronic operon that encodes the two subunits of transketolase TktC (i.e., tktD, tktE) of this bacterium. Transketolases contribute to the nonredox rearrangements of phosphorylated sugars of the pentose phosphate pathway, which provide building blocks for amino acids, nucleotides, cofactors, etc. We also demonstrate that PagR represses the expression of its own gene and define two PagR-binding sites between stm2344 and pagR.

Structural studies of the phosphoribosyltransferase involved in cobamide biosynthesis in methanogenic archaea and cyanobacteria.

Cobamides (Cbas) are coenzymes used by cells across all domains of life, but de novo synthesis is only found in some bacteria and archaea. Five enzymes assemble the nucleotide loop in the alpha phase of the corrin ring. Condensation of the activated ring and nucleobase yields adenosyl-Cba 5'-phosphate, which upon dephosphorylation yields the biologically active coenzyme (AdoCba). Base activation is catalyzed by a phosphoribosyltransferase (PRTase). The structure of the Salmonella enterica PRTase enzyme (i.e., SeCobT) is well-characterized, but archaeal PRTases are not. To gain insights into the mechanism of base activation by the PRTase from Methanocaldococcus jannaschii (MjCobT), we solved crystal structures of the enzyme in complex with substrate and products. We determined several structures: (i) a 2.2 Å structure of MjCobT in the absence of ligand (apo), (ii) structures of MjCobT bound to nicotinate mononucleotide (NaMN) and α-ribazole 5'-phosphate (α-RP) or α-adenylyl-5'-phosphate (α-AMP) at 2.3 and 1.4 Å, respectively. In MjCobT the general base that triggers the reaction is an aspartate residue (Asp 52) rather than a glutamate residue (E317) as in SeCobT. Notably, the dimer interface in MjCobT is completely different from that observed in SeCobT. Finally, entry PDB 3L0Z does not reflect the correct structure of MjCobT.

Localization and interaction studies of the Salmonella enterica ethanolamine ammonia-lyase (EutBC), its reactivase (EutA), and the EutT corrinoid adenosyltransferase.

Some prokaryotes compartmentalize select metabolic capabilities. Salmonella enterica subspecies enterica serovar Typhimurium LT2 (hereafter S. Typhimurium) catabolizes ethanolamine (EA) within a proteinaceous compartment that we refer to as the ethanolamine utilization (Eut) metabolosome. EA catabolism is initiated by the adenosylcobalamin (AdoCbl)-dependent ethanolamine ammonia-lyase (EAL), which deaminates EA via an adenosyl radical mechanism to yield acetaldehyde plus ammonia. This adenosyl radical can be quenched, requiring the replacement of AdoCbl by the ATP-dependent EutA reactivase. During growth on ethanolamine, S. Typhimurium synthesizes AdoCbl from cobalamin (Cbl) using the ATP:Co(I)rrinoid adenosyltransferase (ACAT) EutT. It is known that EAL localizes to the metabolosome, however, prior to this work, it was unclear where EutA and EutT localized, and whether they interacted with EAL. Here, we provide evidence that EAL, EutA, and EutT localize to the Eut metabolosome, and that EutA interacts directly with EAL. We did not observe interactions between EutT and EAL nor between EutT and the EutA/EAL complex. However, growth phenotypes of a ΔeutT mutant strain show that EutT is critical for efficient ethanolamine catabolism. This work provides a preliminary understanding of the dynamics of AdoCbl synthesis and its uses within the Eut metabolosome.

Acinetobacter baumannii Catabolizes Ethanolamine in the Absence of a Metabolosome and Converts Cobinamide into Adenosylated Cobamides.

Acinetobacter baumannii is an opportunistic pathogen typically associated with hospital-acquired infections. Our understanding of the metabolism and physiology of A. baumannii is limited. Here, we report that A. baumannii uses ethanolamine (EA) as the sole source of nitrogen and can use this aminoalcohol as a source of carbon and energy if the expression of the eutBC genes encoding ethanolamine ammonia-lyase (EAL) is increased. A strain with an ISAba1 element upstream of the eutBC genes efficiently used EA as a carbon and energy source. The A. baumannii EAL (AbEAL) enzyme supported the growth of a strain of Salmonella lacking the entire eut operon. Remarkably, the growth of the above-mentioned Salmonella strain did not require the metabolosome, the reactivase EutA enzyme, the EutE acetaldehyde dehydrogenase, or the addition of glutathione to the medium. Transmission electron micrographs showed that when Acinetobacter baumannii or Salmonella enterica subsp. enterica serovar Typhimurium strain LT2 synthesized AbEAL, the protein localized to the cell membrane. We also report that the A. baumannii genome encodes all of the enzymes needed for the assembly of the nucleotide loop of cobamides and that it uses these enzymes to synthesize different cobamides from the precursor cobinamide and several nucleobases. In the absence of exogenous nucleobases, the most abundant cobamide produced by A. baumannii was cobalamin. IMPORTANCE Acinetobacter baumannii is a Gram-negative bacterium commonly found in soil and water. A. baumannii is an opportunistic human pathogen, considered by the CDC to be a serious threat to human health due to the multidrug resistance commonly associated with this bacterium. Knowledge of the metabolic capabilities of A. baumannii is limited. The importance of the work reported here lies in the identification of ethanolamine catabolism occurring in the absence of a metabolosome structure. In other bacteria, this structure protects the cell against damage by acetaldehyde generated by the deamination of ethanolamine. In addition, the ethanolamine ammonia-lyase (EAL) enzyme of this bacterium is unique in that it does not require a reactivase enzyme to remain active. Importantly, we also demonstrate that the A. baumannii genome encodes the functions needed to assemble adenosylcobamide, the coenzyme of EAL, from the precursor cobinamide.

A method for the isolation of α-ribazole from vitamin B12, and its enzymatic conversion to α-ribazole 5'-phosphate.

Cobamides (Cbas) are the largest coenzymes known and are used by cells in all domains of life. These molecules are characterized by a central cobalt-containing tetrapyrrole ring with two opposing axial ligands on the α and ß faces of the ring. All biologically active forms of Cbas have a 5'-deoxyadenosyl group as the upper (Coß) ligand that is covalently attached to the cobalt ion of the ring. In contrast, the lower ligand is a nucleobase of diverse chemical structure; however, nucleobases are usually derivatives of benzimidazole or purine. Phenol and p-cresol can also serve as the nucleobase, but they cannot form a coordination bond with the cobalt ion of the ring because they lack a free pair of electrons. The Cba incorporating 5,6-dimethylbenzimidazole (DMB) is known as cobalamin (Cbl), and the coenzymic form of cobalamin is known as adenosylcobalamin (AdoCbl). A common vitamer of cobalamin has a cyano group as the upper ligand. This vitamer is known as cyanocobalamin (CNCbl), which is commercially marketed as vitamin B12. Here, we describe a combination of chemical hydrolysis of cobalamin with the enzymatic dephosphorylation of the resulting α-R-3'-phosphate to yield α-R, which we enzymically convert to the pathway intermediate α-R-5'-phosphate (α-RP). The methods describe herein can be readily scaled up to generate large amounts of α-RP.

A method for the production, purification and liposome reconstitution of cobamide synthase.

Cobamides are essential for the performance of a variety of reactions such methyl transfers, carbon skeleton rearrangements, and eliminations in both prokaryotes and eukaryotes. However, cobamide biosynthesis is limited to a subset of bacteria and archaea. The biosynthesis pathway culminates with the activation and attachment of a lower ligand to the corrin ring; this branch of the pathway is known as nucleotide loop assembly (NLA) pathway. The cobamide synthase (CobS) enzyme is the penultimate step in NLA pathway, and catalyzes the attachment of an α-ribotide to the activated corrin ring. While other NLA enzymes have been well-studied, studies of CobS have proven difficult to date. CobS is an integral membrane protein, and limitations have been largely due to difficulties in protein purification. Here we provide a method to purify CobS, reconstitute protein in proteoliposomes, and assay for its activity.

A method for the efficient adenosylation of corrinoids.

Adenosylcobamides (AdoCbas) are coenzymes required by organisms from all domains of life to perform challenging chemical reactions. AdoCbas are characterized by a cobalt-containing tetrapyrrole ring, where an adenosyl group is covalently attached to the cobalt ion via a unique Co-C organometallic bond. During catalysis, this bond is homolytically cleaved by AdoCba-dependent enzymes to form an adenosyl radical that is critical for intra-molecular rearrangements. The formation of the Co-C bond is catalyzed by a family of enzymes known as ATP:Co(I)rrinoid adenosyltransferases (ACATs). ACATs adenosylate Cbas in two steps: (I) they generate a planar, Co(II) four-coordinate Cba to facilitate the reduction of Co(II) to Co(I), and (II) they transfer the adenosyl group from ATP to the Co(I) ion. To synthesize adenosylated corrinoids in vitro, it is imperative that anoxic conditions are maintained to avoid oxidation of Co(II) or Co(I) ions. Here we describe a method for the enzymatic synthesis and quantification of specific AdoCbas.

Elevated Levels of an Enzyme Involved in Coenzyme B12 Biosynthesis Kills Escherichia coli.

Cobamides are cobalt-containing cyclic tetrapyrroles involved in the metabolism of organisms from all domains of life but produced de novo only by some bacteria and archaea. The pathway is thought to involve up to 30 enzymes, five of which comprise the so-called "late" steps of cobamide biosynthesis. Two of these reactions activate the corrin ring, one activates the nucleobase, a fourth one condenses activated precursors, and a phosphatase yields the final product of the pathway. The penultimate step is catalyzed by a polytopic integral membrane protein, namely, the cobamide (5'-phosphate) synthase, also known as cobamide synthase. At present, the reason for the association of all putative and bona fide cobamide synthases to cell membranes is unclear and intriguing. Here, we show that, in Escherichia coli, elevated levels of cobamide synthase kill the cell by dissipating the proton motive force and compromising membrane stability. We also show that overproduction of the phosphatase that catalyzes the last step of the pathway or phage shock protein A prevents cell death when the gene encoding cobamide synthase is overexpressed. We propose that in E. coli, and probably all cobamide producers, cobamide synthase anchors a multienzyme complex responsible for the assembly of vitamin B12 and other cobamides. IMPORTANCE E. coli is the best-studied prokaryote, and some strains of this bacterium are human pathogens. We show that when the level of the enzyme that catalyzes the penultimate step of vitamin B12 biosynthesis is elevated, the viability of E. coli decreases. These findings are of broad significance because the enzyme alluded to is an integral membrane protein in all cobamide-producing bacteria, many of which are human pathogens. Our results may provide new avenues for the development of antimicrobials, because none of the enzymes involved in vitamin B12 biosynthesis are present in mammalian cells.

Protein N-terminal acylation: An emerging field in bacterial cell physiology.

N-terminal (Nt)-acylation is the irreversible addition of an acyl moiety to the terminal alpha amino group of a peptide chain. This type of modification alters the nature of the N terminus, which can interfere with the function of the modified protein by disrupting protein interactions, function, localization, degradation, hydrophobicity, or charge. Nt acylation is found in all domains of life and is a highly common occurrence in eukaryotic cells. However, in prokaryotes very few cases of Nt acylation have been reported. It was once thought that Nt acylation of proteins, other than ribosomal proteins, was uncommon in prokaryotes, but recent evidence suggests that this modification may be more common than once realized. In this review, we discuss what is known about prokaryotic Nt acetylation and the acetyltransferases that are responsible, as well as recent advancements in this field and currently used methods to study Nt acetylation.

Sirtuin-Dependent Reversible Lysine Acetylation Controls the Activity of Acetyl Coenzyme A Synthetase in Campylobacter jejuni.

Posttranslational modifications are mechanisms for rapid control of protein function used by cells from all domains of life. Acetylation of the epsilon amino group (Nε) of an active-site lysine of the AMP-forming acetyl coenzyme A (acetyl-CoA) synthetase (Acs) enzyme is the paradigm for the posttranslational control of the activity of metabolic enzymes. In bacteria, this active-site lysine of Acs enzymes can be modified by a number of different GCN5-type N-acetyltransferases (GNATs). Acs activity is lost as a result of acetylation and is restored by deacetylation. Using a heterologous host, we show that Campylobacter jejuni NCTC11168 synthesizes enzymes that control Acs function by reversible lysine acetylation (RLA). This work validates the function of gene products encoded by the cj1537c, cj1715, and cj1050c loci, namely, the AMP-forming acetate-CoA ligase (CjAcs), a type IV GCN5-type lysine acetyltransferase (GNAT [CjLatA]), and a NAD+-dependent (class III) sirtuin deacylase (CjCobB), respectively. To our knowledge, these are the first in vivo and in vitro data on C. jejuni enzymes that control the activity of CjAcs. IMPORTANCE This work provides the experimental evidence needed to support the assignment of function to three key enzymes, two of which control the reversible posttranslational modification of an active-site lysyl residue of the central metabolic enzyme acetyl-CoA synthetase (CjAcs). We can now generate Campylobacter jejuni mutant strains defective in these functions, so we can establish the conditions in which this mode of regulation of CjAcs is triggered in this bacterium. Such knowledge may provide new therapeutic strategies for the control of this pathogen.

Functional Studies of α-Riboside Activation by the α-Ribazole Kinase (CblS) from Geobacillus kaustophilus.

We report the initial characterization of the α-ribazole (α-R) kinase enzyme of Geobacillus kaustophilus (GkCblS), which converts α-R to α-R-phosphate (α-RP) during the synthesis of cobamides. We implemented a continuous spectrophotometric assay to obtain kinetic parameters for several potential substrates and to study the specificity of the enzyme for α-N-linked ribosides. The apparent Km values for α-R and ATP were 358 and 297 µM, respectively. We also report methods for synthesizing and quantifying non-commercially available α-ribosides and ß-ribazole (ß-R). Purified GkCblS activated α-R and other α-ribosides, including α-adenosine (α-Ado). GkCblS did not phosphorylate ß-N-linked glycosides like ß-adenosine or ß-R. Expression of G. kaustophilus cblS+ in a Salmonella enterica subsp. enterica sv Typhimurium LT2 (S. enterica) strain lacking the nicotinate mononucleotide:5,6-dimethylbenzimidazole phosphoribosyl transferase (CobT) enzyme resulted in the activation of various benzimidazole α-ribosides, and the synthesis of benzimidazolyl cobamides to levels that supported robust growth. Notably, α-Ado did not support growth under similar conditions, in spite of the fact that GkCblS phosphorylated α-Ado in vitro. When α-Ado was provided at a very high concentration, growth was observed. This result suggested that in S. enterica α-Ado transport may be inefficient. We conclude that GkCblS has specificity for α-N-glycosidic bonds, but not for the base in α-ribosides.

Insights into the Relationship between Cobamide Synthase and the Cell Membrane.

Cobamides are cobalt-containing cyclic tetrapyrroles used by cells from all domains of life but only produced de novo by some bacteria and archaea. The "late steps" of the adenosylcobamide biosynthetic pathway are responsible for the assembly of the nucleotide loop and are required during de novo synthesis and precursor salvaging. These steps are characterized by activation of the corrin ring and lower ligand base, condensation of the activated precursors to adenosylcobamide phosphate, and removal of the phosphate, yielding a complete adenosylcobamide molecule. The condensation of the activated corrin ring and lower ligand base is performed by an integral membrane protein, cobamide (5' phosphate) synthase (CobS), and represents an important convergence of two pathways necessary for nucleotide loop assembly. Interestingly, membrane association of this penultimate step is conserved among all cobamide producers, yet the physiological relevance of this association is not known. Here, we present the purification and biochemical characterization of the CobS enzyme of the enterobacterium Salmonella enterica subsp. enterica serovar Typhimurium strain LT2, investigate its association with liposomes, and quantify the effect of the lipid bilayer on its enzymatic activity and substrate affinity. We report a purification scheme that yields pure CobS protein, allowing in vitro functional analysis. Additionally, we report a method for liposome reconstitution of CobS, allowing for physiologically relevant studies of this inner membrane protein in a phospholipid bilayer. In vitro and in vivo data reported here expand our understanding of CobS and the implications of membrane-associated adenosylcobamide biosynthesis.IMPORTANCESalmonella is a human pathogen of worldwide importance, and coenzyme B12 is critical for the pathogenic lifestyle of this bacterium. The importance of the work reported here lies on the improvements to the methodology used to isolate cobamide synthase, a polytopic integral membrane protein that catalyzes the penultimate step of coenzyme B12 biosynthesis. This advance is an important step in the analysis of the proposed multienzyme complex responsible for the assembly of the nucleotide loop during de novo coenzyme B12 biosynthesis and for the assimilation of incomplete corrinoids from the environment. We proposed that cobamide synthase is likely localized to the cell membrane of every coenzyme B12-producing bacterium and archaeum sequenced to date. The new knowledge of cobamide synthase advances our understanding of the functionality of the enzyme in the context of the lipid bilayer and sets the foundation for the functional-structural analysis of the aforementioned multienzyme complex.

Modulation of the bacterial CobB sirtuin deacylase activity by N-terminal acetylation.

In eukaryotic cells, the N-terminal amino moiety of many proteins is modified by N-acetyltransferases (NATs). This protein modification can alter the folding of the target protein; can affect binding interactions of the target protein with substrates, allosteric effectors, or other proteins; or can trigger protein degradation. In prokaryotes, only ribosomal proteins are known to be N-terminally acetylated, and the acetyltransferases responsible for this modification belong to the Rim family of proteins. Here, we report that, in Salmonella enterica, the sirtuin deacylase CobB long isoform (CobBL) is N-terminally acetylated by the YiaC protein of this bacterium. Results of in vitro acetylation assays showed that CobBL was acetylated by YiaC; liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to confirm these results. Results of in vitro and in vivo experiments showed that CobBL deacetylase activity was negatively affected when YiaC acetylated its N terminus. We report 1) modulation of a bacterial sirtuin deacylase activity by acetylation, 2) that the Gcn5-related YiaC protein is the acetyltransferase that modifies CobBL, and 3) that YiaC is an NAT. Based on our data, we propose the name of NatA (N-acyltransferase A) in lieu of YiaC to reflect the function of the enzyme.

Small-Molecule Acetylation by GCN5-Related N-Acetyltransferases in Bacteria.

Acetylation is a conserved modification used to regulate a variety of cellular pathways, such as gene expression, protein synthesis, detoxification, and virulence. Acetyltransferase enzymes transfer an acetyl moiety, usually from acetyl coenzyme A (AcCoA), onto a target substrate, thereby modulating activity or stability. Members of the GCN5- N -acetyltransferase (GNAT) protein superfamily are found in all domains of life and are characterized by a core structural domain architecture. These enzymes can modify primary amines of small molecules or of lysyl residues of proteins. From the initial discovery of antibiotic acetylation, GNATs have been shown to modify a myriad of small-molecule substrates, including tRNAs, polyamines, cell wall components, and other toxins. This review focuses on the literature on small-molecule substrates of GNATs in bacteria, including structural examples, to understand ligand binding and catalysis. Understanding the plethora and versatility of substrates helps frame the role of acetylation within the larger context of bacterial cellular physiology.

Mutational and Functional Analyses of Substrate Binding and Catalysis of the Listeria monocytogenes EutT ATP:Co(I)rrinoid Adenosyltransferase.

ATP:Co(I)rrinoid adenosyltransferases (ACATs) catalyze the transfer of the adenosyl moiety from co-substrate ATP to a corrinoid substrate. ACATs are grouped into three families, namely, CobA, PduO, and EutT. The EutT family of enzymes is further divided into two classes, depending on whether they require a divalent metal ion for activity (class I and class II). To date, a structure has not been elucidated for either class of the EutT family of ACATs. In this work, results of bioinformatics analyses revealed several conserved residues between the C-terminus of EutT homologues and the structurally characterized Lactobacillus reuteri PduO (LrPduO) homologue. In LrPduO, these residues are associated with ATP binding and formation of an intersubunit salt bridge. These residues were substituted, and in vivo and in vitro data support the conclusion that the equivalent residues in the metal-free (i.e., class II) Listeria monocytogenes EutT (LmEutT) enzyme affect ATP binding. Results of in vivo and in vitro analyses of LmEutT variants with substitutions at phenylalanine and tryptophan residues revealed that replacement of the phenylalanine residue at position 72 affected access to the substrate-binding site and replacement of a tryptophan residue at position 238 affected binding of the Cbl substrate to the active site. Unlike the PduO family of ACATs, a single phenylalanine residue is not responsible for displacement of the α-ligand. Together, these data suggest that while EutT enzymes share a conserved ATP-binding motif and an intersubunit salt bridge with PduO family ACATs, class II EutT family ACATs utilize an unidentified mechanism for Cbl lower-ligand displacement and reduction that is different from that of PduO and CobA family ACATs.

New AMP-forming acid:CoA ligases from Streptomyces lividans, some of which are posttranslationally regulated by reversible lysine acetylation.

In nature, organic acids are a commonly used source of carbon and energy. Many bacteria use AMP-forming acid:CoA ligases to convert organic acids into their corresponding acyl-CoA derivatives, which can then enter metabolism. The soil environment contains a broad diversity of organic acids, so it is not surprising that bacteria such as Streptomyces lividans can activate many of the available organic acids. Our group has shown that the activity of many acid:CoA ligases is posttranslationally controlled by acylation of an active-site lysine. In some cases, the modification is reversed by deacylases of different types. We identified eight new acid:CoA ligases in S. lividans TK24. Here, we report the range of organic acids that each of these enzymes can activate, and determined that two of the newly identified CoA ligases were under NAD+ -dependent sirtuin deacylase reversible lysine (de)acetylation control, four were not acetylated by two acetyltransferases used in this work, and two were acetylated but not deacetylated by sirtuin. This work provides insights into the broad organic-acid metabolic capabilities of S. lividans, and sheds light into the control of the activities of CoA ligases involved in the activation of organic acids in this bacterium.

The l-Thr Kinase/l-Thr-Phosphate Decarboxylase (CobD) Enzyme from Methanosarcina mazei Gö1 Contains Metallocenters Needed for Optimal Activity.

The MM2060 (cobD) gene from Methanosarcina mazei strain Gö1 encodes a protein (MmCobD) with l-threonine kinase (PduX) and l-threonine-O-3-phosphate decarboxylase (CobD) activities. In addition to the unexpected l-Thr kinase activity, MmCobD has an extended carboxy-terminal (C-terminal) region annotated as a putative metal-binding zinc finger-like domain. Here, we demonstrate that the C-terminus of MmCobD is a ferroprotein containing ∼25 non-heme iron atoms per monomer of protein. The absence of the C-terminus substantially reduces, but does not abolish, enzymatic activities in vitro and in vivo. Single-residue substitutions of C-terminal putative Fe-binding cysteinyl and histidinyl residues resulted in the loss of Fe and changes in enzyme activity levels. Salmonella enterica ΔpduX and ΔcobD strains were used as heterologous hosts to assess coenzyme B12 biosynthesis as a function of 17 MmCobD variants tested. Some of the latter displayed 5-fold higher enzymatic activity in vitro and enhanced the growth rate of the S. enterica strains that synthesized them. Most of the MmCobD variants tested were up to 6-fold less active in vitro and supported slow growth rates of the S. enterica strains that synthesized them; some substitutions abolished enzyme activity. MmCobD exhibited an ultraviolet-visible absorption spectrum consistent with [4Fe-4S] clusters that appeared to be susceptible to oxidation by H2O2 and reduction by sodium dithionite. The presence of FeS clusters in MmCobD was corroborated by electron paramagnetic resonance and magnetic circular dichroism studies. Collectively, our results suggest that MmCobD contains one or more diamagnetic [4Fe-4S]2+ center(s) that may play a structural or regulatory role.