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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. [ABSTRACT FROM AUTHOR]

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 [ABSTRACT FROM AUTHOR]

Acetylation is a posttranslational modification conserved in all domains of life that is carried out by N-acetyltransferases. While acetylation can occur on Nα-amino groups, this review will focus on Nε-acetylation of lysyl residues and how the posttranslational modification changes the cellular physiology of bacteria. Up until the late 1990s, acetylation was studied in eukaryotes in the context of chromatin maintenance and gene expression. At present, bacterial protein acetylation plays a prominent role in central and secondary metabolism, virulence, transcription, and translation. Given the diversity of niches in the microbial world, it is not surprising that the targets of bacterial protein acetyltransferases are very diverse, making their biochemical characterization challenging. The paradigm for acetylation in bacteria involves the acetylation of acetyl-CoA synthetase, whose activity must be tightly regulated to maintain energy charge homeostasis. While this paradigm has provided much mechanistic detail for acetylation and deacetylation, in this review we discuss advances in the field that are changing our understanding of the physiological role of protein acetylation in bacteria. [ABSTRACT FROM AUTHOR]

Acylation of epsilon amino groups of lysyl side chains is a widespread modification of proteins and small molecules in cells of all three domains of life. Recently, we showed that Bacillus subtilis and Bacillus anthracis encode the Gcn5-related N-acetyltransferase (GNAT) SatA that can acetylate and inactive streptothricin, which is a broad-spectrum antibiotic produced by actinomycetes in the soil. To determine functionally relevant residues of B. subtilis SatA, a mutational screen was performed, highlighting the importance of a conserved area near the C-terminus. Upon inspection of the crystal structure of the B. anthracis Ames SatA (PDB 3PP9), this area appears to form a pocket with multiple conserved aromatic residues; we hypothesized this region could contain the streptothricin-binding site. Chemical and site- directed mutagenesis were used to introduce missense mutations into satA, and the functionality of the variants was assessed using a heterologous host (Salmonella enterica). Results of isothermal titration calorimetry (ITC) experiments showed that residue Y164 of BaSatA was important for binding streptothricin. Result of size-exclusion chromatography analyses showed that residue D160 was important for dimerization. Together, these data advance our understanding of how SatA interacts with streptothricin. [ABSTRACT FROM AUTHOR]

Alpha-ribazole (α-R) is a unique riboside found in the nucleotide loop of coenzyme B 12 (CoB 12 ). α-R is not an intermediate of the de novo biosynthetic pathway of coenzyme B 12 , but some bacteria of the phylum Firmicutes have evolved a two-protein system (transporter, kinase) that scavenges α-R from the environment and converts it to the pathway intermediate α-RP. Since α-R is not commercially available, one must either synthesize α-R, or isolate it from hydrolysates of vitamin B 12 (cyano-B 12 , CNB 12 ), so the function of the above-mentioned proteins can be studied. Here we report a facile protocol for the isolation of α-R from CNB 12 hydrolysates. CNB 12 dissolved in NaOH (5 M) was heated to 85 °C for 75 min, then cooled to 4 °C for 30 min. The solution was neutralized with HCl (5 M), and the hydrolysate was diluted with an equal volume of ammonium acetate (0.3 M, pH 8.8). Alkaline phosphatase was added and the mixture was incubated at 37 °C for 16 h. After incubation, the sample was loaded onto a boronate affinity resin column, washed with ammonium sulfate (0.3 M, pH 8.8), water (to remove residual corrinoids) and finally with formic acid (0.1 M) to release (α-R). Formic acid was removed by lyophilization, and the final yield of α-R was 85% from the theoretically recoverable amount. Methods for quantifying the concentration of α-R are reported. [ABSTRACT FROM AUTHOR]

Summary: Protein acetylation is a rapid mechanism for control of protein function. Acetyl‐CoA synthetase (AMP‐forming, Acs) is the paradigm for the control of metabolic enzymes by lysine acetylation. In many bacteria, type I or II protein acetyltransferases acetylate Acs, however, in actinomycetes type III protein acetyltransferases control the activity of Acs. We measured changes in the activity of the Streptomyces lividans Acs (SlAcs) enzyme upon acetylation by PatB using in vitro and in vivo analyses. In addition to the acetylation of residue K610, residue S608 within the acetylation motif of SlAcs was also acetylated (PKTRSGK610). S608 acetylation rendered SlAcs inactive and non‐acetylatable by PatB. It is unclear whether acetylation of S608 is enzymatic, but it was clear that this modification occurred in vivo in Streptomyces. In S. lividans, an NAD+‐dependent sirtuin deacetylase from Streptomyces, SrtA (a homologue of the human SIRT4 protein) was needed to maintain SlAcs function in vivo. We have characterized a sirtuin‐dependent reversible lysine acetylation system in Streptomyces lividans that targets and controls the Acs enzyme of this bacterium. These studies raise questions about acetyltransferase specificity, and describe the first Acs enzyme in any organism whose activity is modulated by O‐Ser and Nɛ‐Lys acetylation. [ABSTRACT FROM AUTHOR]

Soil is a complex niche, where survival of microorganisms is at risk due to the presence of antimicrobial agents. Many microbes chemically modify cytotoxic compounds to block their deleterious effects. Streptothricin is a broad-spectrum antibiotic produced by streptomycetes that affects Gram-positive and Gram-negative bacteria alike. Here we identify the SatA (for streptothricin acetyltransferase A, formerly YyaR) enzyme of Bacillus subtilis as the mechanism used by this soil bacterium to detoxify streptothricin. B. subtilis strains lacking satA were susceptible to streptothricin. Ectopic expression of satA+ restored streptothricin resistance to B. subtilis satA (BsSatA) strains. Purified BsSatA acetylated streptothricin in vitro at the expense of acetylcoenzyme A (acetyl-CoA). A single acetyl moiety transferred onto streptothricin by SatA blocked the toxic effects of the antibiotic. SatA bound streptothricin with high affinity (Kd [dissociation constant] = 1 μM), and did not bind acetyl-CoA in the absence of streptothricin. Expression of B. subtilis satA+ in Salmonella enterica conferred streptothricin resistance, indicating that SatA was necessary and sufficient to detoxify streptothricin. Using this heterologous system, we showed that the SatA homologue from Bacillus anthracis also had streptothricin acetyltransferase activity. Our data highlight the physiological relevance of lysine acetylation for the survival of B. subtilis in the soil. IMPORTANCE Experimental support is provided for the functional assignment of gene products of the soil-dwelling bacilli Bacillus subtilis and Bacillus anthracis. This study focuses on one enzyme that is necessary and sufficient to block the cytotoxic effects of a common soil antibiotic. The enzyme alluded to is a member of a family of proteins that are broadly distributed in all domains of life but poorly studied in B. subtilis and B. anthracis. The initial characterization of the enzyme provides insights into its mechanism of catalysis. [ABSTRACT FROM AUTHOR]

5,6-Dimethylbenzimidazolyl-(DMB)- α-ribotide [ α-ribazole-5′-phosphate ( α-RP)] is an intermediate in the biosynthesis of adenosylcobalamin (AdoCbl) in many prokaryotes. In such microbes, α-RP is synthesized by nicotinate mononucleotide (NaMN):DMB phosphoribosyltransferases (CobT in Salmonella enterica), in a reaction that is considered to be the canonical step for the activation of the base of the nucleotide present in adenosylcobamides. Some Firmicutes lack CobT-type enzymes but have a two-protein system comprised of a transporter ( i.e., CblT) and a kinase ( i.e., CblS) that can salvage exogenous α-ribazole ( α-R) from the environment using CblT to take up α-R, followed by α-R phosphorylation by CblS. We report that Geobacillus kaustophilus CblT and CblS proteins restore α-RP synthesis in S. enterica lacking the CobT enzyme. We also show that a S. enterica cobT strain that synthesizes GkCblS ectopically makes only AdoCbl, even under growth conditions where the synthesis of pseudoCbl is favored. Our results indicate that S. enterica synthesizes α-R, a metabolite that had not been detected in this bacterium and that GkCblS has a strong preference for DMB-ribose over adenine-ribose as substrate. We propose that in some Firmicutes DMB is activated to α-RP via α-R using an as-yet-unknown route to convert DMB to α-R and CblS to convert α-R to α-RP. [ABSTRACT FROM AUTHOR]

S almonella enterica catabolizes ethanolamine inside a compartment known as the metabolosome. The ethanolamine utilization ( eut) operon of this bacterium encodes all functions needed for the assembly and function of this structure. To date, the roles of EutQ and EutP were not known. Herein we show that both proteins have acetate kinase activity and that EutQ is required during anoxic growth of S . enterica on ethanolamine and tetrathionate. EutP and EutQ-dependent ATP synthesis occurred when enzymes were incubated with ADP, Mg( II) ions and acetyl-phosphate. EutQ and EutP also synthesized acetyl-phosphate from ATP and acetate. Although EutP had acetate kinase activity, Δ eut P strains lacked discernible phenotypes under the conditions where Δ eut Q strains displayed clear phenotypes. The kinetic parameters indicate that EutP is a faster enzyme than EutQ. Our evidence supports the conclusion that EutQ and EutP represent novel classes of acetate kinases. We propose that EutQ is necessary to drive flux through the pathway under physiological conditions, preventing a buildup of acetaldehyde. We also suggest that ATP generated by these enzymes may be used as a substrate for EutT, the ATP-dependent corrinoid adenosyltransferase and for the EutA ethanolamine ammonia-lyase reactivase. [ABSTRACT FROM AUTHOR]

Acylation of biomolecules (e.g., proteins and small molecules) is a process that occurs in cells of all domains of life and has emerged as a critical mechanism for the control of many aspects of cellular physiology, including chromatin maintenance, transcriptional regulation, primary metabolism, cell structure, and likely other cellular processes. Although this review focuses on the use of acetyl moieties to modify a protein or small molecule, it is clear that cells can use many weak organic acids (e.g., short-, medium-, and long-chain mono- and dicarboxylic aliphatics and aromatics) to modify a large suite of targets. Acetylation of biomolecules has been studied for decades within the context of histone-dependent regulation of gene expression and antibiotic resistance. It was not until the early 2000s that the connection between metabolism, physiology, and protein acetylation was reported. This was the first instance of a metabolic enzyme (acetyl coenzyme A [acetyl-CoA] synthetase) whose activity was controlled by acetylation via a regulatory system responsive to physiological cues. The abovementioned system was comprised of an acyltransferase and a partner deacylase. Given the reversibility of the acylation process, this system is also referred to as reversible lysine acylation (RLA). A wealth of information has been obtained since the discovery of RLA in prokaryotes, and we are just beginning to visualize the extent of the impact that this regulatory system has on cell function. [ABSTRACT FROM AUTHOR]

Protein and small-molecule acylation reactions are widespread in nature. Many of the enzymes catalyzing acylation reactions belong to the Gcn5-related N-acetyltransferase (GNAT; PF00583) family, named after the yeast Gcn5 protein. The genome of Salmonella enterica serovar Typhimurium LT2 encodes 26 GNATs, 11 of which have no known physiological role. Here, we provide in vivo and in vitro evidence for the role of the MddA (methionine derivative detoxifier; formerly YncA) GNAT in the detoxification of oxidized forms of methionine, including methionine sulfoximine (MSX) and methionine sulfone (MSO). MSX and MSO inhibited the growth of an S. enterica γmddA strain unless glutamine or methionine was present in the medium. We used an in vitro spectrophotometric assay and mass spectrometry to show that MddA acetylated MSX and MSO. An mddA strain displayed biphasic growth kinetics in the presence of MSX and glutamine. Deletion of two amino acid transporters (GlnHPQ and MetNIQ) in a mddA strain restored growth in the presence of MSX. Notably, MSO was transported by GlnHPQ but not by MetNIQ. In summary, MddA is the mechanism used by S. enterica to respond to oxidized forms of methionine, which MddA detoxifies by acetyl coenzyme A-dependent acetylation. [ABSTRACT FROM AUTHOR]

Reversible lysine acetylation (RLA) is a widespread regulatory mechanism that modulates the function of proteins involved in diverse cellular processes. A strong case has been made for RLA control exerted by homologues of the Salmonella enterica protein acetyltransferase (SePat) enzyme on the broadly distributed AMP-forming CoA ligase (a.k.a. acyl-CoA synthetases) family of metabolic enzymes, with acetyl-CoA synthetase (Acs) being the paradigm in the field. Here we investigate why the Acs homologue in Streptomyces lividans (SlAcs) is poorly acetylated in vitro by the S. lividans protein acetyltransferase (SlPat) enzyme. Chimeras of S. enterica Acs (SeAcs) and S. lividans Acs (SlAcs) constructed during the course of this work were acetylated by SlPatA in vitro, retained most of their activity, and were under RLA control in a heterologous host. We identified SeAcs residues N- and C-terminal to the target lysine that when introduced into SlAcs, rendered the latter under RLA control. These results lend further support to the idea that Pat enzymes interact with extensive surfaces of their substrates. Finally, we suggest that acetylation of SlAcs depends on factors or conditions other than those present in our in vitro system. We also discuss possible explanations why SlAcs is not controlled by RLA as defined in other bacterial species. [ABSTRACT FROM AUTHOR]

The AMP-forming acyl coenzyme A (acyl-CoA) synthetases are a large class of enzymes found in both anabolic and catabolic pathways that activate fatty acids to acyl-CoA molecules. The protein acetyltransferase (Pat) from Rhodopseudomonas palustris (RpPat) inactivates AMP-forming acyl-CoA synthetases by acetylating the ε-amino group of a conserved, catalytic lysine residue. In all of the previously described RpPat substrates, this lysine residue is located within a PX4GK motif that has been proposed to be a recognition motif for RpPat. Here, we report five new substrates for RpPat, all of which are also AMP-forming acyl-CoA synthetases. This finding supports the idea that Pat enzymes may have evolved to control the activity of this family of enzymes. Notably, RpPat did not acetylate the wild-type long-chain acyl-CoA synthetase B (RpLcsB; formerly Rpa2714) enzyme of this bacterium. However, a single amino acid change two residues upstream of the acetylation site was sufficient to convert RpLcsB into an RpPat substrate. The results of mutational and functional analyses of RpLcsB and RpPimA variants led us to propose PK/RTXS/T/V/NGKX2K/R as a substrate recognition motif. The underlined positions within this motif are particularly important for acetylation by RpPat. The first residue, threonine, is located 4 amino acids upstream of the acetylation site. The second residue can be S/T/V/N and is located two positions upstream of the acetylation site. Analysis of published crystal structures suggests that the side chains of these two residues are very close to the acetylated lysine residue, indicating that they may directly interact with RpPat. [ABSTRACT FROM AUTHOR]

GCN5-type N-acetyltransferases ( GNATs) are enzymes that catalyse the transfer of the acetyl group from acetyl- CoA to a primary amine. GNATs are conserved in all domains of life. Some members of this family of enzymes acetylate the side-chain of specific lysine residues in proteins of diverse function. In bacteria, GNAT-catalysed protein acetylation regulates carbon metabolism, RNA metabolism and transcriptional regulation. Metabolic regulation in Streptomyces species is of interest due to the role of these organisms in natural product synthesis. Here we identify SlPatA, a GNAT in Streptomyces lividans with unique domain organization, and a new acetylation target, namely acetoacetyl- CoA synthetase ( SlAacS). The latter has homologues in all domains of life. In vitro and in vivo evidence show that SlAacS is a bona fide acetoacetyl- CoA synthetase. SlPatA acetylates SlAacS more efficiently than it does acetyl- CoA synthetase, an enzyme known to be under acetylation control. SlPatA acetylates SlAacS at the active-site residue Lys617 and acetylation inactivates SlAacS. Acetylated SlAacS was deacetylated by a sirtuin-type protein deacetylase. SlAacS acetylation/deacetylation may represent a conserved mechanism for regulation of acetoacetyl- CoA synthetase activity in all domains of life. [ABSTRACT FROM AUTHOR]

Abstract: In Salmonella enterica, the protein acetyltransferase (Pat) enzyme is part of the sirtuin-dependent acylation/deacylation system (SDPADS) that modulates the activity of several proteins via the acylation of lysine residues critical to their activities. Pat is a ∼98 kDa protein with two distinct domains, an N-terminal acyl-CoA synthetase (NDP-forming) domain (∼700 aa) and a C-terminal acetyltransferase domain (∼160 aa), with homology to proteins of the Gcn5-related N-acetyltransferase (GNAT) superfamily. Although the role of the GNAT-like domain is likely responsible for the catalytic activity of Pat, the role of the N-terminal domain remains unclear. Here we report the use of positive selection for identification of residues critical for Pat enzyme activity. This approach revealed seven residues that, when changed, resulted in drastic loss of Pat activity in vitro which caused a discernable loss-of-function phenotype. Five of the seven residues were located in the N-terminal region of Pat and two were located in the GNAT-like domain. Each single-amino-acid variant had a circular dichroism spectrum that differed from that of the wild-type Pat protein, suggesting that loss of enzymatic activity in the mutant proteins was likely due to an inability to acquire its biologically active fold. [Copyright &y& Elsevier]

Summary In the homoacetogenic bacterium Sporomusa ovata, phenol and p-cresol are converted into α-ribotides, which are incorporated into biologically active cobamides (Cbas) whose lower ligand bases do not form axial co-ordination bonds with the cobalt ion of the corrin ring. Here we report the identity of two S. ovata genes that encode an enzyme that transfers the phosphoribosyl group of nicotinate mononucleotide (NaMN) to phenol or p-cresol, yielding α- O-glycosidic ribotides. The alluded genes were named arsA and arsB (for alpha-ribotide synthesis), arsA and arsB were isolated from a genomic DNA library of S. ovata. A positive selection strategy using an Escherichia coli strain devoid of NaMN:5,6-dimethylbenzimidazole (DMB) phosphoribosyltransferase (CobT) activity was used to isolate a fragment of S. ovata DNA that contained arsA and arsB, whose nucleotide sequences overlapped by 8 bp. SoArsAB was isolated to homogeneity, shown to be functional as a heterodimer, and to have highest activity at pH 9. SoArsAB also activated DMB to its α- N-glycosidic ribotide. Previously characterized CobT-like enzymes activate DMB but do not activate phenolics. NMR spectroscopy was used to confirm the incorporation of phenol into the cobamide, and mass spectrometry was used to identify SoArsAB reaction products. [ABSTRACT FROM AUTHOR]

Recently published work indicates that reversible N ɛ-lysine (N ɛ-Lys) acetylation of proteins in bacteria may be as diverse, and as important for cellular function, as it has been reported in eukaryotes for the last five decades. In addition to biochemical and genetic approaches, proteomic studies have identified N ɛ-Lys acetylation of proteins and enzymes involved in diverse cellular activities such as transcription, translation, stress response, detoxification, and especially carbohydrate and energy metabolism. These findings provide a platform for elucidating the molecular mechanisms behind modulation of enzyme activity by N ɛ-Lys acetylation, as well as for understanding how the prokaryotic cell maintains homeostasis in a changing environment. [Copyright &y& Elsevier]

The genomes of Listeria spp. encode all but one of 25 enzymes required for the biosynthesis of adenosylcobalamin (AdoCbl; coenzyme B12). Notably, all Listeria genomes lack CobT, the nicotinamide mononucleotide:5,6-dimethylbenzimidazole (DMB) phosphoribosyltransferase (EC 2.4.2.21) enzyme that synthesizes the unique α-linked nucleotide N1-(5-phospho-α-d-ribosyl)-DMB (α-ribazole-5′-P, α-RP), a precursor of AdoCbl. We have uncovered a new pathway for the synthesis of α-RP in Listeria innocua that circumvents the lack of CobT. The cblT and cblS genes (locus tags lin1153 and lin1110) of L. innocua encode an α-ribazole (α-R) transporter and an α-R kinase respectively. Results from in vivo experiments indicate that L. innocua depends on CblT and CblS activities to salvage exogenous α-R, allowing conversion of the incomplete corrinoid cobinamide (Cbi) into AdoCbl. Expression of the L. innocua cblT and cblS genes restored AdoCbl synthesis from Cbi and α-R in a Salmonella enterica cobT strain. LinCblT transported α-R across the cell membrane, but not α-RP or DMB. UV-visible spectroscopy and mass spectrometry data identified α-RP as the product of the ATP-dependent α-R kinase activity of LinCblS. Bioinformatics analyses suggest that α-R salvaging occurs in important Gram-positive human pathogens. [ABSTRACT FROM AUTHOR]

Strains of Salmonella enterica serovar Typhimurium LT2 lacking a functional 2-methylcitric acid cycle (2-MCC) display increased sensitivity to propionate. Previous work from our group indicated that this sensitivity to propionate is in part due to the production of 2-methylcitrate (2-MC) by the Krebs cycle enzyme citrate synthase (GltA). Here we report in vivo and in vitro data which show that a target of the 2-MC isomer produced by GltA (2-MCGltA) is fructose-1,6-bisphosphatase (FBPase), a key enzyme in gluconeogenesis. Lack of growth due to inhibition of FBPase by 2-MCGltA was overcome by increasing the level of FBPase or by micromolar amounts of glucose in the medium. We isolated an fbp allele encoding a single amino acid substitution in FBPase (S123F), which allowed a strain lacking a functional 2-MCC to grow in the presence of propionate. We show that the 2-MCGltA and the 2-MC isomer synthesized by the 2-MC synthase (PrpC; 2-MCPrpC) are not equally toxic to the cell, with 2-MCGltA being significantly more toxic than 2-MCPrpC. This difference in 2-MC toxicity is likely due to the fact that as a si-citrate synthase, GltA may produce multiple isomers of 2-MC, which we propose are not substrates for the 2-MC dehydratase (PrpD) enzyme, accumulate inside the cell, and have deleterious effects on FBPase activity. Our findings may help explain human inborn errors in propionate metabolism. [ABSTRACT FROM AUTHOR]

The identity of the source of the biological reductant needed to convert cobalamin to its biologically active form adenosylco-balamin has remained elusive. Here we show that free or protein-bound dihydroflavins can serve as the reductant of Co2+Cbl bound in the active site of PduO-type ATP-dependent corrinoid adenosyltransferase enzymes. Free dihydroflavins (dihydroriboflavin, FMNH2, and FADH2) effectively drove the adenosylation of Co2+Cbl by the human and bacterial PduO-type enzymes at very low concentrations (1 μM). These data show that adenosyltransferase enzymes lower the thermodynamic barrier of the Co2+ → Co+ reduction needed for the formation of the unique organometalic Co-C bond of adenosylco-balamin. Collectively, our in vivo and in vitro data suggest that cobalamin reductases identified thus far are most likely electron transfer proteins, not enzymes. [ABSTRACT FROM AUTHOR]

The chemical structures of cobamides [cobalamin (Cbl)-like compounds] are the same, except for the lower ligand, which in adenosylcobalamin (AdoCbl) is 5,6-dimethylbenzimidazole, and in adenosylpseudocobalamin (AdopseudoCbl) is adenine. Why the lower ligand of cobamides varies and what the mechanism of lower ligand replacement is are long-standing questions in the field of B12 biosynthesis. Work reported here uncovers the strategy used by the photosynthetic α-proteobacterium Rhodobacter sphaeroides to procure the cobamide it needs to grow on acetate as a carbon and energy source. On the basis of genetic and biochemical evidence we conclude that, in R. sphaeroides, the activity of the cobyric acid-producing amidohydrolase CbiZ enzyme is essential for the conversion of AdopseudoCbl into AdoCbl, the cobamide needed for the catabolism of acetate. The CbiZ enzyme uses AdopseudoCbl as a substrate, but not AdoCbl. Implications of these findings for cobamide remodelling in R. sphaeroides and in other CbiZ-containing microorganisms are discussed. [ABSTRACT FROM AUTHOR]

The archaeal cobY gene encodes the nonorthologous replacement of the bacterial NTP:AdoCbi kinase (EC 2.7.7.62)/GTP:AdoCbi-P guanylyltransferase (EC 3.1.3.73) and is required for de novo synthesis of AdoCbi (coenzyme B12). Here we show that ORF MJ1117 of the hyperthermophilic, methanogenic archaeon Methanocaldococcus jannaschii encodes a CobY protein (MjCobY) that transfers the GMP moiety of GTP to AdoCbi-P to form AdoCbi-GDP. Results from isothermal titration calorimetry (ITC) experiments show that MjCobY binds GTP (Kd = 5 μM), but it does not bind the GTP analogues GMP-PNP (guanosine 5′-(β,γ)- imidotriphosphate) or GMP-PCP (guanylyl 5′-(β,γ)-methylenediphosphonate) nor GDP. Results from ETC experiments indicate, that MjCobY binds one GTP per dimer. Results from in vivo studies support the conclusion that the 5′-deoxyadenosyl upper ligand of AdoCbi-P is required for MjCobY function. Consistent with these findings, MjCobY displayed high affinity for AdoCbi-P (Kd = 0.76 μM) but did not bind nonadenosylated Cbi-P. Kinetic parameters for the MjCobY reaction were determined. Results from circular dichroism studies indicate that, in isolation, MjCobY denatures at 80 °C with a concomitant loss of activity. We propose that ORF MJ1117 of M. jannaschii be annotated as cobY to reflect its involvement in AdoCbl biosynthesis. [ABSTRACT FROM AUTHOR]

The genome of Rhodobacter sphaeroides encodes the components of two distinct pathways for salvaging cobinamide (Cbi), a precursor of adenosylcobalamin (AdoCbl, coenzyme B12). One pathway, conserved among bacteria, depends on a bifunctional kinase/guanylyltransferase (CobP) enzyme to convert adenosylcobinamide (AdoCbi) to AdoCbi-phosphate (AdoCbi-P), an intermediate in de novo AdoCbl biosynthesis. The other pathway, of archaeal origin, depends on an AdoCbi amidohydrolase (CbiZ) enzyme to generate adenosylcobyric acid (AdoCby), which is converted to AdoCbi-P by the AdoCbi-P synthetase (CobD) enzyme. Here we report that R. sphaeroides strain 2.4.1 synthesizes AdoCbl de novo and that it salvages Cbi using both of the predicted Cbi salvaging pathways. AdoCbl produced by R. sphaeroides was identified and quantified by high-performance liquid chromatography and bioassay. The deletion of cobB (encoding an essential enzyme of the de novo corrin ring biosynthetic pathway) resulted in a strain of R. sphaeroides that would not grow on acetate in the absence of exogenous corrinoids. The results from a nutritional analysis showed that the presence of either CbiZ or CobP was necessary and sufficient for Cbi salvaging, that CbiZ-dependent Cbi salvaging depended on the presence of CobD, and that CobP-dependent Cbi salvaging occurred in a cbiZ+ strain. Possible reasons why R. sphaeroides maintains two distinct pathways for Cbi salvaging are discussed. [ABSTRACT FROM AUTHOR]

This report provides in vivo evidence for the posttranslational control of the acetyl coenzyme A (Ac-CoA) synthetase (AcsA) enzyme of Bacillus subtilis by the acuA and acuC gene products. In addition, both in vivo and in vitro data presented support the conclusion that the yhdZ gene of B. subtilis encodes a NAD+-dependent protein deacetylase homologous to the yeast Sir2 protein (also known as sirtuin). On the basis of this new information, a change in gene nomenclature, from yhdZ to srtN (for sirtuin), is proposed to reflect the activity associated with the YdhZ protein. In vivo control of B. subtilis AcsA function required the combined activities of AcuC and SrtN. Inactivation of acuC or srtN resulted in slower growth and cell yield under low-acetate conditions than those of the wild-type strain, and the acuC srtN strain grew under low-acetate conditions as poorly as the acsA strain. Our interpretation of the latter result was that both deacetylases (AcuC and SrtN) are needed to maintain AcsA as active (i.e., deacetylated) so the cell can grow with low concentrations of acetate. Growth of an acuA acuC srtN strain on acetate was improved over that of the acuA+ acuC srtN strain, indicating that the AcuA acetyltransferase enzyme modifies (i.e., inactivates) AcsA in vivo, a result consistent with previously reported in vitro evidence that AcsA is a substrate of AcuA. [ABSTRACT FROM AUTHOR]

The acuABC genes of Bacillus subtilis comprise a putative posttranslational modification system. The AcuA protein is a member of the Gcn5-related N-acetyltransferase (GNAT) superfamily, the AcuC protein is a class I histone deacetylase, and the role of the AcuB protein is not known. AcuA controls the activity of acetyl coenzyme A synthetase (AcsA; EC 6.2.1.1) in this bacterium by acetylating residue Lys549. Here we report the kinetic analysis of wild-type and variant AcuA proteins. We contrived a genetic scheme for the identification of AcuA residues critical for activity. Changes at residues H177 and G187 completely inactivated AcuA and led to its rapid turnover. Changes at residues R42 and T169 were less severe. In vitro assay conditions were optimized, and an effective means of inactivating the enzyme was found. The basic kinetic parameters of wild-type and variant AcuA proteins were obtained and compared to those of eukaryotic GNATs. Insights into how the isolated mutations may exert their deleterious effect were investigated by using the crystal structure of an AcuA homolog. [ABSTRACT FROM AUTHOR]

Tricarballylate, a citrate analogue, is considered the causative agent of grass tetany, a ruminant disease characterized by acute magnesium deficiency. Although the normal rumen flora cannot catabolize tricarballylate, the Gram-negative enterobacterium Salmonella enterica can. An operon dedicated to tricarballylate utilization (tcuABC) present in this organism encodes all functions required for tricarballylate catabolism. Tricarballylate is converted to the cis-aconitate in a single oxidative step catalyzed by the FAD-dependent tricarballylate dehydrogenase (TcuA) enzyme. We hypothesized that the uncharacterized TcuB protein was required to reoxidize the flavin cofactor in vivo. Here, we report the initial biochemical characterization of TcuB. TcuB is associated with the cell membrane and contains two 4Fe-4S clusters and heme. Site-directed mutagenesis of cysteinyl residues putatively required as ligands of the 4Fe-4S clusters completely inactivated TcuB function. TcuB greatly increased the Vmax of the TcuA reaction from 69 ± 2 to 8200 ± 470 nmol min-1 mg-1 the Km of TcuA for tricarballylate was unaffected. Inhibition of TcuB activity by an inhibitor of ubiquinone oxidation, 2,5-dibromo-3-methyl-6-isoproylbenzoquinone (DBMIB), implicated the quinone pool as the ultimate acceptor of electrons from FADH2. We propose a model for the electron flow from FADH2, to the 4Fe-45 clusters, to the heme, and finally to the quinone pool. [ABSTRACT FROM AUTHOR]

The function of the N-terminal domain (~350 residues) of the Pta (phosphotransacetylase) enzyme of Salmonella enterica is unclear. Results from in vivo genetic and in vitro studies suggest that the N-terminal domain of Pta is a sensor for NADH and pyruvate. We isolated 10 single-amino acid variants of Pta that, unlike the wild-type protein, supported growth of a strain of S. enterica devoid of Acs (acetyl-CoA synthetase; AMP-forming) activity on 10 mM acetate. All mutations were mapped within the N-terminal domain of the protein. Kinetic analyses of the wild type and three variant Pta proteins showed that two of the variant proteins were faster enzymes (kcat 2.5-3-fold > kcat PtaWT. Results from sedimentation equilibrium experiments are consistent with PtaWT being a trimer. Pta variants formed more hexamer than the PtaWT protein. NADH inhibited PtaWT activity by inducing a conformational change detectable by limited trypsin proteolysis; NADH did not inhibit variant protein PtaR252H. Pyruvate stimulated PtaWT activity, and its effect was potentiated in the variants, being most pronounced on PtaR252H. [ABSTRACT FROM AUTHOR]

The synthesis of 5,6-dimethylbenzimidazole (DMB), the lower ligand of coenzyme B12, has remained elusive. We report in vitro and in vivo evidence that the BIuB protein of the photosynthetic bacterium Rhodospirillum rubrum is necessary and sufficient for catalysis of the O2-dependent conversion of FMNH2 to DMB. The product of the reaction (DMB) was isolated by using reverse-phase high-pressure liquid chromatography, and its identity was established by UV-visible spectroscopy and MS. No metals were detected in homogeneous preparations of BIuB, and the enzyme did not affect DMB synthesis from 4,5-dimethyiphenylenediamine and ribose-5-phosphate. The effect of the lack of bluB function in R. rubrum was reflected by the impaired ability of a ΔbIuB strain to convert Mg-protoporphyrin IX monomethyl ester (MPE) into protochlorophylide, a reaction of the bacteriochlorophyll biosynthetic pathway catalyzed by the MPE-cyclase enzyme present in this bacterium (BchE. EC 1.14.13.81), a predicted coenzyme B12-dependent enzyme. The growth defect of the ΔbIuB strain observed under anoxic photoheterotrophic conditions was corrected by the addition of DMB or B12 to the culture medium or by introducing into the strain a plasmid encoding the wild-type allele of bluB. The findings reported here close an important gap in our understanding of the enzymology of the assembly of coenzyme B12. [ABSTRACT FROM AUTHOR]

ATP:cob(l)alamin adenosyltransferase (EutT) of Salmonella enterica was overproduced and enriched to ~70% homogeneity, and its basic kinetic parameters were determined. Abundant amounts of EutT protein were produced, but all of it remained insoluble. Soluble active EutT protein (`70% homogeneous) was obtained after treatment with detergent. Under conditions in which cobalamin (Cbl) was saturating, Km(ATP) = 10 µM, kcat = 0.03 s-1, and Vmax = 54.5 nM min-1. Similarly, under conditions in which MgATP was saturating, Km(Cbl) = 4.1 µM, kcat = 0.06 s-1, and Vmax = 105 nM min-1. Unlike other ATP:co(l)rrinoid adenosyltransferases in the cell (i.e. CobA and PduO), EutT activity was ≥50-fold higher with ATP versus GTP, and EutT retained 80% of its activity with ADP substituted for ATP and was completely inactive with AMP as substrate, indicating that the enzyme requires the β-phosphate group of the nucleotide substrate. The data suggest that the amino group of adenine might play a role in nucleotide recognition and/or binding. Unlike the housekeeping CobA enzyme, EutT was not inhibited by inorganic tripolyphosphate (PPPi). Results from 31P NMR spectroscopy studies identified PPi and P, as by-products of the EutT reaction. In the absence of Cbl, EutT cleaved ATP into adenosine and PPPi, suggesting that PPPi is broken down into PPi and Pi. Electron transfer protein partners for EutT were not encoded by the eut operon. EutT-dependent activity was detected in cell-free extracts of cobA strains enriched for EutT when FMN and NADH were used to reduce cob(III)alamin to cob(I)alamin. [ABSTRACT FROM AUTHOR]

The activity of the housekeeping ATP:co(I)rrinoid adenosyltransferase (CobA) enzyme of Salmonella enterica sv. Typhimurium is required to adenosylate de novo biosynthetic intermediates of adenosylcobalamin and to salvage incomplete and complete corrinoids from the environment of this bacterium. In vitro, reduced flavodoxin (FldA) provides an electron to generate the co(1)rrinoid substrate in the CobA active site. To understand how CobA and FldA interact, a computer model of a CobA·FldA complex was generated. This model was used to guide the introduction of mutations into CobA using site-directed mutagenesis and the synthesis of a peptide mimic of FldA. Residues Arg-9 and Arg-165 of CobA were critical for FldA-dependent adenosylation but were catalytically as competent as the wild-type protein when cob(1)alamin was provided as substrate. These results indicate that Arg-9 and Arg-165 are important for CobA·FldA docking but not to catalysis. A truncation of the 9-amino acid N-terminal helix of CobA reduced its FldA-dependent cobalamin adenosyltransferase activity by 97.4%. The same protein, however, had a 4-fold higher specific activity than the native enzyme when cob(I)alamin was generated chemically in situ. [ABSTRACT FROM AUTHOR]

The function of the PrpR protein of Salmonella enterica serovar Typhimurium LT2 was studied In vitro and in vivo. The PrpR protein is a sensor of 2-methylcitrate (2-MC), an intermediate of the 2-methylcitric acid cycle used by this bacterium to convert propionate to pyruvate. PrpR was unresponsive to citrate (a close structural analogue of 2-MC) and to propionate, suggesting that 2-MC, not propionate, is the metabolite that signals the presence of propionate in the environment to S. enterica. prpR alleles encoding mutant proteins with various levels of 2-MC-independent activity were isolated. All lesions causing constitutive PrpR activity were mapped to the N-terminal domain of the protein. Removal of the entire sensing domain resulted in a protein (PrpRc) with the highest 2-MC-independent activity. Residue A162 is critical to 2-MC sensing, since the mutant PrpR protein PrpRA162T was as active as the PrpRc protein in the absence of 2-MC. DNA footprinting studies identified the site in the region between prpR and the prpBCDE operon to which the PrpR protein binds. Analysis of the binding-site sequence revealed two sites with dyad symmetry. Results from DNase I footprinting assays suggested that the PrpR protein may have higher affinity for the site proximal to the PprpBCDE promoter. [ABSTRACT FROM AUTHOR]

Post-translational modification of proteins is an efficient way cells use to control the activity of structural proteins, gene expression regulatory proteins, and enzymes. In eukaryotes, the Sir2-dependent system of protein acetylation/deacetylation controls a number of processes that affect cell longevity. Sir2 proteins have NAD+-dependent protein deacetylase activity and are found in all forms of life. Although the identity of the acetyltransferases that partner with Sir2 enzymes is known in eukaryotes, the identity of the prokaryotic acetyltransferases is not. We report the identification of the gene of Salmonella enterica serovar Typhimurium LT2 encoding the major protein acetyltransferase (Pat) enzyme that, in concert with the CobB sirtuin of this bacterium, regulates the activity of the central metabolic enzyme acetyl-coenzyme A synthetase (Acs). The Pat enzyme uses acetyl-CoA as substrate to modify residue Lys609 of Acs. The Pat/CobB system of S. enterica should serve as the paradigm to further investigate the contributions of this system to the physiology of prokaryotes. [Copyright &y& Elsevier]

The existence of a pathway for salvaging the coenzyme B12 precursor dicyanocobinamide (Cbi) from the environment was established by genetic and biochemical means. The pathway requires the function of a previously unidentified amidohydrolase enzyme that converts adenosylcobinamide to adenosylcobyric acid, a bona tide intermediate of the de novo coenzyme B12 biosynthetic route. The cbiZ gene of the methanogenic archaeon Methanosarcina mazei strain Göl was dotted, was overproduced in Escherichia coli, and the recombinant protein was isolated to homogeneity. HPLC, UV-visible spectroscopy, MS, and bioassay data established adenosylcobyric as the corrinoid product of the CbiZ-catalyzed reaction. Inactivation of the cbiZ gene in the extremely halophilic archaeon Halobactrium sp. strain NRC-1 blocked the ability of this archaeon to salvage Cbi. cbiZ function restored Cbi salvaging in a strain of the bacterium Salmonella enterica, whose Cbi savaging pathway was blocked. The salvaging of Cbi through the CbiZ enzyme appears to be an archaeal strategy because all of the genomes of B12-producing archaea have a cbiZ ortholog. Reasons for the evolution of two distinct pathways for Cbi salvaging in prokaryotes are discussed. [ABSTRACT FROM AUTHOR]

The propionate utilization operons of several bacteria differ from each other in the occurrence of two genes, acnD and prpF, in place of or in addition to the prpD gene encoding an Fe/S-independent 2-methylcitrate dehydratase enzyme. We cloned the acnD and prpF genes from two organisms, Shewanella oneidensis and Vibrio cholerae, and found that, together, the AcnD and PrpF proteins restored the ability of a prpD mutant strain of Salmonella enterica to grow on propionate as a source of carbon and energy. However, neither acnD nor prpF alone was able to substitute for prpD. The AcnD and PrpF proteins were isolated and biochemically analyzed. The AcnD protein required reconstitution of an Fe/S cluster for activity. All detectable AcnD activity was lost after incubation with iron-chelating agents, and no AcnD activity was observed after attempted reconstitution without iron. Nuclear magnetic resonance spectroscopy and in vitro activity assay data showed that AcnD dehydrated 2-methylcitrate and citrate to 2-methyl-cis-aconitate and cis-aconitate, respectively; AcnD also hydrated cis-aconitate. However, 2-methylisocitrate and isocitrate were not substrates for AcnD, indicating that AcnD only catalyzes the first half of the aconitase-like dehydration reactions. No aconitase-like activity was found for PrpF. It is hypothesized that, in vivo, PrpF is an accessory protein required to prevent oxidative damage of the Fe/S center of active AcnD enzyme or that it may be involved in synthesis or repair of the Fe/S cluster present in AcnD. [ABSTRACT FROM AUTHOR]

The CobT enzyme of Salmonella typhimurium was shown in vitro to have NAD[sup +]-dependent ADPribosyltransferase activity. The CobT enzyme transferred the ADPribosyl moiety of NAD[sup +] onto 5,6-dimethylbenzimidazole (DMB) yielding a new dinucleotide, namely α-5,6-dimethylbenzimidazole adenine dinucleotide (α-DAD), whose identity was established by mass spectrometry. The N¹- (α-D-ribosyl)-5,6-dimethylbenzimidazoyl moiety (α-ribazole) of α-DAD was incorporated into adenosylcobalamin (AdoCbl) by cell-free extracts of S. typhimurium, indicating that α-DAD served as an intermediate of AdoCbl biosynthesis. The rate of transfer of the ADPribosyl moiety was slower than the rate of transfer of the phosphoribosyl moiety of nicotinate mononucleotide (NaMN) to DMB. The CobT enzyme displayed a Iow K[sub m] for NaMN (0.51 mM) relative to the one for NAD[sup +] (9 mM); nicotinate adenine dinucleotide (NaAD) and nicotinamide mononucleotide (NMN) also served as substrates for CobT. In spite of the high K[sub m] of CobT for NAD[sup +], the latter is proposed to be a relevant physiological substrate of CobT, given that the intracellular concentrations of NaMN, NMN and NaAD in actively growing S. typhimurium are undetectable. Evidence shows that extracts of S. typhimurium contain an as-yet unidentified dinucleotide pyrophosphatase that can cleave α-DAD into α-ribazole-5'-P and AMP; α-ribazole-5'-P can then enter the AdoCbl biosynthetic pathway. [ABSTRACT FROM AUTHOR]

Examines the structure of threonine phosphate decarboxylase enzyme from Salmonella enterica. Link between nucleotide loop and corrin ring in cobalamin; Decarboxylation of the external aldimine; Relationship between decarboxylase and aspartate aminotransferase.

Examines the characterization of propionyl-CoA Synthetase (PrpE) from Salmonella enterica. Catalysis on propionate catabolism; Determination on kinetic properties of PrpE; Use of site-directed mutagenesis to change the primary sequence of wild-type proteins.

Investigates the in vitro conversion of propionate to pyruvate by Salmonella enterica enzymes. 2-methylcitrate dehydratase activity of PrpD; Role of PrpD in the hydration of 2-methy;-cisconite; In vivo assessment of the requirement for aconitase A during propionate.

Reports on the isolation of a cob(II)alamin reductase activity in Salmonella enterica serovar Typhimurium LT2 to homogeneity. Identification of NAD(P)H:flavin oxidoreductase (Fre) as the enzyme responsible for the activity; Inability of Fre to cause the generation of cob(I)alamin in vivo.

Reports on the identification of an archaeal gene encoding a nucleotidyltransferase, which is proposed to be the nonorthologous replacement of the Salmonella enterica cobU gene. Amplification of the corresponding open reading frame from Methanobacterium thermoautotrophicum deltaH; Role of NTP:adenosylcobinamide kinase activity.

Reports on the isolation of insertions in prpR from Salmonella enterica. Importance of prpR for growth on propionate as a carbon and energy source; Expression of the operon prpBCDE; Coactivator-sensing domain of the protein; PrpR N terminus; Deleterious effect of the prpR[sup c] allele.

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. [ABSTRACT FROM AUTHOR]

Examines the structure of nicotinate mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase (CobT) from Salmonella typhimurium. Importance of CobT in the synthesis of alpha-ribazole; Role of CobT in the activation of cobalamin lower ligand; Electron density for 5,6-dimethylbenzimidazole.

Presents a study on the in vitro synthesis of the nucleotide loop of adenosylcobalamin by Salmonella typhimurium enzymes. Methodology of the study; Results and discussion; Conclusion.

Demonstrates the catabolism of propionate by Salmonella typhimurium LT2 via the 2-methylcitric acid cycle. Oxidation of the alpha-carbon of propionate; Catalysis of reactions by propionyl coenzyme A synthetase, 2-methylcitrate synthase, 2-methylcitrate dehydratase, 2-methylisocitrate hydratase, and 2-methylisocitrate lyase.

Addresses the function of the prpE gene of Salmonella typhimurium. Identification of prpE as a part of the prp operon; Overexpression of prpE; Acyl-CoA synthetase activity.

Presents information on an experiment which shows that an integration host factor is required for the activation of the transcription of the cobalamin biosynthetic and 1,2-propanediol utilization operons in Salmonella typhimurium LT2. Reasons for the experiment; Details on the methodology used; Results of the experiment; Implication of the results; Benefit of the findings.

Presents a study on the initial characterization of the propionate locus at 8.5 Cs (12). Results of the study; Methods used to carry out the study; Conclusion drawn from the study.

Presents evidence for the existence of a substitute function for CobB within the 1,2-propanediol utilization (pdu) operon of Salmonella typhimurium. Definition of the cobB function of S. typhimurium LT2 in vivo; Characterization of gene product that allows cobB mutant to metabolize propionate.