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.

Protein Acetylation in Bacteria.

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.

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.

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.

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.

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.

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.

Staphylococcus aureus modulates the activity of acetyl-Coenzyme A synthetase (Acs) by sirtuin-dependent reversible lysine acetylation.

Lysine acylation is a posttranslational modification used by cells of all domains of life to modulate cellular processes in response to metabolic stress. The paradigm for the role of lysine acylation in metabolism is the acetyl-coenzyme A synthetase (Acs) enzyme. In prokaryotic and eukaryotic cells alike, Acs activity is downregulated by acetylation and reactivated by deacetylation. Proteins belonging to the bacterial GCN5-related N-acetyltransferase (bGNAT) superfamily acetylate the epsilon amino group of an active site lysine, inactivating Acs. A deacetylase can remove the acetyl group, thereby restoring activity. Here we show the Acs from Staphylococcus aureus (SaAcs) activates acetate and weakly activates propionate, but does not activate >C3 organic acids or dicarboxylic acids (e.g. butyrate, malonate and succinate). SaAcs activity is regulated by AcuA (SaAcuA); a type-IV bGNAT. SaAcuA can acetylate or propionylate SaAcs reducing its activity by >90% and 95% respectively. SaAcuA also succinylated SaAcs, with this being the first documented case of a bacterial GNAT capable of succinylation. Inactive SaAcsAc was deacetylated (hence reactivated) by the NAD+ -dependent (class III) sirtuin protein deacetylase (hereafter SaCobB). In vivo and in vitro evidence show that SaAcuA and SaCobB modulate the level of SaAcs activity in S. aureus.

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.

A New Class of Phosphoribosyltransferases Involved in Cobamide Biosynthesis Is Found in Methanogenic Archaea and Cyanobacteria.

Cobamides are coenzymes used by cells from all domains of life but made de novo by only some bacteria and archaea. The last steps of the cobamide biosynthetic pathway activate the corrin ring and the lower ligand base, condense the activated intermediates, and dephosphorylate the product prior to the release of the biologically active coenzyme. In bacteria, a phosphoribosyltransferase (PRTase) enyzme activates the base into its α-mononucleotide. The enzyme from Salmonella enterica ( SeCobT) has been extensively biochemically and structurally characterized. The crystal structure of the putative PRTase from the archaeum Methanocaldococcus jannaschii ( MjCobT) is known, but its function has not been validated. Here we report the in vivo and in vitro characterization of MjCobT. In vivo, in vitro, and phylogenetic data reported here show that MjCobT belongs to a new class of NaMN-dependent PRTases. We also show that the Synechococcus sp. WH7803 CobT protein has PRTase activity in vivo. Lastly, results of isothermal titration calorimetry and analytical ultracentrifugation analysis show that the biologically active form of MjCobT is a dimer, not a trimer, as suggested by its crystal structure.

In Streptomyces lividans, acetyl-CoA synthetase activity is controlled by O-serine and Nɛ -lysine acetylation.

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.

Rhodobacterales use a unique L-threonine kinase for the assembly of the nucleotide loop of coenzyme B12.

Several of the enzymes involved in the conversion of adenosylcobyric acid (AdoCby) to adenosylcobamide (AdoCba) are yet to be identified and characterized in some cobamide (Cba)-producing prokaryotes. Using a bioinformatics approach, we identified the bluE gene (locus tag RSP_0788) of Rhodobacter sphaeroides 2.4.1 as a putative functional homolog of the L-threonine kinase enzyme (PduX, EC 2.7.1.177) of S. enterica. In AdoCba, (R)-1-aminopropan-2-ol O-phosphate (AP-P) links the nucleotide loop to the corrin ring; most known AdoCba producers derive AP-P from L-Thr-O-3-phosphate (L-Thr-P). Here, we show that RsBluE has L-Thr-independent ATPase activity in vivo and in vitro. We used 31 P-NMR spectroscopy to show that RsBluE generates L-Thr-P at the expense of ATP and is unable to use L-Ser as a substrate. BluE from R. sphaeroides or Rhodobacter capsulatus restored AdoCba biosynthesis in S. enterica ΕpduX and R. sphaeroides ΕbluE mutant strains. R. sphaeroides ΕbluE strains exhibited a decreased pigment phenotype that was restored by complementation with BluE. Finally, phylogenetic analyses revealed that bluE was restricted to the genomes of a few Rhodobacterales that appear to have a preference for a specific form of Cba, namely Coá´½-(á´½-5,6-dimethylbenzimidazolyl-Coᵦ-adenosylcobamide (a.k.a. adenosylcobalamin, AdoCbl; coenzyme B12 , CoB12 ).

Insights into the Function of the N-Acetyltransferase SatA That Detoxifies Streptothricin in Bacillus subtilis and Bacillus anthracis.

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 inactivate streptothricin, which is a broad-spectrum antibiotic produced by actinomycetes in the soil. To determine functionally relevant residues of B. subtilis SatA (BsSatA), 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 (BaSatA; PDB entry 3PP9), this area appears to form a pocket with multiple conserved aromatic residues; we hypothesized this region contains the streptothricin-binding site. Chemical and site-directed mutagenesis was 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 experiments showed that residue Y164 of BaSatA was important for binding streptothricin. Results 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.IMPORTANCE This work provides insights into how an abundant antibiotic found in soil is bound to the enzyme that inactivates it. This work identifies residues for the binding of the antibiotic and probes the contributions of substituting side chains for those in the native protein, providing information regarding hydrophobicity, size, and flexibility of the antibiotic binding site.

A New Class of EutT ATP:Co(I)rrinoid Adenosyltransferases Found in Listeria monocytogenes and Other Firmicutes Does Not Require a Metal Ion for Activity.

ATP:Co(I)rrinoid adenosyltransferases (ACATs) are involved in de novo adenosylcobamide (AdoCba) biosynthesis and in salvaging complete and incomplete corrinoids from the environment. The ACAT enzyme family is comprised of three classes of structurally and evolutionarily distinct proteins (i.e., CobA, PduO, and EutT). The structure of EutT is unknown, and an understanding of its mechanism is incomplete. The Salmonella enterica EutT ( SeEutT) enzyme is the best-characterized member of its class and is known to be a ferroprotein. Here, we report the identification and initial biochemical characterization of an enzyme representative of a new class of EutTs that does not require a metal ion for activity. In vivo and in vitro evidence shows that the metal-free EutT homologue from Listeria monocytogenes ( LmEutT) has ACAT activity and that, unlike other ACATs, the biologically active form of LmEutT is a tetramer. In vitro studies revealed that LmEutT was more efficient than SeEutT and displayed positive cooperativity. LmEutT adenosylated cobalamin, but not cobinamide, showed specificity for ATP and 2'-deoxyATP and released a triphosphate byproduct. Bioinformatics analyses suggest that metal-free EutT ACATs are also present in other Firmicutes.

The Methanosarcina mazei MM2060 Gene Encodes a Bifunctional Kinase/Decarboxylase Enzyme Involved in Cobamide Biosynthesis.

Cobamides (Cbas) are synthesized by many archaea, but some aspects of Cba biosynthesis in these microorganisms remain unclear. Here, we demonstrate that open reading frame MM2060 in the archaeum Methanosarcina mazei strain Gö1 encodes a bifunctional enzyme with l-threonine- O-3-phosphate (l-Thr-P) decarboxylase (EC 4.1.1.81) and l-Thr kinase activities (EC 2.7.1.177). In Salmonella enterica, where Cba biosynthesis has been extensively studied, the activities mentioned above are encoded by separate genes, namely, cobD and pduX, respectively. The activities associated with the MM2060 protein ( MmCobD) were validated in vitro and in vivo. In vitro, MmCobD used ATP and l-Thr as substrates and generated ADP, l-Thr-P, and ( R)-1-aminopropan-2-ol O-phosphate as products. Notably, MmCobD has a 111-amino acid C-terminal extension of unknown function, which contains a putative metal-binding motif. This C-terminal domain alone did not display activity either in vivo or in vitro. Although the C-terminal MmCobD domain was not required for l-Thr-P decarboxylase or l-Thr kinase activities in vivo, its absence negatively affected both activities. In vitro results suggested that this domain may have a regulatory or substrate-gating role. When purified under anoxic conditions, MmCobD displayed Michaelis-Menten kinetics and had a 1000-fold higher affinity for ATP and a catalytic efficiency 1300-fold higher than that of MmCobD purified under oxic conditions. To the best of our knowledge, MmCobD is the first example of a new class of l-Thr-P decarboxylases that also have l-Thr kinase activity. An archaeal protein with l-Thr kinase activity had not been identified prior to this work.

Spectroscopic Study of the EutT Adenosyltransferase from Listeria monocytogenes: Evidence for the Formation of a Four-Coordinate Cob(II)alamin Intermediate.

The EutT enzyme from Listeria monocytogenes ( LmEutT) is a member of the family of ATP:cobalt(I) corrinoid adenosyltransferase (ACAT) enzymes that catalyze the biosynthesis of adenosylcobalamin (AdoCbl) from exogenous Co(II)rrinoids and ATP. Apart from EutT-type ACATs, two evolutionary unrelated types of ACATs have been identified, termed PduO and CobA. Although the three types of ACATs are nonhomologous, they all generate a four-coordinate cob(II)alamin (4C Co(II)Cbl) species to facilitate the formation of a supernucleophilic Co(I)Cbl intermediate capable of attacking the 5'-carbon of cosubstrate ATP. Previous spectroscopic studies of the EutT ACAT from Salmonella enterica ( SeEutT) revealed that this enzyme requires a divalent metal cofactor for the conversion of 5C Co(II)Cbl to a 4C species. Interestingly, LmEutT does not require a divalent metal cofactor for catalytic activity, which exemplifies an interesting phylogenetic divergence among the EutT enzymes. To explore if this disparity in the metal cofactor requirement among EutT enzymes correlates with differences in substrate specificity or the mechanism of Co(II)Cbl reduction, we employed various spectroscopic techniques to probe the interaction of Co(II)Cbl and cob(II)inamide (Co(II)Cbi+) with LmEutT in the absence and presence of cosubstrate ATP. Our data indicate that LmEutT displays a similar substrate specificity as SeEutT and can bind both Co(II)Cbl and Co(II)Cbi+ when complexed with MgATP, though it exclusively converts Co(II)Cbl to a 4C species. Notably, LmEutT is the most effective ACAT studied to date in generating the catalytically relevant 4C Co(II)Cbl species, achieving a >98% 5C → 4C conversion yield on the addition of just over one mol equiv of cosubstrate MgATP.

Salmonella enterica synthesizes 5,6-dimethylbenzimidazolyl-(DMB)-α-riboside. Why some Firmicutes do not require the canonical DMB activation system to synthesize adenosylcobalamin.

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.

Spectroscopic Studies of the EutT Adenosyltransferase from Salmonella enterica: Evidence of a Tetrahedrally Coordinated Divalent Transition Metal Cofactor with Cysteine Ligation.

The EutT enzyme from Salmonella enterica, a member of the family of ATP:cobalt(I) corrinoid adenosyltransferase (ACAT) enzymes, requires a divalent transition metal ion for catalysis, with Fe(II) yielding the highest activity. EutT contains a unique cysteine-rich HX11CCX2C(83) motif (where H and the last C occupy the 67th and 83rd positions, respectively, in the amino acid sequence) not found in other ACATs and employs an unprecedented mechanism for the formation of adenosylcobalamin. Recent kinetic and spectroscopic studies of this enzyme revealed that residues in the HX11CCX2C(83) motif are required for the tight binding of the divalent metal ion and are critical for the formation of a four-coordinate (4c) cob(II)alamin [Co(II)Cbl] intermediate in the catalytic cycle. However, it remained unknown which, if any, of the residues in the HX11CCX2C(83) motif bind the divalent metal ion. To address this issue, we have characterized Co(II)-substituted wild-type EutT (EutTWT/Co) by using electronic absorption, electron paramagnetic resonance, and magnetic circular dichroism (MCD) spectroscopies. Our results indicate that the reduced catalytic activity of EutTWT/Co relative to that of the Fe(II)-containing enzyme arises from the incomplete incorporation of Co(II) ions and, thus, a decrease in the relative population of 4c Co(II)Cbl. Our MCD data for EutTWT/Co also reveal that the Co(II) ions reside in a distorted tetrahedral coordination environment with direct cysteine sulfur ligation. Additional spectroscopic studies of EutT/Co variants possessing a single alanine substitution of either His67, His75, Cys79, Cys80, or Cys83 indicate that Cys80 coordinates to the Co(II) ion, while the additional residues are important for maintaining the structural integrity and/or high affinity of the metal binding site.

The EutQ and EutP proteins are novel acetate kinases involved in ethanolamine catabolism: physiological implications for the function of the ethanolamine metabolosome in Salmonella enterica.

Salmonella 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, ΔeutP strains lacked discernible phenotypes under the conditions where ΔeutQ 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.