A new pathway for the synthesis of α‐ribazole‐phosphate in Listeria innocua

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.     

Functional Analysis of the Nicotinate Mononucleotide:5,6-Dimethylbenzimidazole Phosphoribosyltransferase (CobT) Enzyme,Involved in the Late Steps of Coenzyme B12 Biosynthesis in Salmonella enterica

In Salmonella enterica, the CobT enzyme activates the lower ligand base during the assembly of the nucleotide loop of adenosylcobalamin (AdoCbl) and other cobamides. Previously, mutational analysis identified a class of alleles (class M) that failed to restore AdoCbl biosynthesis during intragenic complementation studies. To learn why class M cobT mutations were deleterious, we determined the nature of three class M cobT alleles and performed in vivo and in vitro functional analyses guided by available structural data on the wild-type CobT (CobT^WT) enzyme. We analyzed the effects of the variants CobT(G257D), CobT(G171D), CobT(G320D), and CobT(C160A). The latter was not a class M variant but was of interest because of the potential role of a disulfide bond between residues C160 and C256 in CobT activity. Substitutions G171D, G257D, and G320D had profound negative effects on the catalytic efficiency of the enzyme. The C160A substitution rendered the enzyme fivefold less efficient than CobT^WT. The CobT(G320D) protein was unstable, and results of structure-guided site-directed mutagenesis suggest that either variants CobT(G257D) and CobT(G171D) have less affinity for 5,6-dimethylbenzimidazole (DMB) or access of DMB to the active site is restricted in these variant proteins. The reported lack of intragenic complementation among class M cobT alleles is caused in some cases by unstable proteins, and in others it may be caused by the formation of dimers between two mutant CobT proteins with residual activity that is so low that the resulting CobT dimer cannot synthesize sufficient product to keep up with even the lowest demand for AdoCbl.Cobalamin (Cbl, also known as B₁₂) is a structurally complex cyclic tetrapyrrole with a cobalt ion coordinated by equatorial bonds with pyrrolic nitrogen atoms and is unique among cyclic tetrapyrroles (e.g., heme, chlorophylls, coenzyme F₄₃₀) in that it has an upper and a lower axial ligand (Fig. ​(Fig.1).1). The coenzymic form of Cbl is known as adenosylcobalamin (AdoCbl) or coenzyme B₁₂.Open in a separate windowFIG. 1.Role of CobT in the late steps of AdoCbl biosynthesis. This branch of the AdoCbl biosynthetic pathway is known as the NLA pathway. The black box in the inner membrane represents the corrinoid-specific ABC transporter BtuCD. The inset shows the chemical structure of AdoCbl; the coring ring in the scheme is represented by the rhomboid cartoon with the Co ion in the middle.Some bacteria and archaea synthesize AdoCbl de novo or from preformed precursors such as cobyric acid (Cby) or cobinamide (Cbi) (Fig. ​(Fig.1)1) (11, 32). The enterobacterium Salmonella enterica serovar Typhimurium LT2 (hereafter referred to as S. Typhimurium) synthesizes the corrin ring of AdoCbl de novo only under anoxic conditions (15). Although oxygen blocks de novo corrin ring biosynthesis in this bacterium, it does not block the assembly of adenosylcobalamin (AdoCbl, coenzyme B₁₂) if cells are provided with preformed, incomplete corrinoids such as Cbi or Cby (11, 13-15).The late steps in AdoCbl biosynthesis can be divided into two different branches that comprise the nucleotide loop assembly (NLA) pathway (19). One of the branches of the NLA pathway activates the lower ligand base, while the other one activates adenosylcobinamide (AdoCbi) to AdoCbi-GDP (Fig. ​(Fig.11).In this paper, we focus on the activation of 5,6-dimethylbenzimidazole (DMB), the lower ligand base of AdoCbl. There are two ways in which DMB can be activated. In both cases, the CobT enzyme (EC 2.4.2.21) catalyzes the reaction, but the product of the reaction varies, depending on whether the cosubstrate of CobT is NAD⁺ or its precursor nicotinate mononucleotide (NaMN). If NaMN is the substrate, CobT synthesizes α-ribazole-phosphate (α-RP) (reaction: DMB + NaMN → α-RP + Na) (30). If NAD⁺ substitutes for NaMN, CobT synthesizes α-DMB adenine dinucleotide (α-DAD) (reaction: DMB + NAD⁺ → α-DAD + Nm) (18). α-RP is a good substrate for the AdoCbl-5′-phosphate (AdoCbl-P) synthase (CobS; EC 2.7.8.26) enzyme (19, 34), suggesting that an unidentified enzyme cleaves α-DAD into α-RP and AMP (reaction: α-DAD → α-RP + AMP). Although it is possible that CobS may use α-DAD as a substrate, to date, data are not available to support this idea. The AdoCbl-P phosphatase (CobC; EC 3.1.3.73) enzyme catalyzes the last step of the NLA pathway to yield AdoCbl (Fig. ​(Fig.1)1) (34).Early genetic studies identified four classes of cobT alleles, namely, classes J, K, L, and M (12); class M was an intriguing class of mutations because they did not display intragenic complementation (12). Here we identify the nature of class M cobT mutations, report the initial in vitro and in vivo characterization of class M CobT variant proteins, and propose structural explanations for the observed deficiencies in CobT activity caused by class M mutations.     

ArsAB, a novel enzyme from Sporomusa ovata activates phenolic bases for adenosylcobamide biosynthesis

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.     

The end of the cob operon: evidence that the last gene (cobT) catalyzes synthesis of the lower ligand of vitamin B12, dimethylbenzimidazole.

The cob operon of Salmonella typhimurium includes 20 genes devoted to the synthesis of adenosyl-cobalamin (coenzyme B12). Mutants with lesions in the promoter-distal end of the operon synthesize vitamin B12 only if provided with 5,6-dimethylbenzimidazole (DMB), the lower ligand of vitamin B12. In the hope of identifying a gene(s) involved in synthesis of DMB, the DNA base sequence of the end of the operon has been determined; this completes the sequence of the cob operon. The cobT gene is the last gene in the operon. Four CobII (DMB-) mutations mapping to different deletion intervals of the CobII region were sequenced; all affect the cobT open reading frame. Both the CobT protein of S. typhimurium and its Pseudomonas homolog have been shown in vitro to catalyze the transfer of ribose phosphate from nicotinate mononucleotide to DMB. This reaction does not contribute to DMB synthesis but rather is the first step in joining DMB to the corrin ring compound cobinamide. Thus, the phenotype of Salmonella cobT mutants conflicts with the reported activity of the affected enzyme, while Pseudomonas mutants have the expected phenotype. J. R. Trzebiatowski, G. A. O'Toole, and J. C. Escalante Semerena have suggested (J. Bacteriol. 176:3568-3575, 1994) that S. typhimurium possesses a second phosphoribosyltransferase activity (CobB) that requires a high concentration of DMB for its activity. We support that suggestion and, in addition, provide evidence that the CobT protein catalyzes both the synthesis of DMB and transfer of ribose phosphate. Some cobT mutants appear defective only in DMB synthesis, since they grow on low levels of DMB and retain their CobII phenotype in the presence of a cobB mutation. Other mutants including those with deletions, appear defective in transferase, since they require a high level of DMB (to activate CobB) and, in combination with a cobB mutation, they eliminate the ability to join DMB and cobinamide. Immediately downstream of the cob operon is a gene (called ORF in this study) of unknown function whose mutants have no detected phenotype. Just counterclockwise of ORF is an asparagine tRNA gene (probably asnU). Farther counterclockwise, a serine tRNA gene (serU or supD) is weakly cotransducible with the cobT gene.     

One pathway can incorporate either adenine or dimethylbenzimidazole as an alpha-axial ligand of B12 cofactors in Salmonella enterica

Corrinoid (vitamin B₁₂-like) cofactors contain various α-axial ligands, including 5,6-dimethylbenzimidazole (DMB) or adenine. The bacterium Salmonella enterica produces the corrin ring only under anaerobic conditions, but it can form “complete” corrinoids aerobically by importing an “incomplete” corrinoid, such as cobinamide (Cbi), and adding appropriate α- and β-axial ligands. Under aerobic conditions, S. enterica performs the corrinoid-dependent degradation of ethanolamine if given vitamin B₁₂, but it can make B₁₂ from exogenous Cbi only if DMB is also provided. Mutants isolated for their ability to degrade ethanolamine without added DMB converted Cbi to pseudo-B₁₂ cofactors (having adenine as an α-axial ligand). The mutations cause an increase in the level of free adenine and install adenine (instead of DMB) as an α-ligand. When DMB is provided to these mutants, synthesis of pseudo-B₁₂ cofactors ceases and B₁₂ cofactors are produced, suggesting that DMB regulates production or incorporation of free adenine as an α-ligand. Wild-type cells make pseudo-B₁₂ cofactors during aerobic growth on propanediol plus Cbi and can use pseudo-vitamin B₁₂ for all of their corrinoid-dependent enzymes. Synthesis of coenzyme pseudo-B₁₂ cofactors requires the same enzymes (CobT, CobU, CobS, and CobC) that install DMB in the formation of coenzyme B₁₂. Models are described for the mechanism and control of α-axial ligand installation.     

The cobinamide amidohydrolase (cobyric acid‐forming) CbiZ enzyme: a critical activity of the cobamide remodelling system of Rhodobacter sphaeroides

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.     

Transketolase activity modulates glycerol‐3‐phosphate levels in Escherichia coli

Transketolase activity provides an important link between the metabolic pathways of glycolysis and pentose phosphate shunt and catalyzes inter‐conversions between pentose phosphates and glycolytic intermediates. It is widely conserved in life forms. A genetic screen for suppression of the growth defect of Escherichia coli tktA tktB mutant in LB medium revealed two mutations, one that rendered the glpK expression constitutive and another that inactivated deoB. Characterizing these mutations aided in uncovering the role of ribose‐5‐P (a transketolase substrate) as an inhibitor of glycerol assimilation and de novo glycerol‐3‐P synthesis. Using lacZ fusions, we show that ribose‐5‐P enhances GlpR–mediated repression of the glpFKX operon and inhibits glycerol assimilation. Electrophoretic Mobility Shift Assay (EMSA) showed ribose‐5‐P made the DNA‐GlpR complex less sensitive to the inducer glycerol‐3‐P. In addition to inhibition of glycerol assimilation, obstruction of ribose‐5‐P metabolism retards growth from glycerol‐3‐P limitation. Glucose helps to overcome this limitation through a mechanism involving catabolite repression. To our knowledge, this report is the first to show ribose‐5‐P can modulate glycerol‐3‐P concentration in the cell by regulation of glycerol assimilation as well as its de novo synthesis. This regulation could be prevalent in other organisms.     

Structural determinants in bacterial 2‐keto‐3‐deoxy‐D‐gluconate dehydrogenase KduD for dual‐coenzyme specificity

Short‐chain dehydrogenase/reductase (SDR) is distributed in many organisms, from bacteria to humans, and has significant roles in metabolism of carbohydrates, lipids, amino acids, and other biomolecules. An important intermediate in acidic polysaccharide metabolism is 2‐keto‐3‐deoxy‐d ‐gluconate (KDG). Recently, two short and long loops in Sphingomonas KDG‐producing SDR enzymes (NADPH‐dependent A1‐R and NADH‐dependent A1‐R′) involved in alginate metabolism were shown to be crucial for NADPH or NADH coenzyme specificity. Two SDR family enzymes—KduD from Pectobacterium carotovorum (PcaKduD) and DhuD from Streptococcus pyogenes (SpyDhuD)—prefer NADH as coenzyme, although only PcaKduD can utilize both NADPH and NADH. Both enzymes reduce 2,5‐diketo‐3‐deoxy‐d ‐gluconate to produce KDG. Tertiary and quaternary structures of SpyDhuD and PcaKduD and its complex with NADH were determined at high resolution (approximately 1.6 Å) by X‐ray crystallography. Both PcaKduD and SpyDhuD consist of a three‐layered structure, α/β/α, with a coenzyme‐binding site in the Rossmann fold; similar to enzymes A1‐R and A1‐R′, both arrange the two short and long loops close to the coenzyme‐binding site. The primary structures of the two loops in PcaKduD and SpyDhuD were similar to those in A1‐R′ but not A1‐R. Charge neutrality and moderate space at the binding site of the nucleoside ribose 2′ coenzyme region were determined to be structurally crucial for dual‐coenzyme specificity in PcaKduD by structural comparison of the NADH‐ and NADPH‐specific SDR enzymes. The corresponding site in SpyDhuD was negatively charged and spatially shallow. This is the first reported study on structural determinants in SDR family KduD related to dual‐coenzyme specificity. Proteins 2016; 84:934–947. © 2016 Wiley Periodicals, Inc.     

L‐2,3‐diaminopropionate generates diverse metabolic stresses in Salmonella enterica

Unchecked amino acid accumulation in living cells has the potential to cause stress by disrupting normal metabolic processes. Thus, many organisms have evolved degradation strategies that prevent endogenous accumulation of amino acids. L‐2,3‐diaminopropionate (Dap) is a non‐protein amino acid produced in nature where it serves as a precursor to siderophores, neurotoxins and antibiotics. Dap accumulation in Salmonella enterica was previously shown to inhibit growth by unknown mechanisms. The production of diaminopropionate ammonia‐lyase (DpaL) alleviated Dap toxicity in S. enterica by catalyzing the degradation of Dap to pyruvate and ammonia. Here, we demonstrate that Dap accumulation in S. enterica elicits a proline requirement for growth and specifically inhibits coenzyme A and isoleucine biosynthesis. Additionally, we establish that the DpaL‐dependent degradation of Dap to pyruvate proceeds through an unbound 2‐aminoacrylate (2AA) intermediate, thus contributing to 2AA stress inside the cell. The reactive intermediate deaminase, RidA, is shown to prevent 2AA damage caused by DpaL‐dependent Dap degradation by enhancing the rate of 2AA hydrolysis. The results presented herein inform our understanding of the effects Dap has on metabolism in S. enterica, and likely other organisms, and highlight the critical role played by RidA in preventing 2AA stress stemming from Dap detoxification.     

Determination of the enantiomeric purity of the selective dopamine transporter inhibitor (+)‐R,R‐4‐(2‐benzhydryloxyethyl)‐1‐(4‐fluorobenzyl)piperidin‐3‐ol

(+)‐R ,R ‐D‐84 ((+)‐R ,R ‐4‐(2‐benzhydryloxyethyl)‐1‐(4‐fluorobenzyl)piperidin‐3‐ol) is a promising pharmacological tool for the dopamine transporter (DAT), due to its high affinity and selectivity for this target. In this study, an analytical method to ascertain the enantiomeric purity of this compound was established. For this purpose, a high‐performance liquid chromatographic (HPLC) method, based on a cellulose derived chiral stationary phase (CSP) was developed. The method was characterized concerning its specificity, linearity, and range. It was shown that the method is suitable to determine an enantiomeric excess of up to 99.8%. With only a few adjustments, this analytical CSP‐HPLC method is also well suited to separate (+)‐R ,R ‐D‐84 from its enantiomer in a semipreparative scale.     

Comparison of intestinal permeability and p‐glycoprotein effects on the intestinal absorption of enantiomers of 2‐(2‐hydroxypropanamido) benzoic acid in rats

The purpose of this study was to compare intestinal permeability between enantiomers of 2‐(2‐hydroxypropanamido) benzoic acid ((R )‐/ (S )‐ HPABA), a marine‐derived antiinflammatory drug, using an in situ single‐pass intestinal perfusion (SPIP) model in rats. Concentrations, isolated regions of small intestine, and p ‐glycoprotein (P‐gp) inhibitor were performed to investigate their influences on the intestinal absorption of (R )‐/ (S )‐ HPABA. In addition, a molecular docking method was performed to illustrate our prediction. The absorption rate coefficients (K _a ) and permeability values (P _eff ) of (R )‐/ (S )‐ HPABA were calculated. The permeability of (S )‐HPABA was significantly (P <  0.01) higher than that of (R )‐HPABA in jejunum, and ileum permeability of (R )‐/ (S )‐ HPABA appeared best in ileum; the investigated concentrations ranged from 20 to 80 μg/mL, K _a and P _eff values of (R )‐/ (S )‐ HPABA increased linearly; in the presence of P‐gp inhibitor (verapamil), P _eff values of two enantiomers were increased significantly; and the effect of P‐gp on absorption of (R )‐HPABA is stronger than that of (S )‐HPABA in ileum segment. Based on these results, carrier‐mediated transport or passive transport combined with carrier‐mediated transport seems to be the mechanism for intestinal absorption of (R )‐/ (S )‐ HPABA, and (R )‐/ (S )‐ HPABA may be recognized as the P‐gp substrate. In addition, the intestinal permeability of (S )‐HPABA is higher than that of (R )‐HPABA.     

Spectral study of interaction between poly [acrylamide‐(2‐methyl acryloxyethyl) dimethyl buthylammonium bromide] and nucleic acids

A polycation, poly[acrylamide‐(2‐methylacryloxyethyl) dimethyl buthylammonium bromide] [P (AM‐DMB)], was synthesized. The interaction between P (AM‐DMB) and DNA was studied by spectral methods. Resonance light scattering spectra, FTIR‐spectra and UV‐spectra were used to study the mechanisms of the interaction. The results indicate that the conformation of the DNA alters during the interaction because the microenvironment of DNA changes. The binding constant K between P (AM‐DMB) and DNA is 8.0 × 10³ L mol^?1. Copyright © 2009 John Wiley & Sons, Ltd.     

TGF‐β1 rs1982073 polymorphism contributes to radiation pneumonitis in lung cancer patients: a meta‐analysis

Transforming growth factor beta 1(TGF‐β1) polymorphism was associated with radiation pneumonitis (RP) susceptibility, but their results have been inconsistent. The PubMed and CNKI were searched for case‐control studies published up to Januray 01, 2016 was Data were extracted and pooled odds ratios (OR) with 95% confidence intervals (CI) were calculated. In this meta‐analysis, we assessed eight publications involving 368 radiation pneumonitis cases and 855 controls of the association between TGF‐β1 T869C (rs1982073) and G915C (rs1800471) polymorphism and RP susceptibility. Our analysis suggested that TGF‐β1 T869C rs1982073 polymorphism was associated with lower RP risk for CT combined CC versus TT model (OR = 0.58, 95% CI = 0.43–0.77). However, for the G915C rs1800471 polymorphism, no association was found between the polymorphism and the susceptibility to RP in GC combined CC versus GG model (OR = 0.82, 95% CI = 0.50–1.35). These results from the meta‐analysis suggest that T869C rs1982073 polymorphism of TGF‐β1 may be associated with RP risk, and there may be no association between G915C polymorphism and RP risk.     

Crystallographic studies for the chiral recognition of the novel chiral stationary phase derived from (+)‐(R)‐18‐crown‐6 tetracarboxylic acid

Chiral discrimination observed in high‐performance liquid chromatography (HPLC) with the novel chiral stationary phase (CSP‐18C6I) derived from (+)‐(R)‐18‐crown‐6 tetracarboxylic acid [(+)‐18C6H₄] was investigated by X‐ray crystallographic analysis of the complex composed of the R‐enantiomer of 1‐(1‐naphthyl)ethylamine (1‐NEA) and (+)‐18C6H₄. Mixtures of 1‐NEA (the R‐ or S‐enantiomer) and (+)‐18C6H₄ were dissolved in methanol‐water (1:1) solution and allowed to stand for crystallization. The R‐enantiomer crystallized with (+)‐18C6H₄ as a co‐crystal, although the S‐enantiomer did not. This result was in good agreement with the enantiomer elution order of 1‐NEA in CSP‐18C6I. The apparent binding constants (K_a) of the enantiomers to the (+)‐18C6H₄ obtained from ¹H‐NMR experiments also supported the above‐mentioned result. The X‐ray crystal structure of the 1:1 complex of the R‐enantiomer and (+)‐18C6H₄ indicated the four sets of hydrogen bond association between the naphthylethylammonium cation and oxygen of polyether ring or carbonyl group of (+)‐18C6H₄. Chirality 11:173–178, 1999. © 1999 Wiley‐Liss, Inc.     

Dissecting cobamide diversity through structural and functional analyses of the base-activating CobT enzyme of Salmonella enterica

Background

Cobamide diversity arises from the nature of the nucleotide base. Nicotinate mononucleotide (NaMN):base phosphoribosyltransferases (CobT) synthesize α-linked riboside monophosphates from diverse nucleotide base substrates (e.g., benzimidazoles, purines, phenolics) that are incorporated into cobamides.

Methods

Structural investigations of two members of the CobT family of enzymes in complex with various substrate bases as well as in vivo and vitro activity analyses of enzyme variants were performed to elucidate the roles of key amino acid residues important for substrate recognition.

Results

Results of in vitro and in vivo studies of active-site variants of the Salmonella enterica CobT (SeCobT) enzyme suggest that a catalytic base may not be required for catalysis. This idea is supported by the analyses of crystal structures that show that two glutamate residues function primarily to maintain an active conformation of the enzyme. In light of these findings, we propose that proper positioning of the substrates in the active site triggers the attack at the C1 ribose of NaMN.

Conclusion

Whether or not a catalytic base is needed for function is discussed within the framework of the in vitro analysis of the enzyme activity. Additionally, structure-guided site-directed mutagenesis of SeCobT broadened its substrate specificity to include phenolic bases, revealing likely evolutionary changes needed to increase cobamide diversity, and further supporting the proposed mechanism for the phosphoribosylation of phenolic substrates.

General Significance

Results of this study uncover key residues in the CobT enzyme that contribute to the diversity of cobamides in nature.     

Racemic and enantiopure forms of 3‐ethyl‐3‐phenylpyrrolidin‐2‐one adopt very different crystal structures

3‐Ethyl‐3‐phenylpyrrolidin‐2‐one ( EPP) is an experimental anticonvulsant based on the newly proposed α‐substituted amide group pharmacophore. These compounds show robust activity in animal models of drug‐resistant epilepsy and are thus promising for clinical development. In order to understand pharmaceutically relevant properties of such compounds, we are conducting an extensive investigation of their structures in the solid state. In this article, we report chiral high‐performance liquid chromatography (HPLC) separation, determination of absolute configuration of enantiomers, and crystal structures of EPP. Preparative resolution of EPP enantiomers by chiral HPLC was accomplished on the Chiralcel OJ stationary phase in the polar‐organic mode. Using a combination of electronic CD spectroscopy and anomalous dispersion of X‐rays we established that the first‐eluted enantiomer corresponds to (+)‐(R )‐EPP, while the second‐eluted enantiomer corresponds to (−)‐(S )‐EPP. We also demonstrated that, in the crystalline state, enantiopure and racemic forms of this anticonvulsant have considerable differences in their supramolecular organization and patterns of hydrogen bonding. These stereospecific structural differences can be related to the differences in melting points and, correspondingly, solubility and bioavailability.     

The three-dimensional structures of nicotinate mononucleotide:5,6- dimethylbenzimidazole phosphoribosyltransferase (CobT) from Salmonella typhimurium complexed with 5,6-dimethybenzimidazole and its reaction products determined to 1.9 A resolution

Nicotinate mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase (CobT) from Salmonella typhimurium plays a central role in the synthesis of alpha-ribazole, which is a key component of the lower ligand of cobalamin. Two X-ray structures of CobT are reported here at 1.9 A resolution. First, a complex of CobT with 5,6-dimethylbenzimidazole, and second, a complex of CobT with its reaction products, nicotinate and alpha-ribazole-5'-phosphate. CobT was cocrystallized with 5,6-dimethylbenzimidazole (DMB) in the space group P2(1)2(1)2 with unit cell dimensions of a = 72.1 A, b = 90.2 A, and c = 47.5 A and one protomer per asymmetric unit. Subsequently, the crystals containing DMB were soaked in nicotinate mononucleotide whereupon the physiological reaction occurred in the crystal lattice to yield nicotinate and alpha-ribazole-5'-phosphate. These studies show that CobT is a dimer where each subunit consists of two domains. The large domain is dominated by a parallel six-stranded beta-sheet with connecting alpha-helices that exhibit the topology of a Rossmann fold. The small domain is made from components of the N- and C-terminal sections of the polypeptide chain and contains a three-helix bundle. The fold of CobT is unrelated to the type I and II phosphoribosylpyrophosphate dependent transferases and does not appear to be related to any other protein whose structure is known. The enzyme active site is located in a large cavity formed by the loops at the C-terminal ends of the beta-strands and the small domain of the neighboring subunit. DMB binds in a hydrophobic pocket created in part by the neighboring small domain. This is consistent with the broad specificity of this enzyme for aromatic substrates [Trzebiatowski, J. R., Escalante-Semerena (1997) J. Biol. Chem. 272, 17662-17667]. The binding site for DMB suggests that Glu317 is the catalytic base required for the reaction. The remainder of the cavity binds the nicotinate and ribose-5'-phosphate moieties, which are nestled within the loops at the ends of the beta-strands. Interestingly, the orientation of the substrate and products are opposite from that expected for a Rossmann fold.     

Uncovering mechanisms of rubber biosynthesis in Taraxacum koksaghyz – role of cis‐prenyltransferase‐like 1 protein

The Russian dandelion Taraxacum koksaghyz synthesizes considerable amounts of high‐molecular‐weight rubber in its roots. The characterization of factors that participate in natural rubber biosynthesis is fundamental for the establishment of T. koksaghyz as a rubber crop. The cis‐1,4‐isoprene polymers are stored in rubber particles. Located at the particle surface, the rubber transferase complex, member of the cis‐prenyltransferase (cisPT) enzyme family, catalyzes the elongation of the rubber chains. An active rubber transferase heteromer requires a cisPT subunit (CPT) as well as a CPT‐like subunit (CPTL), of which T. koksaghyz has two homologous forms: TkCPTL1 and TkCPTL2, which potentially associate with the rubber transferase complex. Knockdown of TkCPTL1, which is predominantly expressed in latex, led to abolished poly(cis‐1,4‐isoprene) synthesis but unaffected dolichol content, whereas levels of triterpenes and inulin were elevated in roots. Analyses of latex from these TkCPTL1‐RNAi plants revealed particles that were similar to native rubber particles regarding their particle size, phospholipid composition, and presence of small rubber particle proteins (SRPPs). We found that the particles encapsulated triterpenes in a phospholipid shell stabilized by SRPPs. Conversely, downregulating the low‐expressed TkCPTL2 showed no altered phenotype, suggesting its protein function is redundant in T. koksaghyz. MS‐based comparison of latex proteomes from TkCPTL1‐RNAi plants and T. koksaghyz wild‐types discovered putative factors that convert metabolites in biosynthetic pathways connected to isoprenoids or that synthesize components of the rubber particle shell.     

The cobT gene of Salmonella typhimurium encodes the NaMN: 5,6-dimethylbenzimidazole phosphoribosyltransferase responsible for the synthesis of N1-(5-phospho-alpha-D-ribosyl)-5,6-dimethylbenzimidazole, an intermediate in the synthesis of the nucleotide loop of cobalamin.

We present in vitro evidence which demonstrates that CobT is the nicotinate nucleotide:5,6-dimethylbenzimidazole (DMB) phosphoribosyltransferase (EC 2.4.2.21) that catalyzes the synthesis of N1-(5-phospho-alpha-D-ribosyl)-5,6-dimethylbenzimidazole, a biosynthetic intermediate of the pathway that assembles the nucleotide loop of cobalamin in Salmonella typhimurium. Mutants previously isolated as DMB auxotrophs are shown by physical and genetic mapping studies and complementation studies to carry lesions in cobT. Explanations for this unexpected phenotype of cobT mutants are discussed. The expected nucleotide loop assembly phenotype of cobT mutants can be observed only in a specific genetic background, i.e., cobB deficient, an observation that is consistent with the existence of an alternative CobT function (G. A. O'Toole, M. R. Rondon, and J. C. Escalante-Semerena, J. Bacteriol. 175:3317-3326, 1993). Computer analysis of CobT homologs showed that at the amino acid level, enteric CobT proteins were 80% identical whereas Pseudomonas denitrificans and Rhizobium meliloti CobT proteins were 95% identical. Interestingly, the degree of identity between enteric and nonenteric CobT homologs was only 30%. The same pattern of homologies was reported for the S. typhimurium CobA, Escherichia coli BtuR, and P. denitrificans CobO proteins (S.-J. Suh and J.C. Escalante-Semerena, Gene 129:93-97, 1993), suggesting evolutionary divergence between the cob genes found in the enteric bacteria E. coli and S. typhimurium and those found in P. denitrificans and R. meliloti.