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. 2010 Sep;77(6):1429-38.
doi: 10.1111/j.1365-2958.2010.07294.x. Epub 2010 Aug 5.

A New Pathway for the Synthesis of α-Ribazole-Phosphate in Listeria Innocua

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Free PMC article

A New Pathway for the Synthesis of α-Ribazole-Phosphate in Listeria Innocua

Michael J Gray et al. Mol Microbiol. .
Free PMC article

Abstract

The genomes of Listeria spp. encode all but one of 25 enzymes required for the biosynthesis of adenosylcobalamin (AdoCbl; coenzyme B(12) ). 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 N(1) -(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.

Figures

Figure 1
Figure 1. De novo assembly of the lower ligand of coenzyme B12 in S. enterica
The α-R moiety is indicated with grey boxes. Abbreviations: CobT, nicotinate mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase; CobS, adenosylcobalamin-phosphate synthase; CobC, adenosylcobalamin-phosphate phosphatase; NaMN, nicotinate mononucleotide; Ado, 5′-deoxyadenosine; P, phosphate; GDP, guanosine diphosphate.
Figure 2
Figure 2. α-R salvaging in S. enterica expressing cblT and cblS alleles from L. innocua
Corrinoid-dependent aerobic growth of S. enterica JE1244 (metE205 ara-9 cobT10::Tn10d[tet+]) derivatives in NCE containing glycerol (22 mM), MgSO4 (1 mM), trace minerals, ampicillin (100 μg ml−1), and arabinose (as indicated). Optical density at 650 nm was measured for 24 h at 37°C. Corrinoids were added at 15 nM. DMB, α-R, and α-RP were added at 250 nM. Plasmids used were: vector, pBAD24; pcobT+, pCOBT48 (S. enterica cobT+); pcblTS+, pCBLTS1 (Lin cblT+ cblS+); pcblT+, pCBLT1 (Lin cblT+); pcblS+, pCBLS4 (Lin cblS+). Growth curves were obtained using an ELx808 Ultra Microplate reader (Bio-Tek Instruments). Each growth curve was performed in triplicate. Error bars of one standard deviation are indicated.
Figure 3
Figure 3. Corrinoid-dependent ethanolamine utilization in L. innocua
Aerobic growth of L. innocua DD680 in MLM containing ethanolamine (100 mM). Optical density at 650 nm was measured for 12 h at 37°C. Corrinoids were added at 15 nM, α-R was added at 500 nM, and NH4Cl was added at 1 g l−1. Growth curves were obtained using an ELx808 Ultra Microplate reader (Bio-Tek Instruments). Each growth curve was performed in triplicate. Error bars of one standard deviation are indicated.
Figure 4
Figure 4. LinCblT is an α-R transporter
Accumulation of radiolabeled compounds by S. enterica strains JE8511 (vector; open symbols; metE205 ara-9 cobT10::Tn10d[tet+]/pBAD24 [bla+]) and JE12550 (pcblT+; filled symbols; metE205 ara-9 cobT10::Tn10d[tet+]/pCBLT1 [cblT+ bla+]) in NCE containing glycerol (22 mM), MgSO4 (1 mM), trace minerals, ampicillin (100 μg ml−1), and arabinose (250 μM). At time zero, [14C]DMB (top panel), [14C]α-R (middle panel), or [14C]α-RP (bottom panel) was added to exponentially growing cells to a concentration of 250 nM. Each accumulation curve was performed in triplicate. Error bars of one standard deviation are indicated.
Figure 5
Figure 5. LinCblS is an ATP-dependent α-R kinase
Reactions containing LinCblS (10 μg), Tris-HCl (100mM, pH 7.5), MgCl2 (1 mM), KCl (50 mM), α-R (30 μM), ATP (1 mM), and TCEP (1 mM) were incubated 3 h at 37°C. A. Products were separated by RP-HPLC with a 10-min linear gradient from 97% ammonium acetate (20 mM, pH 4.5) + 3% acetonitrile (CH3CN) to 40% CH3CN. Control reactions lacking LinCblS, ATP, or TCEP are indicated. The authentic α-RP control is shown in grey. B. UV-visible spectra of the LinCblS product (black trace) and authentic α-RP (grey trace). C. Structure and formula weight of α-RP. D. ESI-TOF LC/MS elution time of the LinCblS product, separated with a 20-min linear gradient from 100% formic acid in H2O (0.1% v/v), to 15% formic acid (0.1% v/v) in CH3CN. E. Mass spectrum of the LinCblS product, which was identical to that of authentic α-RP (not shown).
Figure 6
Figure 6. Comparison of lower ligand assembly in S. enterica and L. innocua
Abbreviations: LinCblT, α-R transporter; LinCblS, α-R kinase; AdoCbl-P, adenosylcobalamin-phosphate. The right panel reflects the findings reported in figure 4, which indicate that, most likely, in L. innocua CblT does not transport DMB nor α-ribazole-P (α-RP) into the cell.

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