BluB/CobT2 fusion enzyme activity reveals mechanisms responsible for production of active form of vitamin B12 by Propionibacterium freudenreichii
© Deptula et al. 2015
Received: 15 September 2015
Accepted: 18 October 2015
Published: 23 November 2015
Propionibacterium freudenreichii is a food grade bacterium that has gained attention as a producer of appreciable amounts of cobalamin, a cobamide with activity of vitamin B12. Production of active form of vitamin is a prerequisite for attempts to naturally fortify foods with B12 by microbial fermentation. Active vitamin B12 is distinguished from the pseudovitamin by the presence of 5,6-dimethylbenzimidazole (DMBI) as the lower ligand. Genomic data indicate that P. freudenreichii possesses a fusion gene, bluB/cobT2, coding for a predicted phosphoribosyltransferase/nitroreductase, which is presumably involved in production of vitamin B12. Understanding the mechanisms affecting the synthesis of different vitamin forms is useful for rational strain selection and essential for engineering of strains with improved B12 production properties.
Here, we investigated the activity of heterologously expressed and purified fusion enzyme BluB/CobT2. Our results show that BluB/CoBT2 is responsible for the biosynthesis of the DMBI base and its activation into α-ribazole phosphate, preparing it for attachment as the lower ligand of cobalamin. The fusion enzyme was found to be efficient in metabolite channeling and the enzymes’ inability to react with adenine, a lower ligand present in the pseudovitamin, revealed a mechanism favoring the production of the active form of the vitamin. P. freudenreichii did not produce cobalamin under strictly anaerobic conditions, confirming the requirement of oxygen for DMBI synthesis. In vivo experiments also revealed a clear preference for incorporating DMBI over adenine into cobamide under both microaerobic and anaerobic conditions.
The herein described BluB/CobT2 is responsible for the production and activation of DMBI. Fusing those two activities results in high pressure towards production of the true vitamin B12 by efficiently activating DMBI formed within the same enzymatic complex. This indicates that BluB/CobT2 is the crucial enzyme in the B12 biosynthetic pathway of P. freudenreichii. The GRAS organism status and the preference for synthesizing active vitamin form make P. freudenreichii a unique candidate for the in situ production of vitamin B12 within food products.
In recent years the interest in fortification of food products in vitamin B12 by in situ fermentation has been growing [4–8]. Vitamin B12 production by P. freudenreichii is of special interest because of the Generally Recognized as Safe (GRAS) status of the organism, which allows its direct use in food and feed preparations . Additionally, P. freudenreichii is an efficient producer, reaching cobalamin levels of 15 µg/mL , and furthermore, reportedly produces only trace amounts of pseudocobalamin . In the pseudo form of the vitamin the lower ligand, adenine (Fig. 1a), renders its affinity to intrinsic factor 500-fold lower than cobalamin . Some other GRAS organisms, mainly Lactobacillus, have also been reported to produce cobalamin [12–15]; however, it is questioned whether these species produce the active or the pseudo form of vitamin. Lactobacillus reuteri, which possesses the genes for B12 synthesis , was recently shown to produce pseudocobalamin exclusively due to its inability to incorporate other bases than adenine as the lower ligand of the cobamide [16, 17]. Streptomyces griseus produces vitamin B12 for which the GRAS status has been granted (§184.1945), however since “Streptomyces spp. produce antibiotics and are therefore inappropriate for QPS (EFSA opinion 2008)”  the bacterium itself is not eligible for such status. With Lactobacilli unable to produce active vitamin B12, P. freudenreichii remains the only known producer with GRAS status granted by the FDA, and QPS granted by EFSA  allowing for its direct use in food and feed preparations.
The complete biosynthesis of cobalamin requires approximately 30 gene products , but it is the final steps of the pathway, namely production, activation and attachment of the lower ligand that decide whether the final product will be an active vitamin B12 or an analog. In aerobic and aerotolerant microorganisms, DMBI is synthesized in an oxygen-dependent manner from reduced FMN by the action of the BluB enzyme [19–21], while in strict anaerobes gene cluster bzaABCDE has been recently associated with this function .
Synthesized DMBI is then activated by the CobT enzyme (CobU in aerobes) into α-ribazole-phosphate (α-RP) (Fig. 1c), preparing the lower ligand for attachment to form the complete cobalamin molecule . Recent studies determined that the selectivity of CobT is responsible for the range of lower ligands that can be attached to cobamide . A study with seven CobT homologues from diverse organisms revealed that DMBI is a preferred substrate (with the exception of ArsAB of Veillonella parvula preferentially activating phenolic bases), although some of the homologues, such as CobT from L. reuteri, were unable to activate DMBI in vivo . In a related report, it was suggested that the ability to exclude certain lower ligands from incorporation into cobamides provides a mechanism preventing the production of compounds that cannot be used by the organism .
The genetics of the complete cobalamin synthesis pathway of P. freudenreichii have been previously studied and described [25–30]. The genome sequence of P. freudenreichii CIRM-BIA1 indicated that in this organism, the bluB gene is fused with cobT2  resulting in a putative phosphoribosyl- transferase/nitroreductase that could both synthesize and activate DMBI for attachment into the cobalamin molecule. Although homologues of the fusion enzyme BluB/CobT2 have been found in multiple organisms on the genetic level, the enzyme has not yet been studied. In this work, we used the heterologously expressed and purified BluB/CobT2 enzyme of the type strain P. freudenreichii subsp. shermanii DSM 4902 to study its predicted novel ability to both synthesize and activate DMBI for attachment as a lower ligand in the final steps of biosynthesis of the active vitamin B12.
Results and discussion
Heterologous expression and purification of the BluB/CobT2 enzyme
Sequence analysis on BluB/CobT2
According to a Blastp search, the closest homolog of BluB/CobT2 is P. acidifaciens, which encodes a 72 % identical protein [NCBI: WP_028706153.1]. Notably, BluB/CobT2 fusion proteins are not found in all members of propionibacteria. Outside propionibacteria, a Blastp search revealed the highest homology against proteins from Propionimicrobium lymphophilum ACS-093-V-SCH5 and Austwickia chelonae, showing 59 and 58 % identity with BluB/CobT2 of P. freudenreichii, respectively. Another Actinobacterium and known producer of vitamin B12, Streptomyces griseus, also posseses a fusion BluB/CobT enzyme, however it is additionally fused with a protein of unknown function at the N-terminus. S. griseus fusion protein shows 34 % identity over the whole region of homology to P. freudenreichii BluB/CobT2. BluB/CobT appears to be rather widespread among Actinobacteria, but only a few isolated entries from other classes, including Spirochaetia, Flavobacteria, Cytophagia and Deltaproteobacteria, were found.
BluB/CobT2 produces DMBI in presence of FMN and NADH
For enzymatic characterization, BluB/CobT2 of P. freudenreichii was heterologously produced in E. coli KRX and purified. The obtained recombinant BluB/CobT2 was used in enzyme assays. Reactions performed with BluB/CobT2 included the formation of DMBI and its subsequent activation into α-ribazole-phosphate. We addressed these two predicted activities of BluB/CobT2 both separately and jointly.
Effect of NADH concentration and presence of NaMN on levels of DMBI and α-RP in reactions with the BluB/CobT2
FMN (100 µM)
FMN (100 µM) + NaMN (200 µM)
120 ± 6
1362 ± 12
4458 ± 200
0.5 ± 0.2
2301 ± 9
5095 ± 215
2 ± 0.5
BluB/CobT2 activates DMBI into α-RP in the presence of NaMN
CobT catalyzes the activation of the lower ligand base to form an α-ribosylated product. For the BluB/CobT2 fusion enzyme, the lower ligand base DMBI is provided directly by the BluB part to feed the activation into α-RP by the CobT2 part of the enzyme. After confirming that BluB/CobT2 produces DMBI from FMNH2, we prepared a two-step reaction containing the substrates necessary for the biosynthesis of DMBI and its subsequent activation into α-RP, namely 100 µM FMN, 20 or 40 mM NADH and 200 µM of nicotinic acid mononucleotide (NaMN). LC–MS/MS analysis of the reaction products revealed a peak with the retention time of 2.75 min at m/z 359.1 that corresponds to α-RP (Fig. 4b). An MS/MS experiment on the ion at m/z 359.1 showed a fragment of 147.3, which corresponds to DMBI (Additional file 1: Figure S1). DMBI was observed only in trace amounts, and this result was not affected by NADH concentration, suggesting efficient activation of the produced DMBI into α-RP by BluB/CobT2 (Table 1). It has been noted previously that benzimidazoles do not appear to be used for any other purposes than the lower ligands of cobamides . The fusion of BluB with CobT allows the efficient use of produced DMBI, which is not required for any other processes in the cell.
In general, the fusion of two activities in a single polypeptide may be beneficial for the enzyme activity because of the increased efficiency of substrate transfer, protection of the intermediates, facilitation of the interactions between domains and establishment of a fixed stoichiometric ratio of the enzymatic activities of sequential reactions . In our experiments the molar concentrations of α-RP obtained from reaction coupling synthesis and activation of DMBI were ~2–3-fold higher than the DMBI concentrations measured from the DMBI synthesis reaction alone (Table 1). This may indicate that the presence of NaMN stimulates the production of DMBI, possibly through imposing certain conformational changes on the BluB/CobT2 enzyme, resulting either in more efficient release of DMBI from the active site of the BluB part or an increase in BluB enzyme activity. The increased formation of α-ribazole-phosphate in the presence of NaMN relative to the formation of DMBI can be attributed to either of these mechanisms, leading to efficient usage of the scarce substrates. This issue could be addressed further through crystallography studies.
Effect of substrate and environmental pH on DMBI activation by BluB/CobT2
Formation of Ade-RP by BluB/CobT2
Cobamide production in vivo
Previous studies have revealed that the range of cobamides produced by a given organism in vivo does not always correlate to the range observed with CobT enzymes in vitro. Furthermore, these studies have shown that the cobamide requirements of the organism largely determine the variety it produces [17, 24]. An attempt to distinguish between cobalamin and pseudocobalamin production under aerobic and strictly anaerobic conditions was previously investigated in S. enterica, showing that the bacterium exclusively synthesizes pseudocobalamin under strictly anaerobic conditions and cobalamin under microaerobic conditions . This finding is consistent with the necessity of oxygen for the formation of DMBI in S. enterica , even though in that case the DMBI formation could have been non-enzymatic . Knowing that P. freudenreichii is an aerotolerant anaerobe and that oxygen is required for the synthesis of DMBI, we decided to test cobamide formation in vivo under both anaerobic and microaerobic conditions.
Finally, we observed no peak corresponding to pseudocobalamin in the samples from cells grown under conditions where both bases were supplemented, indicating a preference towards incorporating DMBI over adenine and thereby a preference for the production of the active vitamin over the pseudovitamin in vivo (Fig. 7). For S. meliloti, pseudocobalamin is not functionally equivalent to cobalamin, as observed from the poor growth of mutant strains producing only the pseudo form of the vitamin in guided biosynthesis . For P. freudenreichii, we observed that the cultures reached the same final cell densities under anaerobic conditions with only the pseudovitamin produced, suggesting that while pseudocobalamin is not the preferred corrinoid it may, under some conditions, be able to substitute for cobalamin.
The herein described fusion enzyme BluB/CobT2 is responsible for the production and activation of DMBI for attachment as a lower ligand for cobalamin. Fusing those two activities results in high pressure towards the production of true vitamin B12 by activating DMBI produced within the same enzymatic complex. Presence of NaMN was shown to stimulate the production of DMBI in vitro, suggesting a regulatory mechanism securing efficient usage of the scarce substrates. This efficiency, combined with the low ability to produce the pseudovitamin B12 form in vivo, suggests that P. freudenreichii may prefer the active vitamin form as a cofactor for its own use. Our results indicate that BluB/CobT2 is the crucial enzyme in the B12 biosynthetic pathway, responsible for the production of active vitamin over pseudovitamin. At present, P. freudenreichii is the only microorganism that has been both granted GRAS status and shown to produce the active form of vitamin B12, making it a unique candidate for the in situ microbial fortification of food products.
Strains and culture conditions
The P. freudenreichii DSM 4902 was grown on PPA medium composed of 5.0 g tryptone (Sigma-Aldrich), 10.0 g yeast extract (DIFCO Becton, Dickinson), 14.0 ml 60 % w/w DL-sodium lactate (Sigma-Aldrich) per litre with/without 1.5 % agar, pH 7.3. For liquid cultures, the pH of the PPA medium was adjusted to 6.7 prior to autoclaving. The strain was routinely grown at 30 °C in anaerobic jar on plates and under microaerobic condition in broth, unless stated otherwise.
Escherichia coli KRX clones carrying pFN18A-bluB/cobT2 constructs were routinely grown in LB medium  with 100 µg/ml ampicillin, at 37 or 25 °C, with 275 rpm shaking.
Overexpression and purification of BluB/CobT2
BluB/CobT2 was expressed in E. coli KRX cells using the Flexi® Vector pFN18A (Promega, Wisconsin, USA). Genomic DNA from P. freudenreichii was isolated as described previously . The bluB/cobT2 coding region (NC_014215.1) was PCR amplified using primer 1 and primer 2 with SgfI and PmeI restriction sites (Additional file 1: Table S2), and cloned as a SgfI-PmeI fragment in the pFN18A vector (Promega, Wisconsin, USA). E. coli KRX clones carrying pFN18A-bluB/cobT2 constructs were screened by PCR using primer 3 and primer 4. Plasmid from a selected clone was further verified by sequencing of the insert at the DNA sequencing laboratory (Institute of Biotechnology, University of Helsinki).
The heterologous expression and purification of the BluB/CobT2 enzyme were performed according to the HaloTag protein expression and purification system manuals (Promega), with 50 mM NaCl, 1 mM DTT, and 0.5 mM EDTA in the purification buffer and 2 mM ATP, 10 mM MgCl and 0.005 % IGEPAL CA-630 added for the binding step at 4 °C for 16 h. The HaloTag protein was removed with HaloTEV protease (Promega, Wisconsin, USA) by incubating the protein preparation for 12 h at 4 °C, followed by 90 min incubation at room temperature. The purity of the recombinant BluB/CobT2 protein was confirmed by SDS-PAGE, stained with PageBlue (Fermentas, Thermo Fisher Scientific, Delaware, USA) and the concentration determined using a NanoDrop 1000 (Thermo Fisher Scientific, Delaware, USA. The protein was stored in aliquots at −20 °C until used.
Similarity searches for BluB/CobT2 were performed using BLAST at the National Center for Biotechnology Information. Protein sequence alignments were performed with the program ClustalW via the ClustalW web service at the European Bioinformatics Institute .
BluB/CobT enzyme activity reactions and the LC–MS method
All the enzyme activity reactions were conducted in triplicate, using 5 µM of the BluB/CobT2 enzyme. The BluB reaction was performed with 1 mM DTT, 100 µM FMN, and 20 or 40 mM NADH in 90 mM Tris–HCl at pH 8.5. CobT activity with DMBI was tested with 1 mM DTT, 100 µM DMBI, and 200 µM NaMN or NMN in 90 mM Tris–HCl at pH 7.5 or 8.5. For the measurement of CobT activity with adenine, DMBI was replaced by adenine (100 µM) in the reaction, and NMN was omitted. The two-step BluB-CobT reaction was performed with 1 mM DTT, 100 µM FMN, 20 or 40 mM NADH, and 200 µM NaMN in 90 mM Tris–HCl at pH 7.5 or 8.5. Control reactions lacking the enzyme were also performed, and in the case of BluB activity, a reaction condition lacking NADH was also tested. For the reactions containing FMN, dark tubes were used. All the reactions were incubated at room temperature, protected from direct light. After the indicated incubation time, the reactions were stopped by the addition of 6.5 % TCA.
The LC–ESI–MS consisted of a Waters Acquity UPLC I class binary solvent manager, sample manager and column thermostat (maintained at 25 °C) and a Waters Xevo TQ-S triple quadrupole mass spectrometer (Waters, Milford, MA, USA). A Waters Acquity UPLC BEH C18 (2.1 × 50 mm, 1.7 μm) column was used at a flow rate of 0.3 mL/min. The mobile phase consisted of 0.1 % formic acid in water (A) and 0.1 % formic acid in methanol (B). The linear gradient elution for DMBI and α-RP was as follows: 0–6.0 min: 5 % B to 50 % B, 6.0–6.5 min: 50 % B to 100 % B, 6.5–8.0 min: 100 % B, 8.0–8.1 min: 100–5 % B, 8.1–10.0 min: 5 % B. The linear gradient elution for adenine and adenosine monophosphate was as follows: 0–1.7 min: 0.5 % B to 1 % B, 1.7–2.2 min: 1 % B to 5 % B, 2.2–2.4 min: 5 % B to 100 % B, 2.4–3.5 min: 100 % B, 3.5–3.6 min: 100–0.5 % B, 3.6–5.0 min: 0.5 % B. The injection volume was 1 μL. The mass spectrometer was operated in the positive electrospray ionization mode: capillary voltage was 0.5 kV, source offset 50 V and cone voltage 30 V. The source temperature was 150 °C, the desolvation temperature 350 °C and the desolvation gas flow was 800 L/h. The LC–MS analyses were conducted using selected reaction monitoring (SRM): ion transitions and collision energies were as follows: DMBI 147 → 132, 25 eV; α-RP 359 → 147, 20 eV; adenine 136 → 119, 20 eV; and adenosine monophosphate 348 → 136, 25 eV.
Stock solutions (5 mM) were prepared by dissolving the analytes in deionized water. Working standard solutions were prepared by diluting the stock solutions to the appropriate concentrations. The standard solutions were used to prepare a calibration curve with the following concentration levels: 0.1, 0.2, 1.0, 2.5, 5.0, 10, 50, 100, 500, 1000, 5000 and 7500 nM. DMBI and adenine were quantitated against authentic standard compounds. Due to the lack of standard compounds, α-RP and ade-RP were quantified using DMBI and adenine as reference standards.
Cobamide production in vivo and the UHPLC-UV/MS method
Fresh colonies of P. freudenreichii DSM 4902 were inoculated into 30 mL of PPA broth supplemented with 5 µg/mL of cobalt chloride and either DMBI (100 µM) or adenine (100 µM), or a combination of both DMBI and adenine. Additionally, control cultures with neither adenine nor DMBI were simultaneously inoculated. The cultures were incubated statically under normal atmosphere (microaerobic condition) or in an anaerobic chamber (Don Whitley Scientific, West Yorkshire, UK) for 7 days. For each condition, three biological replicate cultures were used. Cells were harvested by centrifugation, and the cobamides were analyzed as previously described . Briefly, the cobamides were extracted, in their cyano form, from bacterial pellets by boiling with 10 mL of extraction buffer (pH 4.5) containing 100 µL of 1 % NaCN. After purification through immunoaffinity columns (Easy Extract; R-Biopharma, Glasgow, Scotland), the extracts were analyzed by UHPLC on a Waters UPLC system (Waters, Milford, MA, USA) with separation on a Waters Acquity HSS T3 C18 column (2.1 × 100 mm, 1.8 µm) at a flow rate of 0.32 mL/min and with UV detection by a photo diode array (PDA) detector at 361 nm. The mobile phase was a gradient flow of 0.025 % trifluoroacetic acid in water and 0.025 % trifluoroacetic acid in acetonitrile . The injection volume was 10 μL. Cobamides were identified with their retention times and their absorption spectra, and quantified using a calibration curve prepared with a set of cyanocobalamin standards with concentrations ranging from 0.015‒1.5 ng/µL. The flowthrough from the immunoaffinity purification was also analyzed with UHPLC and MS for the presence of cobamides and cobinamide (See Additional file 1: Figure S4).
To confirm the identity of the cobamides and cobinamide, the extracts were analyzed using a high resolution quadrupole time-of-flight mass spectrometer (QTOF, Synapt G2-Si; Waters, Milford, MA, USA) with an electrospray ionization interface to the UHPLC system. The mobile phase for MS contained 0.1 % formic acid instead of 0.025 % trifluoroacetic acid. The mass spectrometer was operated in positive electrospray ionization mode with a scanning range set for ions with m/z of 50–1500. Parent ions were collected and fragmented (MS/MS) using argon as a collision gas. The capillary voltage was 0.5 kV, the sampling cone voltage 40 V and the source offset 80 V. The source temperature was 150 °C, desolvation temperature 600 °C, desolvation gas flow 1000 L/h, nebulizer gas flow 6.5 bar, and cone gas flow 50 L/h. The trap collision energy ramp was 15‒90 eV, trap gas flow 2 mL/min and scan time 0.2 s. A lock-spray mass correction standard (leucine-enkephalin; m/z 556.2771) was introduced every 10 s.
VP, PV, KS and PD conceived the study. RK provided LC–MS/MS setup. PK developed the LC–MS/MS method and analyzed activity reactions. BC analyzed cobamides in vivo. LH performed bioinformatic analysis of the CobT homologues. PD performed the microbial experiments and database searches. PD, PV, KS, PK, BC, LH wrote the manuscript. All authors read and approved the final manuscript.
This study was supported by the Academy of Finland (Grant No 257333). Pekka Oivanen and Sanna Laaksonen are thanked for the help with operating of the anaerobic chamber.
The authors declare that they have no competing interests.
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