Department of Bacteriology, University of Wisconsin, 1710 University Avenue, Madison, WI 53726-4087, USA
Correspondence
Jorge C. Escalante-Semerena
escalante{at}bact.wisc.edu
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ABSTRACT |
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INTRODUCTION |
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The CobT reaction yields -ribazole-5'-P when the enzyme transfers the phosphoribosyl moiety of NaMN to DMB (Trzebiatowski & Escalante-Semerena, 1997
). Data reported here show that CobT can transfer the ADPribosyl moiety of NAD+ to DMB to yield
-5,6-dimethylbenzimidazole adenine dinucleotide (hereafter referred to as
-DAD) (Fig. 2
). A recent report showed that the Escherichia coli CobB protein used NAD+ as substrate to derivatize DMB. The same report showed that the same activity was associated with the CobT protein from this bacterium (Frye, 1999
). The identity of the products of these reactions was not established.
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METHODS |
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Nucleotide pyrophosphatase assays.
A 50 µl reaction mixture containing 100 mM MOPS buffer pH 7, 2·5 mM MgCl2, 0·05 U snake venom nucleotide pyrophosphatase (Sigma) and purified -DAD (200 µM) was incubated for 4 h at 37 °C. A reaction mixture without enzyme was used as negative control. Reaction mixtures were analysed by ion-exchange HPLC (see below).
In vitro NLA assays.
NLA assays were performed at the 500 µl scale as described (Maggio-Hall & Escalante-Semerena, 1999) except that cell-free extracts were added simultaneously instead of sequentially. Where indicated,
-DAD (120 µM) substituted for NaMN and DMB in the reaction mixture. Plasmids pNLA1 (cobUST+; Maggio-Hall & Escalante-Semerena, 1999
), pJO46 (cobC+; O'Toole et al., 1994
) or pT7-5 (expression vector; Tabor, 1990
) were introduced into strain JE6200 [metE205 ara-9
299(hisGcobT) cobC1175 : : Tn10
16
17 pnuE : : MudQ]. Strain JE6200 was constructed by P22-mediated transduction of the pnuE : : MudQ allele from strain SF456 (pnuE : : MudQ) into strain JE2197 [metE205 ara-9
299(hisGcobT) cobC1175 : : Tn10d(Tc)] using previously described methods (Chan et al., 1972
; Davis et al., 1980
). Reactions (final vol. 500 µl) were stopped by the addition of 100 µl 100 mM KCN followed by heating at 80 °C for 10 min to convert adenosylcorrinoids to cyanocorrinoids. Reaction mixtures were passed over C18 SepPak columns (Waters), vacuumed to dryness in a SpeedVac concentrator (Savant Instruments), resuspended in 200 µl double-distilled water and analysed by reverse-phase HPLC (RP-HPLC) (see below).
Synthesis and purification of [2-14C]DMB.
Radiolabelled [2-14C]DMB was synthesized as described (Trzebiatowski & Escalante-Semerena, 1997).
Purification of NAD+.
NAD+ was resolved from ADPribose and NMN using a fast-flow DEAE anion-exchange resin (2·5x40 cm; Toyopearl; Rohm & Haas) previously equilibrated with 10 mM NaCl at a flow rate of 250 ml h-1; a linear gradient from 10300 mM NaCl resolved NAD+ from the above-mentioned contaminants (Dickinson & Engel, 1977). Fractions containing NAD+ were identified by UV-visible spectroscopy, were concentrated under vacuum and applied onto a reverse-phase C18 HPLC column to desalt NAD+ (see below). The concentration of the purified product was determined using the molar extinction coefficient at 260 nm (
260) of 17 600 M-1 (Dalziel & Dickinson, 1966
).
Synthesis and purification of -DAD.
A 5 ml CobT reaction mixture (pH 10) containing NaAD and 55 µg CobT protein was used to isolate microgram amounts of -DAD. The incubation time was extended to 3 h, and
-DAD was isolated from the mixture by RP-HPLC followed by ion-exchange HPLC (see below);
-DAD was desalted by RP-HPLC and dried under vacuum.
Chromatographic techniques
TLC.
Reagents and products of the CobT reaction were resolved using TLC as described (Maggio-Hall & Escalante-Semerena, 1999).
HPLC.
-DAD was isolated using a previously described RP-HPLC protocol (Maggio-Hall & Escalante-Semerena, 1999
). This procedure was also used to desalt
-DAD (retention time, 12·5 min) and NAD+ (retention time, 10 min). Ion-exchange HPLC was performed on a Spheroclone SAX column (4·6x250 mm; Phenomenex). The mobile phase was a 26 min gradient of potassium phosphate, pH 5·5 (40500 mM). The column was developed at a flow rate of 1 ml min-1. Under these conditions the retention time for
-DAD was 21 min. Cyanocorrinoids were resolved using a previously published RP-HPLC protocol (Blanche et al., 1990
; O'Toole et al., 1993
). Samples (200 µl) were injected onto a Prodigy C18 column (Phenomenex) equilibrated with a mobile phase containing 98 % solvent A (100 mM potassium phosphate buffer pH 8, containing 10 mM KCN), 1 % solvent B (100 mM potassium phosphate buffer pH 6·8, containing 10 mM KCN) and 1 % solvent C (acetonitrile). The column was developed with a 45 min linear gradient that changed the composition of the mobile phase to a 1 : 1 ratio of solvent B : solvent C. The column was equilibrated and developed at a rate of 1 ml min-1.
Mass spectrometry.
-DAD and cyanocobalamin isolated from NLA reactions were subjected to electrospray ionization mass spectrometry (ESIMS) analysis at the Biotechnology Center at the University of Wisconsin-Madison.
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RESULTS |
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DISCUSSION |
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The ability of the S. typhimurium CobT enzyme to use NAD+ and its precursors NaMN, NMN and NaAD as substrates provides flexibility to the synthesis of AdoCbl under diverse physiological conditions
As mentioned above, the NAD+ concentration in exponential-phase cells of S. typhimurium is several orders of magnitude higher than any of the above-mentioned NAD+ precursors (790 µM vs undetectable) (Bochner & Ames, 1982). This difference in the levels of NAD+ and its above-mentioned precursors would be enough to compensate for the 18-fold higher Km of CobT for NAD+ than NaMN (0·51 mM) under physiological conditions of active growth. However, because the concentrations of NAD+ precursors under other physiological conditions have not been measured, it is possible that the concentration of these precursors may rise under specific growth conditions. The ability of the CobT enzyme to use NaMN, NMN, NaAD or NAD+ as substrates would allow S. typhimurium to synthesize
-ribazole-5'-P for the assembly of AdoCbl under any growth conditions. We propose that both enzymic activities of the S. typhimurium CobT enzyme (phosphoribosyltransferase, ADPribosyltransferase) are physiologically relevant.
If NAD+ is a physiological substrate for CobT in vivo, why does the CobT enzyme of S. typhimurium have a lower Km for NaMN?
The answer to this question may lie in the origin of the cob operon. It has been postulated that the entire cob operon (including cobT) was inherited by S. typhimurium from an unknown donor (Lawrence & Roth, 1996). It is reasonable to speculate that the extant CobT enzyme might have evolved in a prokaryote whose intracellular level of NaMN was substantially higher than that found in S. typhimurium. Support for this idea can be found in studies of the CobT homologue of Propionibacterium fruendenreichii subsp. shermanii, which was reported to be unable to use NAD+ as substrate (Friedmann, 1965
). The ability of the S. typhimurium CobT enzyme to use other NAD+ precursors such as NMN as substrate is not shared by all CobT homologues, as reported for the CobT enzyme activity of Clostridium sticklandii which failed to use NMN as substrate (Fyfe & Friedmann, 1969
). The ability of the S. typhimurium CobT enzyme to use NAD+ or its mononucleotide and dinucleotide precursors may be interpreted to mean that the physiological levels of these precursors may vary in this bacterium. Low levels of NAD+ precursors may be the selective pressure for the evolution of CobT enzymes able to use NAD+ as substrate.
Supporting in vivo evidence that NAD+ is a substrate for the CobT enzyme in vivo
The ADPribosyltransferase activity of CobT is not likely to be an in vitro artefact. Recall that the CobB enzyme uses NAD+ as substrate, that CobB has NAD+-dependent ADPribosyltransferase activity, that AdoCbl biosynthesis in cobT mutant strains of S. typhimurium is restored by the addition of DMB to the medium and that the response of cobT mutants to DMB depends on a functional CobB enzyme. Together, these facts strongly suggest that CobB transfers the ADPribose moiety of NAD+ to DMB in vivo, resulting in the synthesis of -DAD, effectively compensating for the lack of CobT enzyme (Fig. 6
). It is important, however, to keep in mind that the CobB enzyme is not part of the AdoCbl biosynthetic pathway; instead CobB function is critical for the post-translational regulation of acyl-coenzyme A synthetase activities and probably of other members of the AMP-forming family of enzymes (Starai et al., 2002
, 2003
). Under growth conditions that require low levels of AdoCbl (e.g. methionine synthesis), the ADPribosyltransferase activity of CobB fully compensates for the lack of CobT (Trzebiatowski et al., 1994
). However, under physiological conditions that require a higher level of the coenzyme (e.g. growth on ethanolamine or 1,2-propanediol as carbon and energy source), CobB does not compensate for the lack of CobT, suggesting that the synthesis of
-DAD by CobB is an inefficient side reaction of this enzyme with limited physiological significance to the cell (J. C. Escalante-Semerena, unpublished results).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 4 October 2002;
revised 23 December 2002;
accepted 6 January 2003.
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