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. 2001 Mar;183(5):1577-84.
doi: 10.1128/JB.183.5.1577-1584.2001.

Functional genomic, biochemical, and genetic characterization of the Salmonella pduO gene, an ATP:cob(I)alamin adenosyltransferase gene

C L Johnson  1 , E Pechonick, S D Park, G D Havemann, N A Leal, T A Bobik
Affiliations

Functional genomic, biochemical, and genetic characterization of the Salmonella pduO gene, an ATP:cob(I)alamin adenosyltransferase gene

C L Johnson et al. J Bacteriol. 2001 Mar.

Abstract

Salmonella enterica degrades 1,2-propanediol by a pathway dependent on coenzyme B12 (adenosylcobalamin [AdoCb1]) VSports手机版. Previous studies showed that 1,2-propanediol utilization (pdu) genes include those for the conversion of inactive cobalamins, such as vitamin B12, to AdoCbl. However, the specific genes involved were not identified. Here we show that the pduO gene encodes a protein with ATP:cob(I)alamin adenosyltransferase activity. The main role of this protein is apparently the conversion of inactive cobalamins to AdoCbl for 1,2-propanediol degradation. Genetic tests showed that the function of the pduO gene was partially replaced by the cobA gene (a known ATP:corrinoid adenosyltransferase) but that optimal growth of S. enterica on 1,2-propanediol required a functional pduO gene. Growth studies showed that cobA pduO double mutants were unable to grow on 1,2-propanediol minimal medium supplemented with vitamin B(12) but were capable of growth on similar medium supplemented with AdoCbl. The pduO gene was cloned into a T7 expression vector. The PduO protein was overexpressed, partially purified, and, using an improved assay procedure, shown to have cob(I)alamin adenosyltransferase activity. Analysis of the genomic context of genes encoding PduO and related proteins indicated that particular adenosyltransferases tend to be specialized for particular AdoCbl-dependent enzymes or for the de novo synthesis of AdoCbl. Such analyses also indicated that PduO is a bifunctional enzyme. The possibility that genes of unknown function proximal to adenosyltransferase homologues represent previously unidentified AdoCbl-dependent enzymes is discussed. .

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Figures

FIG. 1
FIG. 1
Proposed vitamin B12 adenosylation pathway. Coenzyme B12 (a required cofactor for a number of enzymes) is thought to be produced from vitamin B12 by the series of reactions shown. The corrin ring of vitamin B12 is shown lightly shaded, the nucleotide loop is darkly shaded, and the upper ligand of the cobalt is indicated as follows: CN, cyano group; HO, hydroxy group; Ado, 5′-deoxyadenosyl group.
FIG. 2
FIG. 2
Effects of pduO and cobA mutations on growth of S. enterica on 1,2-propanediol–CNCbl minimal medium. ■, Wild type (S. enterica serovar Typhimurium LT2); ▵, cobA; ○, pduO; ◊, pduO cobA; □, pduO cobA/pDP1; ●, LT2/pDP1. The strains used were BE83, BE111, BE121, and BE114. The cells were cultured as described in Materials and Methods. OD600, optical density at 600 nm.
FIG. 3
FIG. 3
Growth of S. enterica strain BE121 (pduO cobA) on 1,2-propanediol minimal medium supplemented with either AdoCbl or CNCbl. □, 2 μg of CNCbl/ml; ■, 0.2 g of AdoCbl/ml; ◊, 2 μg of AdoCbl/ml; ○, 20 μg of AdoCbl/ml. Strain BE121 did not grow with CNCbl at concentrations from 0.2 to 20 μg/ml (not shown). The cells were cultured as described in Materials and Methods. OD600, optical density at 600 nm.
FIG. 4
FIG. 4
SDS-PAGE analysis of pduO expression strains. Lane 1, molecular mass markers; lanes 2 and 3, soluble extracts of strain BE119 (T7 expression vector without insert) and strain BE118 (T7 expression vector with pduO insert), respectively; lanes 4 and 5, inclusion body extracts from strains BE119 and BE118, respectively. The 37-kDa protein band is indicated.
FIG. 5
FIG. 5
Absorption spectra of adenosyltransferase assay mixtures. The spectra indicate the predominant corrinoid present in the assay mixture: prior to the addition of titanium(III) citrate, the spectrum is that of HOCbl (–·–·); after the addition of titanium(III) citrate, the spectrum is that of cob(I)alamin (…………); 10 min after the addition of an inclusion body preparation of the PduO enzyme, the spectrum is that of AdoCbl (–––); after photolysis, the spectrum is that of cob(I)alamin (——).
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