1887

Abstract

degrades 1,2-propanediol (1,2-PD) by a pathway that requires coenzyme B (adenosylcobalamin; AdoCbl). The genes specifically involved in 1,2-PD utilization () are found in a large contiguous cluster, the locus. Earlier studies have indicated that this locus includes genes for the conversion of vitamin B (cyanocobalamin; CNCbl) to AdoCbl and that the gene encodes an ATP : cob(I)alamin adenosyltransferase which catalyses the terminal step of this process. Here, evidence is presented that the gene encodes a bifunctional cobalamin reductase that catalyses two reductive steps needed for the conversion of CNCbl into AdoCbl. The PduS enzyme was produced in high levels in . Enzyme assays showed that cell extracts from the PduS expression strain reduced cob(III)alamin (hydroxycobalamin) to cob(II)alamin at a rate of 91 nmol min mg and cob(II)alamin to cob(I)alamin at a rate of 7·8 nmol min mg. In contrast, control extracts had only 9·9 nmol min mg cob(III)alamin reductase activity and no detectable cob(II)alamin reductase activity. Thus, these results indicated that the PduS enzyme is a bifunctional cobalamin reductase. Enzyme assays also showed that the PduS enzyme reduced cob(II)alamin to cob(I)alamin for conversion into AdoCbl by purified PduO adenosyltransferase. Moreover, studies in which iodoacetate was used as a chemical trap for cob(I)alamin indicated that the PduS and PduO enzymes physically interact and that cob(I)alamin is sequestered during the conversion of cob(II)alamin to AdoCbl by these two enzymes. This is likely to be important physiologically, since cob(I)alamin is extremely reactive and would need to be protected from unproductive by-reactions. Lastly, bioinformatic analyses showed that the PduS enzyme is unrelated in amino acid sequence to enzymes of known function currently present in GenBank. Hence, results indicate that the PduS enzyme represents a new class of cobalamin reductase.

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2005-04-01
2019-08-19
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