PduL is an evolutionarily distinct phosphotransacylase involved in B12-dependent 1,2-propanediol degradation by Salmonella enterica serovar typhimurium LT2.

Salmonella enterica degrades 1,2-propanediol (1,2-PD) in a coenzyme B(12)-dependent manner. Previous enzymatic assays of crude cell extracts indicated that a phosphotransacylase (PTAC) was needed for this process, but the enzyme involved was not identified. Here, we show that the pduL gene encodes an evolutionarily distinct PTAC used for 1,2-PD degradation. Growth tests showed that pduL mutants were unable to ferment 1,2-PD and were also impaired for aerobic growth on this compound. Enzyme assays showed that cell extracts from a pduL mutant lacked measurable PTAC activity in a background that also carried a pta mutation (the pta gene was previously shown to encode a PTAC enzyme). Ectopic expression of pduL corrected the growth defects of a pta mutant. PduL fused to eight C-terminal histidine residues (PduL-His(8)) was purified, and its kinetic constants were determined: the V(max) was 51.7 +/- 7.6 micromol min(-1) mg(-1), and the K(m) values for propionyl-PO(4)(2-) and acetyl-PO(4)(2-) were 0.61 and 0.97 mM, respectively. Sequence analyses showed that PduL is unrelated in amino acid sequence to known PTAC enzymes and that PduL homologues are distributed among at least 49 bacterial species but are absent from the Archaea and Eukarya.

Biochemical evidence that the pduS gene encodes a bifunctional cobalamin reductase.

Salmonella enterica degrades 1,2-propanediol (1,2-PD) by a pathway that requires coenzyme B(12) (adenosylcobalamin; AdoCbl). The genes specifically involved in 1,2-PD utilization (pdu) are found in a large contiguous cluster, the pdu locus. Earlier studies have indicated that this locus includes genes for the conversion of vitamin B(12) (cyanocobalamin; CNCbl) to AdoCbl and that the pduO gene encodes an ATP : cob(I)alamin adenosyltransferase which catalyses the terminal step of this process. Here, in vitro evidence is presented that the pduS 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 Escherichia coli. 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(-1) mg(-1) and cob(II)alamin to cob(I)alamin at a rate of 7.8 nmol min(-1) mg(-1). In contrast, control extracts had only 9.9 nmol min(-1) mg(-1) 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.

Purification and initial characterization of the Salmonella enterica PduO ATP:Cob(I)alamin adenosyltransferase.

The PduO enzyme of Salmonella enterica is an ATP:cob(I)alamin adenosyltransferase that catalyzes the final step in the conversion of vitamin B(12) to coenzyme B(12). The primary physiological role of this enzyme is to support coenzyme B(12)-dependent 1,2-propanediol degradation, and bioinformatic analysis has indicated that it has two domains. Here the PduO adenosyltransferase was produced in Escherichia coli, solubilized from inclusion bodies, purified to apparent homogeneity, and partially characterized biochemically. The K(m) values of PduO for ATP and cob(I)alamin were 19.8 and 4.5 microM, respectively, and the enzyme V(max) was 243 nmol min(-1) mg of protein(-1). Further investigations showed that PduO was active with ATP and partially active with deoxy-ATP, but lacked measurable activity with other nucleotides. (31)P nuclear magnetic resonance established that triphosphate was a product of the PduO reaction, and kinetic studies indicated a ternary complex mechanism. A series of truncated versions of the PduO protein were produced in Escherichia coli, partially purified, and used to show that adenosyltransferase activity is associated with the N-terminal domain. The N-terminal domain was purified to near homogeneity and shown to have biochemical properties and kinetic constants similar to those of the full-length enzyme. This indicated that the C-terminal domain was not directly involved in catalysis or substrate binding and may have another role.