Biochemical evidence that the pduS gene encodes a bifunctional cobalamin reductase

Abstract

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. INTRODUCTIONSalmonella enterica degrades 1,2-propanediol (1,2-PD) in a coenzyme B 12 (adenosylcobalamin; AdoCbl)-dependent fashion (Jeter, 1990). 1,2-PD is a major product of the fermentation of the common plant sugars rhamnose and fucose and is thought to be an important carbon source in anaerobic environments such as the large intestines of higher animals (Obradors et al., 1988;Toraya et al., 1980). Virtually all natural isolates of Salmonella degrade 1,2-PD and in vivo studies with mice have suggested that the degradation of this small molecule may be important for the interaction of S. enterica with its host organisms (Conner et al., 1998;Heithoff et al., 1999).The pathway of 1,2-PD degradation begins with the conversion of 1,2-PD to propionaldehyde by AdoCbldependent diol dehydratase (Bobik et al., 1997;Obradors et al., 1988;Toraya et al., 1979). Subsequently, propionaldehyde is converted to propanol and propionate by alcohol dehydrogenase, CoA-dependent propionaldehyde dehydrogenase, phosphotransacylase and propionate kinase (O...

Identification, Characterization, and Structure/Function Analysis of a Corrin Reductase Involved in Adenosylcobalamin Biosynthesis

“…First, the enzyme is related in both sequence and physical characteristics to the enzyme previously reported by the Rhone Poulenc group (6). Moreover, there is sequence similarity between CobR and a cobalamin reductase (PduS) that has recently been described as part of the propanediol utilization machinery (20). Second, CobR exhibits both co(III)rrinoid and co(II)rrinoid reductase activities and displays a wide substrate specificity, from cobyrinic acid to cobalamin.…”

“…S1) (18,19). Some sequence similarity was also observed with PduS (data not shown), a cobalamin reductase that is required for the activation of the diol dehydratase as part of the propanediol utilization metabolosome (20). To investigate the activity of CobR, the gene was amplified by PCR from the genomic DNA of B. melitensis and cloned into the vector pET-14b to allow the encoded protein to be produced recombinantly with an N-terminal His tag.…”

Phylogeny and physiology of candidate phylum ‘Atribacteria’ (OP9/JS1) inferred from cultivation-independent genomics

“…Three of the genes in these BMC loci encode homologues of PduP (coenzyme A-acylating aldehyde dehydrogenase), PduL (phosphotransacetylase) and PduS (RnfC/quinone oxidoreductase (Nqo)-like NADH dehydrogenase), which are involved in BMC-mediated 1,2-propanediol catabolism in Salmonella spp. (Sampson et al, 2005;Parsons et al, 2010). The presence of PduPL suggests that the putative 'Atribacteria' BMC may sequester metabolically generated, toxic aldehydes as has been proposed for catabolic BMCs in other organisms (Sampson and Bobik, 2008).…”

Diverse Bacterial Microcompartment Organelles

“…Sampson et al first reported that the PduS enzyme of Salmonella is a B 12 reductase that catalyzes the reduction of OH-B 12 to cob(I)alamin in two one-electron steps (80,106,107) (Fig. 7).…”

“…Subsequent studies determined that 4 additional proteins (PduMNQS) are tightly associated with the Pdu MCP and that the PduV protein might be associated with the outer surface of the shell (48,58,79,80,84). Of the 19 polypeptides that are components of the Pdu MCP, 8 (PduABB=JKNTU) are likely shell components, PduM and PduV are of uncertain function, and 9 polypeptides are subunits of six enzymes: B 12 -dependent diol dehydratase (DDH) (PduCDE), DDH reactivase (PduGH), propionaldehyde dehydrogenase (PduP), 1-propanol dehydrogenase (PduQ), cobalamin reductase (PduS), and adenosyltransferase (ATR) (PduO) (6,28,79,80,83,89,(104)(105)(106)(107). DDH, propionaldehyde dehydrogenase, and 1-propanol dehy-…”