Reassessment of the late steps of coenzyme B12 synthesis in Salmonella enterica: evidence that dephosphorylation of adenosylcobalamin-5'-phosphate by the CobC phosphatase is the last step of the pathway.

De novo synthesis of coenzyme B12 (adenosylcobalamin [AdoCbl]) is a process unique to prokaryotes that requires at least 25 gene functions (4, 8, 9, 26, 43, 44, 47, 51, 57, 59-61). Salmonella enterica synthesizes coenzyme B12 de novo under anaerobic conditions but salvages incomplete corrinoids (e.g., cobinamide [Cbi], cobyric acid [Cby]) from its environment under aerobic and anaerobic conditions (30, 61). Cbi and Cby are transported into the cell via the corrinoid-specific transport system comprised of the outer membrane protein BtuB (28), the periplasmic corrinoid shuttle protein BtuF (11, 55), and the inner membrane proteins BtuCD (6, 20, 42). Once inside the cell, Cbi and Cby are adenosylated by the ATP:co(I)rrinoid adenosyltransferase (CobA, EC 2.5.1.17) (10, 24, 48) enzyme, yielding AdoCbi or AdoCby, which enter the biosynthetic pathway at different points (Fig. ​(Fig.11). FIG. 1. Late steps in corrin ring biosynthesis in serovar Typhimurium. When this work was initiated, this was the model of the enzymatic reactions needed for the last step in de novo corrin ring synthesis and nucleotide loop assembly in serovar Typhimurium. Shown ... Adenosylcobinamide (AdoCbi) is converted to AdoCbi-GDP in two steps catalyzed by the CobU enzyme (NTP:AdoCbi kinase, EC 2.7.7.62; GTP:AdoCbi-P guanylyltransferase, EC 2.7.1.156). AdoCby follows a different route. It is converted by the AdoCbi-P synthase (CbiB) enzyme to AdoCbi-P, which is converted by CobU to AdoCbi-GDP. A second set of reactions activates the lower ligand base, which in S. enterica serovar Typhimurium can be either 5,6-dimethylbenzimidazole (DMB) under aerobic conditions (31, 32) or adenine or 2-methyladenine under anaerobic conditions (33). In our current understanding of these reactions, the first step in lower ligand activation is catalyzed by a unique phosphoribosyltransferase enzyme (CobT in serovar Typhimurium; EC 2.7.8.26) (16-18, 52, 53), while the second step is thought to be catalyzed by a phosphatase enzyme (CobC in serovar Typhimurium; EC 3.1.3.73) (40) (Fig. ​(Fig.1).1). The CobT enzyme can use NaMN or NAD+ as substrate. When NAD+ and DMB are the substrates, the product of the CobT reaction is α-5,6-dimethylbenzimidazole adenine dinucleotide (α-DAD) (34), which is postulated to be cleaved by an uncharacterized hydrolase (Fig. ​(Fig.1).1). When DMB and NaMN are the substrates, the product of the CobT reaction is α-DMB-ribose-5′-phosphate (α-ribazole-P [α-RP]) (52), which is dephosphorylated to α-ribazole (α-R) by the phosphatase activity of CobC (40). The CobC enzyme was previously reported to also dephosphorylate AdoCbl-P in vitro, but the physiological meaning of this activity remained unclear (35). According to our current understanding of the late steps of AdoCbl biosynthesis in serovar Typhimurium, the AdoCbl-5′-P synthase (CobS) enzyme is an integral membrane protein that catalyzes the condensation of AdoCbi-GDP and α-R to yield AdoCbl (Fig. ​(Fig.1)1) (35). However, the idea that α-R was the physiological substrate for CobS was first brought into question by our previous in vitro studies of the CobS enzyme, which showed that α-RP was also a substrate for the enzyme (35). In addition, earlier work with Pseudomonas denitrificans indicated that CobV (the CobS homolog in P. denitrificans) had higher affinity for α-RP than for α-R (Kmα-RP, 2.7 μM; Kmα-R, 7.8 μM) (13), suggesting that α-RP might be the preferred substrate for the enzyme in both bacteria. This idea was also consistent with results from earlier in vitro work with Propionibacterium shermanii, which suggested that Cbl-5′-phosphate was the last intermediate of the pathway (25). However, all of the above-mentioned studies were performed in vitro, and in vivo data supporting the conclusions have not been reported. We took genetic and biochemical approaches to further investigating the timing of phosphate removal in serovar Typhimurium during the assembly of the nucleotide loop. This analysis was aided by the uncovering of a new phenotype of serovar Typhimurium cobC strains, which we argue provides the first in vivo support for the preference of the AdoCbl-5′-P synthase (CobS) enzyme for α-RP. We conclude that α-RP is the physiological substrate for CobS, that CobC catalyzes the last step of the AdoCbl biosynthetic pathway, and that removal of the 5′-O-P from AdoCbl-5′-P by CobC is the last step of the pathway.