This review examines deoxyadenosylcobalamin (Ado-B12) biosynthesis, transport, use, and uneven distribution among living forms. We describe how genetic analysis of enteric bacteria has contributed to these issues. Two pathways for corrin ring formation have been found-an aerobic pathway (in P. denitrificans) and an anaerobic pathway (in P. shermanii and S. typhimurium)-that differ in the point of cobalt insertion. Analysis of B12 transport in E. coli reveals two systems: one (with two proteins) for the outer membrane, and one (with three proteins) for the inner membrane. To account for the uneven distribution of B12 in living forms, we suggest that the B12 synthetic pathway may have evolved to allow anaerobic fermentation of small molecules in the absence of an external electron acceptor. Later, evolution of the pathway produced siroheme, (allowing use of inorganic electron acceptors), chlorophyll (O2 production), and heme (aerobic respiration). As oxygen became a larger part of the atmosphere, many organisms lost fermentative functions and retained dependence on newer, B12 functions that did not involve fermentation. Paradoxically, Salmonella spp. synthesize B12 only anaerobically but can use B12 (for degradation of ethanolamine and propanediol) only with oxygen. Genetic analysis of the operons for these degradative functions indicate that anaerobic degradation is important. Recent results suggest that B12 can be synthesized and used during anaerobic respiration using tetrathionate (but not nitrate or fumarate) as an electron acceptor. The branch of enteric taxa from which Salmonella spp. and E. coli evolved appears to have lost the ability to synthesize B12 and the ability to use it in propanediol and glycerol degradation. Salmonella spp., but not E. coli, have acquired by horizontal transfer the ability to synthesize B12 and degrade propanediol. The acquired ability to degrade propanediol provides the selective force that maintains B12 synthesis in this group.
Hundreds of bacterial species produce proteinaceous microcompartments (MCPs) that act as simple organelles by confining the enzymes of metabolic pathways that have toxic or volatile intermediates. A fundamental unanswered question about bacterial MCPs is how enzymes are packaged within the protein shell that forms their outer surface. Here, we report that a short N-terminal peptide is necessary and sufficient for packaging enzymes into the lumen of an MCP involved in B 12 -dependent 1,2-propanediol utilization (Pdu MCP). Deletion of 10 or 14 amino acids from the N terminus of the propionaldehyde dehydrogenase (PduP) enzyme, which is normally found within the Pdu MCP, substantially impaired packaging, with minimal effects on its enzymatic activity. Fusion of the 18 N-terminal amino acids from PduP to GFP, GST, or maltose-binding protein resulted in their encapsulation within MCPs. Bioinformatic analyses revealed N-terminal extensions in two additional Pdu proteins and three proteins from two unrelated MCPs, suggesting that N-terminal peptides may be used to package proteins into diverse MCPs. The potential uses of MCP assembly principles in nature and in biotechnology are discussed.
Salmonella enterica forms polyhedral organelles during coenzyme B 12 -dependent growth on 1,2-propanediol (1,2-PD). Previously, these organelles were shown to consist of a protein shell partly composed of the PduA protein, the majority of the cell's B 12 -dependent diol dehydratase, and additional unidentified proteins. In this report, the polyhedral organelles involved in B 12 -dependent 1,2-PD degradation by S. enterica were purified by a combination of detergent extraction and differential and density gradient centrifugation. The course of the purification was monitored by electron microscopy and gel electrophoresis, as well as enzymatic assay of B 12 -dependent diol dehydratase. Following one-and two-dimensional gel electrophoresis of purified organelles, the identities and relative abundance of their constituent proteins were determined by N-terminal sequencing, protein mass fingerprinting, Western blotting, and densitometry. These analyses indicated that the organelles consisted of at least 15 proteins, including PduABB CDEGHJKOPTU and one unidentified protein. Seven of the proteins identified (PduABB JKTU) have some sequence similarity to the shell proteins of carboxysomes (a polyhedral organelle involved in autotrophic CO 2 fixation), suggesting that the S. enterica organelles and carboxysomes have a related multiprotein shell. In addition, S. enterica organelles contained four enzymes: B 12 -dependent diol dehydratase, its putative reactivating factor, aldehyde dehydrogenase, and ATP cob(I) alamin adenosyltransferase. This complement of enzymes indicates that the primary catalytic function of the S. enterica organelles is the conversion of 1,2-PD to propionyl coenzyme A (which is consistent with our prior proposal that the S. enterica organelles function to minimize aldehyde toxicity during growth on 1,2-PD). The possibility that similar protein-bound organelles may be more widespread in nature than currently recognized is discussed.The vitamin B 12 coenzymes adenosyl-B 12 (Ado-B 12 ) and methyl-B 12 (CH 3 -B 12 ) are required cofactors for at least 15 different enzymes (5, 27, 30). These enzymes have a broad but uneven distribution among living forms and are vital to human health, are essential to the carbon cycle, and have important industrial applications (5, 27, 30). Historically, bacteria have provided excellent model systems for the study of vitamins, and recent investigations with several bacterial systems have found the molecular biology of B 12 -dependent processes to be unexpectedly complex (9,27,29,34,35). One of the most surprising findings in this area has been the identification of a polyhedral organelle involved in coenzyme B 12 -dependent 1,2-propanediol (1,2-PD) degradation by Salmonella enterica (9).Salmonella enterica utilizes 1,2-PD as a carbon and energy source in an Ado-B 12 -dependent fashion (19). Degradation occurs aerobically, or anaerobically if tetrathionate is added as a terminal electron acceptor (26). Based on biochemical studies, a pathway for 1,2-PD degradation has been ...
Salmonella enterica grows on 1,2-propanediol (1,2-PD) in a coenzyme B 12 -dependent fashion. Prior studies showed that a bacterial microcompartment (MCP) is involved in this process and that an MCP-minus mutant undergoes a 20-h period of growth arrest during 1,2-PD degradation. It was previously proposed that growth arrest resulted from propionaldehyde toxicity, but no direct evidence was presented. Here, high-pressure liquid chromatography analyses of culture medium were used to show that the major products of aerobic 1,2-PD degradation are propionaldehyde, propionate, and 1-propanol. A MCP-minus mutant accumulated a level of propionaldehyde 10-fold higher than that of the wild type (1.6 mM compared to 15.7 mM), associating this compound with growth arrest. The addition of propionaldehyde to cultures of S. enterica caused growth arrest from 8 to 20 mM, but not at 4 mM, providing direct evidence for propionaldehyde toxicity. Studies also indicated that propionaldehyde was toxic due to the inhibition of respiratory processes, and the growth arrest ended when propionaldehyde was depleted primarily by conversion to propionate and 1-propanol and secondarily due to volatility. The Ames test was used to show that propionaldehyde is a mutagen and that mutation frequencies are increased in MCP-minus mutants during 1,2-PD degradation. We propose that a primary function of the MCPs involved in 1,2-PD degradation is the mitigation of toxicity and DNA damage by propionaldehyde.
SUMMARY Bacterial microcompartments (MCPs) are sophisticated protein-based organelles used to optimize metabolic pathways. They consist of metabolic enzymes encapsulated within a protein shell, which creates an ideal environment for catalysis and facilitates the channeling of toxic/volatile intermediates to downstream enzymes. The metabolic processes that require MCPs are diverse and widely distributed and play important roles in global carbon fixation and bacterial pathogenesis. The protein shells of MCPs are thought to selectively control the movement of enzyme cofactors, substrates, and products (including toxic or volatile intermediates) between the MCP interior and the cytoplasm of the cell using both passive electrostatic/steric and dynamic gated mechanisms. Evidence suggests that specialized shell proteins conduct electrons between the cytoplasm and the lumen of the MCP and/or help rebuild damaged iron-sulfur centers in the encapsulated enzymes. The MCP shell is elaborated through a family of small proteins whose structural core is known as a bacterial microcompartment (BMC) domain. BMC domain proteins oligomerize into flat, hexagonally shaped tiles, which assemble into extended protein sheets that form the facets of the shell. Shape complementarity along the edges allows different types of BMC domain proteins to form mixed sheets, while sequence variation provides functional diversification. Recent studies have also revealed targeting sequences that mediate protein encapsulation within MCPs, scaffolding proteins that organize lumen enzymes and the use of private cofactor pools (NAD/H and coenzyme A [HS-CoA]) to facilitate cofactor homeostasis. Although much remains to be learned, our growing understanding of MCPs is providing a basis for bioengineering of protein-based containers for the production of chemicals/pharmaceuticals and for use as molecular delivery vehicles.
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