The PduL Phosphotransacylase Is Used To Recycle Coenzyme A within the Pdu Microcompartment

Abstract

In Salmonella enterica, 1,2-propanediol (1,2-PD) utilization (Pdu) is mediated by a bacterial microcompartment (MCP). The Pdu MCP consists of a multiprotein shell that encapsulates enzymes and cofactors for 1,2-PD catabolism, and its role is to sequester a reactive intermediate (propionaldehyde) to minimize cellular toxicity and DNA damage. For the Pdu MCP to function, the enzymes encapsulated within must be provided with a steady supply of substrates and cofactors. In the present study, Western blotting assays were used to demonstrate that the PduL phosphotransacylase is a component of the Pdu MCP. We also show that the N-terminal 20-residue-long peptide of PduL is necessary and sufficient for targeting PduL and enhanced green fluorescent protein (eGFP) to the lumen of the Pdu MCP. We present the results of genetic tests that indicate that PduL plays a role in the recycling of coenzyme A internally within the Pdu MCP. However, the results indicate that some coenzyme A recycling occurs externally to the Pdu MCP. Hence, our results support a model in which a steady supply of coenzyme A is provided to MCP lumen enzymes by internal recycling by PduL as well as by the movement of coenzyme A across the shell by an unknown mechanism. These studies expand our understanding of the Pdu MCP, which has been linked to enteric pathogenesis and which provides a possible basis for the development of intracellular bioreactors for use in biotechnology. IMPORTANCEBacterial MCPs are widespread organelles that play important roles in pathogenesis and global carbon fixation. Here we show that the PduL phosphotransacylase is a component of the Pdu MCP. We also show that PduL plays a key role in cofactor homeostasis by recycling coenzyme A internally within the Pdu MCP. Further, we identify a potential N-terminal targeting sequence using a bioinformatic approach and show that this short sequence extension is necessary and sufficient for directing PduL as well as heterologous proteins to the lumen of the Pdu MCP. These findings expand our general understanding of bacterial MCP assembly and cofactor homeostasis. Bacterial microcompartments (MCPs) are a diverse family of subcellular proteinaceous organelles used to optimize metabolic pathways that have toxic or volatile intermediates (1, 2). MCPs are polyhedral in shape and about 100 to 150 nm in size and consist of a multiprotein shell that encapsulates sequentially acting enzymes of specific metabolic pathways. Overall, they are composed of several thousand protein subunits of 10 to 20 different types and may exceed a gigadalton in mass. Genomic analyses indicate that MCPs are produced by about 20% of bacteria and that there are seven or more different types (which function in diverse metabolic processes) as well as numerous subtypes (3-5). The first MCP identified was the carboxysome (6). This MCP functions to optimize carbon fixation, and it has been estimated that 25% of CO 2 fixation on Earth occurs within carboxysomes (2). Two other MCPs that have been studied are used for...

Engineering bacterial microcompartments with heterologous enzyme cargos

“…Together with previous reports by other groups, peptide‐mediated targeting seems to be suitable to rationally reprogram the Pdu‐BMC with various heterologous proteins. Four native EPs (PduD, PduL, PduP, PduV) are currently available that had been engineered to compete with the native EPs, thus allowing to control the ratio between the heterologous proteins to be encapsulated . However, our observations indicate that the nature of the cargo‐protein might influence targeting efficiency and encapsulation in a so far elusive manner, too, thereby complicating the rational design.…”

Bioinformatic Characterization of Glycyl Radical Enzyme-Associated Bacterial Microcompartments

“…One of these four loci, the GUF locus in D. psychrophila, encodes a PTA_PTB-like PTAC; these are typically associated with only a subset of EUT BMC loci (5). PduL homologs contain an N-terminal EP for encapsulation within the BMC (25). Phylogenetically, the PduL homologs from the same locus type tend to group together and form clades (Fig.…”

Bacterial microcompartments

“…Notably, the amino acid sequence of the AldDH can be used to independently reconstruct BMC phylogeny 6, 8 . An alcohol dehydrogenase and a phosphotransacylase (either a PduL-like/pfam06130 50,85 PTAC, which is found mostly associated with BMCs or a EutD-like/pfam01515 86 PTAC, a type that is ubiquitous and often referred to as housekeeping) recycle the NAD + and the CoA cofactors (Figure 1B). …”