Bioinformatic Characterization of Glycyl Radical Enzyme-Associated Bacterial Microcompartments

Abstract

Bacterial microcompartments (BMCs) are proteinaceous organelles encapsulating enzymes that catalyze sequential reactions of metabolic pathways. BMCs are phylogenetically widespread; however, only a few BMCs have been experimentally characterized. Among them are the carboxysomes and the propanediol-and ethanolamine-utilizing microcompartments, which play diverse metabolic and ecological roles. The substrate of a BMC is defined by its signature enzyme. In catabolic BMCs, this enzyme typically generates an aldehyde. Recently, it was shown that the most prevalent signature enzymes encoded by BMC loci are glycyl radical enzymes, yet little is known about the function of these BMCs. Here we characterize the glycyl radical enzyme-associated microcompartment (GRM) loci using a combination of bioinformatic analyses and active-site and structural modeling to show that the GRMs comprise five subtypes. We predict distinct functions for the GRMs, including the degradation of choline, propanediol, and fuculose phosphate. This is the first family of BMCs for which identification of the signature enzyme is insufficient for predicting function. The distinct GRM functions are also reflected in differences in shell composition and apparently different assembly pathways. The GRMs are the counterparts of the vitamin B 12 -dependent propanediol-and ethanolamine-utilizing BMCs, which are frequently associated with virulence. This study provides a comprehensive foundation for experimental investigations of the diverse roles of GRMs. Understanding this plasticity of function within a single BMC family, including characterization of differences in permeability and assembly, can inform approaches to BMC bioengineering and the design of therapeutics. In the last decade, an accumulation of countervailing evidence has overturned the long-held verdict that bacteria are primitive organisms lacking intracellular organization. In addition to membrane-bound organelles like the magnetosomes of magnetotactic bacteria (1, 2) or the anammoxosomes found in some members of the Planctomycetes, bacteria also form protein-based organelles known as bacterial microcompartments (BMCs) (3-5). The defining feature of all BMCs is a shell composed of homologous proteins containing the Pfam00936 domain. A protein with a single copy of the Pfam00936 domain that forms hexamers is referred to as BMC-H; a second type of shell protein consists of a fusion of two Pfam00936 domains and forms pseudohexameric trimers and is referred to as BMC-T. The hexamers and pseudohexamers are thought to tile into the facets of the shell (3, 6). A third type of shell protein containing the Pfam03319 domain forms pentamers that are assumed to cap the vertices of an apparently icosahedral shell and is referred to as BMC-P (3, 7). The BMC-H, BMC-T, and BMC-P oligomers are typically perforated by pores, through which metabolite exchange with the cytosol is assumed to be mediated. Residues flanking the pores are generally polar and well conserved among orthologs, and mutation of the residu...

Bacterial microcompartments

“…For example, in the glycyl radical enzyme containing microcompartments (GRMs), a subset of the enzymes contain domains with homology to their catalytically active, encapsulated counterparts; this is analogous to the role of the SSLDs of β-carboxysomes. Other examples of such ‘domain mimics’ include catalytically inactive aldehyde dehydrogenase domains or the N-terminal extension of some of the GRM-specific glycyl radical enzymes (GREs) [G] that resemble half of the catalytic domain of the GRE 6,8 . Likewise, encapsulation peptides are now an established feature of many different metabolosome enzymes such as the aldehyde dehydrogenase PduP 72 and the phosphotransacylase PduL 50 of the PDU BMC, and the ethanolamine lyase small subunit (EutC) and ethanol dehydrogenase EutG of the EUT BMC 63 .…”

“…The aldehyde intermediate that is generated by the signature enzyme is subsequently processed by a CoA-dependent aldehyde dehydrogenase. 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).…”

“…Bacterial microcompartments (BMCs) are functional analogs of eukaryotic organelles; a semi-permeable protein shell sequesters an enzymatic core that constitutes a segment of a metabolic pathway 1–8 . Instead of a lipid membrane, the restricting boundary of a BMC is a selectively permeable protein shell.…”

“…GRM loci are the most widespread and consist of functionally distinct BMCs 8 and are widely distributed in human gut isolates 108, 161 …”

The N Terminus of the PduB Protein Binds the Protein Shell of the Pdu Microcompartment to Its Enzymatic Core

“…undreds of species of bacteria produce complex proteinaceous organelles known as bacterial microcompartments (MCPs) (1)(2)(3)(4)(5)(6)(7)(8). The function of MCPs is to optimize metabolic pathways having intermediates that are toxic or poorly retained by the cell envelope (5,7,9).…”