Characterization of a Glycyl Radical Enzyme Bacterial Microcompartment Pathway in Rhodobacter capsulatus

Abstract

Bacterial microcompartments (BMCs) are large (∼100-nm) protein shells that encapsulate enzymes, their substrates, and cofactors for the purposes of increasing metabolic reaction efficiency and protecting cells from toxic intermediates. The best-studied microcompartment is the carbon-fixing carboxysome that encapsulates ribulose-1,5-bisphosphate carboxylase and carbonic anhydrase. Other well-known BMCs include the Pdu and Eut BMCs, which metabolize 1,2-propanediol and ethanolamine, respectively, with vitamin B12-dependent diol dehydratase enzymes. Recent bioinformatic analyses identified a new prevalent type of BMC, hypothesized to utilize vitamin B12-independent glycyl radical enzymes to metabolize substrates. Here we use genetic and metabolic analyses to undertake in vivo characterization of the newly identified glycyl radical enzyme microcompartment 3 (GRM3) class of microcompartment clusters. Transcriptome sequencing analyses showed that the microcompartment gene cluster in the genome of the purple photosynthetic bacterium Rhodobacter capsulatus was expressed under dark anaerobic respiratory conditions in the presence of 1,2-propanediol. High-performance liquid chromatography and gas chromatography-mass spectrometry analyses showed that enzymes coded by this cluster metabolized 1,2-propanediol into propionaldehyde, propanol, and propionate. Surprisingly, the microcompartment pathway did not protect these cells from toxic propionaldehyde under the conditions used in this study, with buildup of this intermediate contributing to arrest of cell growth. We further show that expression of microcompartment genes is regulated by a two-component system located downstream of the microcompartment cluster. IMPORTANCE BMCs are protein shells that are designed to compartmentalize enzymatic reactions that require either sequestration of a substrate or the sequestration of toxic intermediates. Due to their ability to compartmentalize reactions, BMCs have also become attractive targets for bioengineering novel enzymatic reactions. Despite these useful features, little is known about the biochemistry of newly identified classes of BMCs. In this study, we have undertaken genetic and in vivo metabolic analyses of the newly identified GRM3 gene cluster.

Bacterial microcompartments: catalysis-enhancing metabolic modules for next generation metabolic and biomedical engineering

“…Metabolosomes are functionally diverse. The types that have been experimentally characterized are propanediol utilizing (PDU and GRM3) [28–30], ethanolamine utilizing (EUT) [31], fucose and rhamnose utilizing (GRM5 and PVM) [32, 33], 1-amino-2-propanol utilizing (RMM) [34, 35] and choline utilizing (GRM2) [36, 37]. Even though these metabolosomes have different substrates, they share a common core biochemistry which consists of a substrate-defining signature enzyme, an aldehyde dehydrogenase (AldDH), an alcohol dehydrogenase (AlcDH), and a phosphotransacylase (PTAC) (Fig.…”

Genetic Characterization of a Glycyl Radical Microcompartment Used for 1,2-Propanediol Fermentation by Uropathogenic Escherichia coli CFT073

“…Glycyl radical MCPs associated with choline trimethylamine (TMA) lyase mediate anaerobic choline degradation in varied organisms (45)(46)(47). Glycyl radical MCPs associated with glycyl radical diol dehydratases (GR-DDH) mediate the degradation of 1,2-PD, fucose, rhamnose, and possibly glycerol by pathways that are independent of coenzyme B 12 (19,(48)(49)(50). Recent studies partially characterized the type 3 glycyl radical MCP loci of Rhodopseudomonas palustris and Rhodobacter capsulatus (48,49).…”

A Survey of Bacterial Microcompartment Distribution in the Human Microbiome

“…Interestingly, the conversion of choline to TMA is only possible through microbial activity ( Craciun and Balskus, 2012 ; Craciun et al, 2014 ). The GRM3/4/6 organelles use the GRE 1,2-PD dehydratase to convert 1,2-PD into propanol and propionate ( Zarzycki et al, 2017 ; Schindel et al, 2019 ) and GRM5 is involved in the anaerobic degradation of rhamnose/fuculose ( Petit et al, 2013 ; Zarzycki et al, 2015 ). Collectively, we find these BMCs are the most prevalent in available sequence data from human microbiome samples.…”