Bacterial microcompartments (BMCs) are prokaryotic organelles consisting of a protein shell and an encapsulated enzymatic core. BMCs are involved in several biochemical processes, such as choline, glycerol and ethanolamine degradation and carbon fixation. Since non-native enzymes can also be encapsulated in BMCs, an improved understanding of BMC shell assembly and encapsulation processes could be useful for synthetic biology applications. Here we report the isolation and recombinant expression of BMC structural genes from the Klebsiella pneumoniae GRM2 locus, the investigation of mechanisms behind encapsulation of the core enzymes, and the characterization of shell particles by cryo-EM. We conclude that the enzymatic core is encapsulated in a hierarchical manner and that the CutC choline lyase may play a secondary role as an adaptor protein. We also present a cryo-EM structure of a pT = 4 quasi-symmetric icosahedral shell particle at 3.3 Å resolution, and demonstrate variability among the minor shell forms.

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Dihydroxyacetone (DHA), d-glyceraldehyde and l-glyceraldehyde can be reduced using NADPH as a cofactor to form glycerol and NADP. Enzymes catalysing this reaction are generally called NADP:glycerol dehydrogenases. NADP:glycerol dehydrogenase activity is common in moulds and filamentous fungi. Enzymes from different species of filamentous fungi have been purified and characterized. The enzymes purified from Aspergillus niger [1] and Aspergillus nidulans [2] catalyse the reversible reaction from glycerol and NADP to DHA and NADPH. For the A. niger enzyme, an equilibrium constant of 3.1-4.6 · 10 )12 m was estimated for the reaction: The mould Hypocrea jecorina (Trichoderma reesei) has two genes coding for enzymes with high similarity to the NADP-dependent glycerol dehydrogenase. These genes, called gld1 and gld2, were cloned and expressed in a heterologous host. The encoded proteins were purified and their kinetic properties characterized. GLD1 catalyses the conversion of d-glyceraldehyde and l-glyceraldehyde to glycerol, whereas GLD2 catalyses the conversion of dihydroxyacetone to glycerol. Both enzymes are specific for NADPH as a cofactor. The properties of GLD2 are similar to those of the previously described NADP-dependent glycerol-2-dehydrogenases (EC 1.1.1.156) purified from different mould species. It is a reversible enzyme active with dihydroxyacetone or glycerol as substrates. GLD1 resembles EC 1.1.1.72. It is also specific for NADPH as a cofactor but has otherwise completely different properties. GLD1 reduces d-glyceraldehyde and l-glyceraldehyde with similar affinities for the two substrates and similar maximal rates. The activity in the oxidizing reaction with glycerol as substrate was under our detection limit. Although the role of GLD2 is to facilitate glycerol formation under osmotic stress conditions, we hypothesize that GLD1 is active in pathways for sugar acid catabolism such as d-galacturonate catabolism.Abbreviations DHA, dihydroxyacetone; DHAP, dihydroxyacetone phosphate.

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