Characterization of 1,2-Propanediol Dehydratases Reveals Distinct Mechanisms for B₁₂-Dependent and Glycyl Radical Enzymes

Abstract

Propanediol dehydratase (PD), a recently characterized member of the glycyl radical enzyme (GRE) family, uses protein-based radicals to catalyze the chemically challenging dehydration of ( S)-1,2-propanediol. This transformation is also performed by the well-studied enzyme B-dependent propanediol dehydratase (B-PD) using an adenosylcobalamin cofactor. Despite the prominence of PD in anaerobic microorganisms, it remains unclear if the mechanism of this enzyme is similar to that of B-PD. Here we report O labeling experiments that suggest PD and B-PD employ distinct mechanisms. Unlike B-PD, PD appears to catalyze the direct elimination of a hydroxyl group from an initially formed substrate-based radical, avoiding the generation of a 1,1- gem diol intermediate. Our studies provide further insights into how GREs perform elimination chemistry and highlight how nature has evolved diverse strategies for catalyzing challenging reactions.

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“…111,112,116 This mechanism is consistent with the observed retention of the 18 O-label in the product when using a C2- 18 O-isotopically labeled substrate. 120,121 The C2-centered substrate radical then abstracts a hydrogen atom from the same 5 0 -deoxyadenosine molecule that initiated chemistry, 122,123 regenerating the 5 0 -dA c that can reform the Co-C bond with concomitant oxidation of Co 2+ to Co 3+ (Fig. 2c).…”

“…130,131 This elimination mechanism is consistent with the observed loss of the 18 O-label in the product when using a C2- 18 O-isotopically labeled substrate. 121 Hydrogen atom abstraction by the C2 alkyl radical from the catalytic cysteine thiol regenerates the thiyl radical and forms the propionaldehyde product. At the end of each cycle, the radical migrates back to the initial conserved glycine residue.…”

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“…The conversion of choline into TMA and acetaldehyde requires the energetically demanding cleavage of its C–N bond (15). Enzymes catalyzing similar elimination reactions involving C–N (ethanolamine ammonia lyase) or C–O (glycerol dehydratase) bonds typically require low-potential reactive radical species such as the Ado ⋅ derived from adenosylcobalamin in B 12 -dependent enzymes (34). In contrast, the first GRE responsible for catalyzing the cleavage of the C–N bond of choline using a Gly ⋅ was identified in 2012 (16).…”

“…1,2-PD is a common by-product of fucose and rhamnose degradation (37, 38) and can be an abundant source of energy and carbon for microbes in environmental niches such as the human gastrointestinal tract (39). 1,2-PD is the signature substrate of the well-studied propanediol utilization (PDU) BMC (40) and is dehydrated to propionaldehyde by the B 12 -derived Ado · of the 1,2-PD dehydratase signature enzyme (34, 41). In contrast, GRM subtypes 3 (17), 4 (4, 19), and 5 (18, 27) catalyze a B 12 -independent dehydration of 1,2-PD using the protein-based glycyl/thiyl radicals of a GRE (34).…”

“…1,2-PD is the signature substrate of the well-studied propanediol utilization (PDU) BMC (40) and is dehydrated to propionaldehyde by the B 12 -derived Ado · of the 1,2-PD dehydratase signature enzyme (34, 41). In contrast, GRM subtypes 3 (17), 4 (4, 19), and 5 (18, 27) catalyze a B 12 -independent dehydration of 1,2-PD using the protein-based glycyl/thiyl radicals of a GRE (34). To date, this is the only known example of functional redundancy among different types of catabolic BMCs.…”