Hydroxyprolines are highly abundant in nature as they are components of many structural proteins and osmolytes. Anaerobic degradation of trans-4-hydroxy-l-proline (t4L-HP) was previously found to involve the glycyl radical enzyme (GRE) t4L-HP dehydratase (HypD). Here, we report a pathway for anaerobic hydroxyproline degradation that involves a new GRE, trans-4-hydroxy-d-proline (t4D-HP) C–N-lyase (HplG). In this pathway, cis-4-hydroxy-l-proline (c4L-HP) is first isomerized to t4D-HP, followed by radical-mediated ring opening by HplG to give 2-amino-4-ketopentanoate (AKP), the first example of a ring opening reaction catalyzed by a GRE 1,2-eliminase. Subsequent cleavage by AKP thiolase (OrtAB) yields acetyl-CoA and d-alanine. We report a crystal structure of HplG in complex with t4D-HP at a resolution of 2.7 Å, providing insights into its catalytic mechanism. Different from HypD commonly identified in proline-reducing Clostridia, HplG is present in other types of fermenting bacteria, including propionate-producing bacteria, underscoring the diversity of enzymatic radical chemistry in the anaerobic microbiome.

The glycyl radical enzymes (GREs) p-hydroxyphenylacetate (HPA) decarboxylase (HPAD), indoleacetate decarboxylase, and phenylacetate decarboxylase catalyze the radical-mediated decarboxylation of arylacetates, which are products of bacterial aromatic amino acid fermentation. Here, we report the discovery and structural and biochemical investigation of a fourth GRE arylacetate decarboxylase (AAD) from Olsenella scatoligenes that catalyzes HPA decarboxylation. AAD also catalyzes the decarboxylation of p-aminophenylacetate, which is not a substrate of HPAD, and lacks the Fe–S cluster containing small subunit. The structure of AAD in complex with p-hydroxyphenylacetate was determined by X-ray crystallography. The differing substrate ranges and active site structures of AAD and HPAD suggest distinct catalytic mechanisms, underscoring the diversity of radical-mediated decarboxylation reactions.

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