Computational insights into chemoselectivity of Trans-4-Hydroxy-L-Proline dehydratase HypD

The dehydration of trans-4-hydroxy-L-proline (t4L-HP) yielding 1-pyrroline-5-carboxylate in certain gut bacteria requires the glycyl radical enzyme HypD, while isomerisation of trans-4-hydroxy-D-proline (t4D-HP) generating 2-amino-4-ketopentanoate involves a homologous enzyme HplG. Although the former enzyme exhibits high chemoselectivity when catalysing the dehydration and is regarded as a promising target for treating bacteriacaused gut diseases, its catalytic mechanism remains elusive. Herein, we deeply investigated the two reaction pathways by carrying out theoretical studies at the QM/MM level. Our calculations show that, in HypD, a catalytic Cys434 radical facilitates the C delta-H activation in an initially deprotonated zwitterionic t4L-HP, which is followed by a heterolytic cleavage of t4L-HP's C gamma-O bond with a water formed being assisted by the conserved Asp279. In the isomerisation pathway, the Cys434 radical would abstract the hydrogen atom from C gamma of zwitterionic t4L-HP, and then Asp279 cleaves the C delta-N bond by abstracting a proton from the hydroxyl group of t4LHP. The two reaction paths are calculated to have barriers of 18.0 and 22.8 kcal/mol, respectively, which well explain HypD's chemoselectivity. In the mechanisms suggested by our study, Asp279 plays a role as "proton shuttle" and regulates the protonation state of t4L-HP in the selective hydrogen atom abstraction invoked by the cysteine radical. Our findings provide an insight of universal significance into radical-based enzymatic catalysis and expand the understanding of how nature wisely modulates catalytic reactivity and selectivity by switching the substrate's protonation state.