8CV8

Structure of Hyoscyamine 6-beta Hydroxylase in complex with iron, 2-oxoglutarate, and hyoscyamine

Summary for 8CV8

• Entry DOI: 10.2210/pdb8cv8/pdb
• Descriptor: Hyoscyamine 6-beta-hydroxylase, STRONTIUM ION, 1,2-ETHANEDIOL, ... (8 entities in total)
• Functional Keywords: oxacyclase, scopolamine, epoxide, oxidoreductase
• Biological source: Atropa belladonna (belladonna)
• Total number of polymer chains: 1
• Total formula weight: 43669.82

Authors

Wenger, E.W.,Boal, A.K.,Bollinger, J.M.,Krebs, C. (deposition date: 2022-05-18, release date: 2023-11-22, Last modification date: 2024-12-04)

Primary citation

Wenger, E.S.,Martinie, R.J.,Ushimaru, R.,Pollock, C.J.,Sil, D.,Li, A.,Hoang, N.,Palowitch, G.M.,Graham, B.P.,Schaperdoth, I.,Burke, E.J.,Maggiolo, A.O.,Chang, W.C.,Allen, B.D.,Krebs, C.,Silakov, A.,Boal, A.K.,Bollinger Jr., J.M.
Optimized Substrate Positioning Enables Switches in the C-H Cleavage Site and Reaction Outcome in the Hydroxylation-Epoxidation Sequence Catalyzed by Hyoscyamine 6 beta-Hydroxylase.
J.Am.Chem.Soc., 146:24271-24287, 2024

PubMed Abstract: Hyoscyamine 6β-hydroxylase (H6H) is an iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase that produces the prolifically administered antinausea drug, scopolamine. After its namesake hydroxylation reaction, H6H then couples the newly installed C6 oxygen to C7 to produce the drug's epoxide functionality. Oxoiron(IV) (ferryl) intermediates initiate both reactions by cleaving C-H bonds, but it remains unclear how the enzyme switches the target site and promotes (C6)O-C7 coupling in preference to C7 hydroxylation in the second step. In one possible epoxidation mechanism, the C6 oxygen would─analogously to mechanisms proposed for the Fe/2OG halogenases and, in our more recent study, -acetylnorloline synthase (LolO)─coordinate as alkoxide to the C7-H-cleaving ferryl intermediate to enable alkoxyl coupling to the ensuing C7 radical. Here, we provide structural and kinetic evidence that H6H does not employ substrate coordination or repositioning for the epoxidation step but instead exploits the distinct spatial dependencies of competitive C-H cleavage (C6 vs C7) and C-O-coupling (oxygen rebound vs cyclization) steps to promote the two-step sequence. Structural comparisons of ferryl-mimicking vanadyl complexes of wild-type H6H and a variant that preferentially 7-hydroxylates instead of epoxidizing 6β-hydroxyhyoscyamine suggest that a modest (∼10°) shift in the Fe-O-H(C7) approach angle is sufficient to change the outcome. The 7-hydroxylation:epoxidation partition ratios of both proteins increase more than 5-fold in HO, reflecting an epoxidation-specific requirement for cleavage of the alcohol O-H bond, which, unlike in the LolO oxacyclization, is not accomplished by iron coordination in advance of C-H cleavage.

PubMed: 39172701
DOI: 10.1021/jacs.4c04406

Experimental method

X-RAY DIFFRACTION (1.532 Å)