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Publication Abstract from PubMed

Hyoscyamine 6beta-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 horizontal line analogously to mechanisms proposed for the Fe/2OG halogenases and, in our more recent study, N-acetylnorloline synthase (LolO) horizontal line 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 6beta-hydroxyhyoscyamine suggest that a modest ( approximately 10 degrees ) 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 (2)H(2)O, 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.

Optimized Substrate Positioning Enables Switches in the C-H Cleavage Site and Reaction Outcome in the Hydroxylation-Epoxidation Sequence Catalyzed by Hyoscyamine 6beta-Hydroxylase.,Wenger ES, Martinie RJ, Ushimaru R, Pollock CJ, Sil D, Li A, Hoang N, Palowitch GM, Graham BP, Schaperdoth I, Burke EJ, Maggiolo AO, Chang WC, Allen BD, Krebs C, Silakov A, Boal AK, Bollinger JM Jr J Am Chem Soc. 2024 Sep 4;146(35):24271-24287. doi: 10.1021/jacs.4c04406. Epub , 2024 Aug 22. PMID:39172701^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.