- Evidence for Modulation of Oxygen Rebound Rate in Control of Outcome by Iron(II)- And 2-Oxoglutarate-Dependent Oxygenases
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Iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenases generate iron(IV)-oxo (ferryl) intermediates that can abstract hydrogen from aliphatic carbons (R-H). Hydroxylation proceeds by coupling of the resultant substrate radical (Ra€￠) and oxygen of the Fe(III)-OH complex ("oxygen rebound"). Nonhydroxylation outcomes result from different fates of the Fe(III)-OH/R?state; for example, halogenation results from R?coupling to a halogen ligand cis to the hydroxide. We previously suggested that halogenases control substrate-cofactor disposition to disfavor oxygen rebound and permit halogen coupling to prevail. Here, we explored the general implication that, when a ferryl intermediate can ambiguously target two substrate carbons for different outcomes, rebound to the site capable of the alternative outcome should be slower than to the adjacent, solely hydroxylated site. We evaluated this prediction for (i) the halogenase SyrB2, which exclusively hydroxylates C5 of norvaline appended to its carrier protein but can either chlorinate or hydroxylate C4 and (ii) two bifunctional enzymes that normally hydroxylate one carbon before coupling that oxygen to a second carbon (producing an oxacycle) but can, upon encountering deuterium at the first site, hydroxylate the second site instead. In all three cases, substrate hydroxylation incorporates a greater fraction of solvent-derived oxygen at the site that can also undergo the alternative outcome than at the other site, most likely reflecting an increased exchange of the initially O2-derived oxygen ligand in the longer-lived Fe(III)-OH/R?states. Suppression of rebound may thus be generally important for nonhydroxylation outcomes by these enzymes.
- Pan, Juan,Wenger, Eliott S.,Matthews, Megan L.,Pollock, Christopher J.,Bhardwaj, Minakshi,Kim, Amelia J.,Allen, Benjamin D.,Grossman, Robert B.,Krebs, Carsten,Martin Bollinger
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supporting information
p. 15153 - 15165
(2019/10/19)
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- Changes in Regioselectivity of H Atom Abstraction during the Hydroxylation and Cyclization Reactions Catalyzed by Hyoscyamine 6β-Hydroxylase
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Hyoscyamine 6β-hydroxylase (H6H) is an αKG-dependent nonheme iron oxidase that catalyzes the oxidation of hyoscyamine to scopolamine via two separate reactions: hydroxylation followed by oxidative cyclization. Both of these reactions are expected to involve H atom abstraction from each of two adjacent carbon centers (C6 vs C7) in the substrate. During hydroxylation, there is a roughly 85:1 preference for H atom abstraction from C6 versus C7; however, this inverts to a 1:16 preference during cyclization. Furthermore, 18O incorporation experiments in the presence of deuterated substrate are consistent with the catalytic iron(IV)-oxo complex being able to support the coordination of an additional ligand during hydroxylation. These observations suggest that subtle differences in the substrate binding configuration can have significant consequences for the catalytic cycle of H6H.
- Ushimaru, Richiro,Ruszczycky, Mark W.,Liu, Hung-Wen
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p. 1062 - 1066
(2019/01/23)
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- Preparative separation of four isomers of synthetic anisodamine by HPLC and diastereomer crystallization
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Anisodamine (654-1), a well-known cholinergic antagonist, is marketed as synthetic anisodamine (mixture of four isomers, 654-2) in China. To preparative resolution and comparison of the bioactivities of the four isomers of synthetic anisodamine, current work explores an economic and effective separation method by using preparative high performance liquid chromatography (HPLC) and diastereomer crystallization. Their absolute configurations were established by single-crystal X-ray diffraction and circular dichroism method. The purities of each isomer were more than 95%. Among them, 654-2-A2 (6R, 2′S configuration) exhibited better effect on cabachol preconditioned small intestine tension more than 654-2 and other isomers. The direct separation method without using HPLC was tried as well, which was still on progress. This is the first report of the method for preparative separation of four isomers of synthetic anisodamine which could be used for large-scale production in industry.
- Wu, Tong,Zhu, Jiu-Xin,Wei, Qian,Li, Ping,Wang, Li-Bo,Huang, Jian,Wang, Jin-Hui,Tang, Li-Ke,Wu, Li-Jun,Li, Chang,Han, Wei-Na
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- Functional characterization of recombinant hyoscyamine 6β-hydroxylase from Atropa belladonna
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(-)-Hyoscyamine, the enantiomerically pure form of atropine, and its derivative scopolamine are tropane alkaloids that are extensively used in medicine. Hyoscyamine 6β-hydroxylase (H6H, EC 1.14.11.11), a monomeric α-ketoglutarate dependent dioxygenase, converts (-)-hyoscyamine to its 6,7-epoxy derivative, scopolamine, in two sequential steps. In this study, H6H of Atropa belladonna (AbH6H) was cloned, heterologously expressed in Escherichia coli, purified and characterized. The catalytic efficiency of AbH6H, especially for the second oxidation, was found to be low, and this may be one of the reasons why Atropa belladonna produces less scopolamine than other species in the same family. 6,7-Dehydrohyoscyamine, a potential precursor for the last step of epoxidation, was shown not to be an obligatory intermediate in the biosynthesis of scopolamine using purified AbH6H with an in vitro 18O labeling experiment. Moreover, the nitrogen atom in the tropane ring of (-)-hyoscyamine was found to play an important role in substrate recognition.
- Li, Jing,Van Belkum, Marco J.,Vederas, John C.
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experimental part
p. 4356 - 4363
(2012/08/28)
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- Molecular cloning, expression and characterization of hyoscyamine 6β-hydroxylase from hairy roots of Anisodus tanguticus
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Anisodus tanguticus, one of the indigenous Chinese ethnological medicinal plants of the Solanaceae, produces anticholinergic alkaloids such as hyoscyamine, 6β-hydroxyhyoscyamine and scopolamine. Hyoscyamine 6β-hydroxylase (H6H), a key enzyme in the biosynthetic pathway of scopolamine, catalyzes the hydroxylation of hyoscyamine and epoxide formation from 6β-hydroxyhyoscyamine to generate scopolamine. A full-length cDNA of H6H has been isolated from A. tanguticus hairy roots. Nucleotide sequence analysis of the cloned cDNA revealed an open reading frame of 1035 bp encoding 344 amino acids with high homology to other known H6Hs. The equivalent amino acid sequence shows a typical motif of 2-oxoglutarate-dependent dioxygenase. The A. tanguticus H6H was expressed in Escherichia coli and purified for enzyme function analysis. This study characterized the recombinant AtH6H and showed it could generate scopolamine from hyoscyamine.
- Liu, Tao,Zhu, Ping,Cheng, Ke-Di,Meng, Chao,He, Hui-Xia
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p. 249 - 253
(2007/10/03)
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- 7β-HYDROXYHYOSCYAMINE FROM DUBOISIA MYOPOROIDES-D. LEICHHARDTII HYBRID AND HYOSCYAMUS ALBUS
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A new tropane alkaloid, 7β-hydroxyhyoscyamine was isolated, together with 6β-hydroxyhyoscyamine, hyoscyamine, norhyoscyamine, scopolamine and tropine, from the leaves of Duboisia hybrid M-II-8-6 (crossbred between D. myoporoides and D. leichhardtii) and the hairy roots of Hyoscyamus albus.Key Word Index - Duboisia myoporoides; D. leichhardtii; Hyoscyamus albus; Solanaceae; Agrobacterium rhizogenes; tropane alkaloid; 7β-hydroxyhyoscyamine; 6β-hydroxyhyoscyamine.
- Ishimaru, Kanji,Shimomura, Koichiro
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p. 3507 - 3510
(2007/10/02)
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