GilR, an unusual lactone-forming enzyme involved in gilvocarcin biosynthesis

Madan Kumar Kharel, Pallab Pahari, Hui Lian, Jürgen Rohr

Research output: Contribution to journalArticlepeer-review

25 Scopus citations

Abstract

In summary, the above results have clearly established that GilR, which is an FAD-dependent oxidoreductase, is responsible for oxidizing hemiacetal 11 to lactone 1. Because preGV exists as an equilibrium mixture of two diastereomers (R and S configuration at the hemiacetal carbon), one could argue that through ring opening/closing GilR oxidizes the aldehyde to an acid, and the lactone forms spontaneously (via route A). However, this would require a more complex reaction, namely addition of water followed by dehydrogenation. The sequence of the gilR gene shows no hint for such a dual activity of its product, and an opened lactone form of GV has never been detected, not even in traces. Thus, the above-discussed direct dehydrogenation of the hemiacetal to the lactone is far more likely. In the primary metabolic pentose phosphate pathway, glucose-6-phoshate (G6P) dehydrogenase catalyzes a similar reaction by converting G6P to 6-phospho-d-gluconolactone. However, unlike GilR, this enzyme utilizes free NADP+.[27] Thus, GilR represents a new class of oxidoreductase in that it uses covalently bound FAD. Interestingly, no other cofactor was needed for the in vitro assay, and we assume that FADH2 reacts with dissolved oxygen to regenerate FAD. The results also clarify that lactone generation is the last step in GV (1) biosynthesis, and the enzyme is flexible enough to handle both sugar-tethered and sugar-free substrates, but its flexibility is limited by the nature of the linked deoxysugar. The unusual oxidative lactone generation found for the GV pathway might also play a role in the biosyntheses of other naturally occurring lactones, the pathways for which are yet to be elucidated. Intriguingly, the sequence of events for GV biosynthesis also requires another (intermediate) lactone 16 formation, here suggested to be a result of a Baeyer-Villiger oxidation, en route to 11 and 1, when the polyketide synthase product undergoes a series of dehydrations and oxidations, a decarboxylation and O-methylations (Scheme 3). GilGT-mediated glycosylation (12 to 11) followed by the GilR-catalyzed dehydrogenation finish the biosynthesis of 1. In the absence of GilGT, GilR catalyzes dehydrogenation of 12 to yield shunt product 9. Understanding the substrate-binding mode of GilR and widening its substrate specificity through site-directed mutagenesis might be necessary to generate further GV analogues by combinatorial biosynthesis.

Original languageEnglish
Pages (from-to)1305-1308
Number of pages4
JournalChemBioChem
Volume10
Issue number8
DOIs
StatePublished - May 25 2009

Funding

FundersFunder number
National Childhood Cancer Registry – National Cancer InstituteR01CA102102

    Keywords

    • Biosynthesis
    • C-glycosides
    • Dehydrogenases
    • Gilvocarcins
    • Lactones

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Medicine
    • Molecular Biology
    • Organic Chemistry

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