The Crystal Structure and Mechanism of an Unusual Oxidoreductase, GilR, Involved in Gilvocarcin V Biosynthesis

Abstract

GilR is a recently identified oxidoreductase that catalyzes the terminal step of gilvocarcin V biosynthesis and is a unique enzyme that establishes the lactone core of the polyketide-derived gilvocarcin chromophore. Gilvocarcin-type compounds form a small distinct family of anticancer agents that are involved in both photo-activated DNA-alkylation and histone H3 cross-linking. High resolution crystal structures of apoGilR and GilR in complex with its substrate pregilvocarcin V reveals that GilR belongs to the small group of a relatively new type of the vanillyl-alcohol oxidase flavoprotein family characterized by bicovalently tethered cofactors. GilR was found as a dimer, with the bicovalently attached FAD cofactor mediated through His-65 and Cys-125. Subsequent mutagenesis and functional assays indicate that Tyr-445 may be involved in reaction catalysis and in mediating the covalent attachment of FAD, whereas Tyr-448 serves as an essential residue initiating the catalysis by swinging away from the active site to accommodate binding of the 6R-configured substrate and consequently abstracting the proton of the hydroxyl residue of the substrate hemiacetal 6-OH group. These studies lay the groundwork for future enzyme engineering to broaden the substrate specificity of this bottleneck enzyme of the gilvocarcin biosynthetic pathway for the development of novel anti-cancer therapeutics.

Enzymatic Total Synthesis of Defucogilvocarcin M and Its Implications for Gilvocarcin Biosynthesis

“…[1a] The function of biosynthetic enzymes was then confirmed by both in vitro and in vivo studies. [7a, 9, 11–13] The cluster encodes a set of typical type II polyketide synthase (PKS) enzymes, which includes a ketosynthase (GilA), a chain-length factor (GilB), an acyl carrier protein (GilC), two malonyl-CoA:acyl carrier protein transacylase (MCAT) homologues (GilP, GilQ), a PKS-associated keto reductase (GilF), and two cyclases (GilK, GilG). Together, this group of enzymes was able to catalyze the formation of the angucy-clinone UWM6 ( 10 ).…”

“…[8d, 9] Rabelomycin ( 12 ) and homorabelomycin ( 13 ) are the major metabolites of the GilOIV-deleted mutant, whereas 5 is the major metabolite of the GilOIII-deleted mutant. [13b] The production of angucyclinones 12–18 by these oxygenase-deficient mutants led to the hypothesis that the three oxygenases, GilOI, GilOII, and GilOIV, might form a multienzyme complex that is responsible for a concerted reaction pathway. This pathway includes the crucial C–C bond cleavage that eventually leads to the formation of the gilvocarcin scaffold.…”

“…[9] Then, the methylation of the two aromatic hydroxy groups, formation of a hemiacetal ring, decarboxylation, and C-glycosyltransfer are still necessary to generate pregilvocarcins 22 and 23 , which are the substrates of GilR. [13a,c] GilR has recently been shown to catalyze the very last step in the biosynthesis of gilvocarcin by converting 22 and 23 into the final lactone-containing products (Scheme 2). …”

“…An authentic sample of 1 was isolated from the previously characterized GilGT-minus mutant. [13b] Control reactions without acetyl-CoA and malonyl-CoA or without the PKS enzymes did not produce any metabolite (Figure 1, trace A).…”

Delineation of gilvocarcin, jadomycin, and landomycin pathways through combinatorial biosynthetic enzymology

“…The functions of the post-PKS gene products were assigned after gene inactivation, complementation, cross-feeding experiments along with few in vitro and in vivo studies of activity of individual enzymes or enzyme mixtures [75 • ,79–82,83 • ]. However, many of the inactivation mutants accumulated biosynthetic shunt products, and left ambiguity over post-PKS biosynthetic steps (for some examples, see Figure 4) [49,80,82].…”