Sequential Action of Two Flavoenzymes, PgaE and PgaM, in Angucycline Biosynthesis: Chemoenzymatic Synthesis of Gaudimycin C

Abstract

Tailoring steps in aromatic polyketide antibiotic biosynthesis are an important source of structural diversity and, consequently, an intriguing focal point for enzymological studies. PgaE and PgaM from Streptomyces sp. PGA64 are representatives of flavoenzymes catalyzing early post-PKS reactions in angucycline biosynthesis. This in vitro study illustrates that the chemoenzymatic conversion of UWM6 into the metabolite, gaudimycin C, requires multiple closely coupled reactions to prevent intermediate degradation. The NMR structure of gaudimycin C confirms that the reaction cascade involves C12- and C12b-hydroxylation, C2,3-dehydration, and stereospecific ketoreduction at C6. Enzymatic 18O incorporation studies verify that the oxygens at C12 and C12b derive from O2 and H2O, respectively. The results indicate that PgaM deviates mechanistically from flavoprotein monooxygenases, and suggest an alternative catalytic mechanism involving a quinone methide intermediate.

A Nested Gene in Streptomyces Bacteria Encodes a Protein Involved in Quaternary Complex Formation

“…15 Recent in vitro and in vivo functional studies have shown that PgaM is a multifunctional protein involved in angucycline post-PKS tailoring reactions. 16,17 It converts 12-hydroxy UWM6 into gaudimycin C in a complex reaction cascade. 16 The 492 aa N-terminal domain, which is homologous to flavoprotein oxygenases, catalyses hydroxylation of position 12b and C6 ketoreduction of the substrate 16 (Fig.…”

Characterization of JadH as an FAD‐ and NAD(P)H‐Dependent Bifunctional Hydroxylase/Dehydrase in Jadomycin Biosynthesis

“…JadH homologue, PgaE (involved in 6 biosynthesis) was proposed as not having 4a,12b-dehydrase activity by the fact that 4a,12b-dehydrated product was not observed in the in vitro bienzyme assays of PgaE and PgaM. [16] Sequence comparison of JadH and its homologues did not show clear differences between those deduced to catalyze 4a,12b-dehydration and those that probably not. Recently, the three-dimensional structures of PgaE and CabE (involved in the biosynthesis of 7) were determined by X-ray crystallography, [19] which were similar to those structures of previously characterized flavin-dependent aromatic hydroxylases.…”

“…[13,14] It was also shown to convert 2,3-dehydro-UWM6 (3) to dehydrorabelomycin (4) and 1 to rabelomycin (5) in vitro (Scheme 2). [13] Studies on the JadH homologues, (for example, PgaE (55.9 % identity) from the gaudimycin A (6), [15,16] CabE (55.1 % identity) from the gaudimycin B (7), [15,16] OvmOI (54.4 % identity) from the oviedomycin (8), [17] and GilOI (40.1 % identity) from the gilvocarcin V (9) gene clusters, [3,18] in other angucycline pathways also support the C12 monooxygenation function of JadH (Scheme 1).…”

“…This is consistent with the fact that all of these molecules possess a hydroxyl or keto group at their C12 position. These homologues include the aforementioned OvmOI, [17] PgaE, CabE, [14,16] GilOI, [18] and several other proteins such as KinO2 (69.2 % identity) from the kinamycin (13), [27] LanE (54.5 % identity) from the landomycin (14), [28] UrdE (54.9 % identity) from the urdamycin (15), [29] Pd2O (55.5 % identity) from the PD116740 (16), [30] and SimA7 (54.8 % identity) from the simocyclinone D8 (10) [20] gene clusters (Scheme 1).…”

“…The FAD-binding and middle domains are essential for hydroxylation catalysis; whereas the precise function of the C-terminal domain is unknown, which resembled thioredoxin in fold, but lacked two conserved cysteine residues in thioredoxin. [16] Unfortunately, because no protein structure of FAD-dependent hydroxylase/dehydrase is solved yet, investigating the dehydration activity of JadH homologues by structure comparison is still difficult. The mutational analyses of JadH function are underway in our lab; and they should shed some light on the dehydration activities of this group of enzymes.…”