EcdGHK Are Three Tailoring Iron Oxygenases for Amino Acid Building Blocks of the Echinocandin Scaffold

Abstract

The echinocandins are a small group of fungal N-acylated cyclic hexapeptides that are fungicidal for candida strains and fungistatic for aspergilli by targeting cell wall 1,3-β-glucan synthases. The side chains of all six amino acid building blocks have hydroxyl groups, including the nonproteinogenic 4R,5R-dihydroxy-Orn1, 4R-OH-Pro3, 3S, 4S-dihydroxy-homoTyr4 and 3S-OH-4S-Me-Pro6. The echinocandin (ecd) gene cluster contains two predicted nonheme mononuclear iron oxygenase genes (ecdG,K) and one encoding a P450 type heme protein (ecdH). Deletion of the ecdH gene in the producing Emericella rugulosa generates an echinocandin scaffold (echinocandin D) lacking both hydroxyl groups on Orn1. Correspondingly, the ΔecdG strain failed to hydroxylate C3 of the homoTyr residue, and purified EcdG hydroxylated free L-homoTyr at C3. The ΔecdK strain failed to generate mature echinocandin unless supplemented with either 4R-Me-Pro or 3S-OH-4S-Me-Pro, indicating blockage of a step upstream of Me-Pro formation. Purified EcdK is a Leu 5-hydroxylase, acting iteratively at C5 to yield γ-Me-Glu-γ-semialdehyde in equilibrium with the cyclic imine product. Evaluation of deshydroxyechinocandin scaffolds in in vitro anticandidal assays revealed up to 3-fold loss of potency for the ΔecdG scaffolds, but a 3-fold gain of potency for the ΔecdH scaffold, in line with prior results on deoxyechinocandin homologs.

Structural diversity in echinocandin biosynthesis: the impact of oxidation steps and approaches toward an evolutionary explanation

“…After oxidative decarboxylation, an α-keto acid is obtained, which is finally transaminated to provide homotyrosine. In a subsequent 2013 study, Tang, Walsh, and colleagues revealed the activity of the three oxygenases in the Ecd section of Ecd/Hty, which will be discussed in the next section [16].…”

“…Figure 3) [16]. Nevertheless, the order of biosynthetic steps, especially of the oxidations catalyzed by up to six dioxygenases, remains enigmatic.…”

“…Figure 3 and Table 4) [16]. Deletion of the αKG-dependent LDO EcdK resulted in a breakdown of echinocandin B biosynthesis; however, a minimal activity was detected in vitro with the heterologously expressed enzyme: the pro-(S)-methyl group of leucine was first hydroxylated and then further oxidized to the aldehyde, which undergoes spontaneous condensation to the cyclic imine.…”

“…It is assumed that, in vivo, the imine is reduced to trans-4-methylproline by a dehydrogenase whose gene is not included in the cluster. Genomic deletion and in vitro experiments have consistently shown that the αKG-dependent non-heme dioxygenase EcdG is a homotyrosine 3-hydroxylase [16]. It was found that EcdG acts upon the free amino acid, but not upon the homotyrosine residue in echinocandin D (cf.…”

“…Further information about the order of oxidation steps was deduced from deletion of ecdG and ecdH [16]. Echinocandins produced by these strains not only lack the Brought to you by | MIT Libraries Authenticated Download Date | 5/12/18 7:55 PM hydroxyl groups introduced by the corresponding gene, the hydroxylation at other positions was also incomplete.…”