Purification and biochemical characterization of methanobactin biosynthetic enzymes

Abstract

Methanobactin (Mbn) is a ribosomally synthesized and post-translationally modified peptide (RiPP) natural product that binds Cu(I) with high affinity. The copper-chelating thioamide/oxazolone groups in Mbn are installed on the precursor peptide MbnA by the core enzyme complex, MbnBC, which includes the multinuclear non-heme iron-dependent oxidase (MNIO) MbnB and its RiPP recognition element-containing partner protein MbnC. For the extensively characterized Mbn biosynthetic gene cluster (BGC) from the methanotroph Methylosinus trichosporium OB3b, the tailoring aminotransferase MbnN further modifies MbnA after leader sequence cleavage by an unknown mechanism. Here we detail methods to express and purify M. trichosporium OB3b MbnBC and MbnN along with protocols for assessing MbnA modification by MbnBC and MbnN aminotransferase activity. In addition, we describe crystallization and structure determination of MbnBC. These procedures can be adapted for other MNIOs and partner proteins encoded in Mbn and Mbn-like BGCs. Furthermore, these methods provide a first step toward in vitro biosynthesis of Mbns and related natural products as potential therapeutics.

Keywords: Aminotransferase; Chalkophore; Copper homeostasis; DUF692; Heterologous expression; MNIO; Methanobactin; Methanotroph; Multinuclear iron enzyme; RiPP natural product.

Fig. 1

Biosynthetic pathway of M. trichosporium OB3b Mbn. The post-translational modifications installed by MbnBC and MbnN are shown in orange and maroon, respectively. The two nitrogen and two sulfur atoms from the oxazolone/thioamide pairs form a fourcoordinate binding site for Cu(I).

Fig. 2

Denaturing SDS-PAGE (16% Tris-glycine) of purified M. trichosporium OB3b MbnBC (~85 μM).

Fig. 3

Reaction of 100 μM ascorbate-reduced MbnBC with 300 μM MbnA over 30 min. Inset depicts the difference spectrum between the first and last spectrum.

Fig. 4

Crystallization and structure of MbnBC. (A) MbnBC crystals visualized by light microscope. (B) Asymmetric unit of the MbnBC crystal lattice containing 2 copies of MbnBC colored in two shades of orange (MbnB) and green (MbnC) (PDB: 7TCR). The Fe ions are colored in teal.

Fig. 5

Purification of M. trichosporium OB3b MbnN. (A) Denaturing 16% Tris-glycine SDS-PAGE of ~20 μM MbnN. (B) UV-Vis spectrum of ~3 μM MbnN. (C) Native PAGE (4–16% Bis-Tris) of ~20 μM MbnN.