Elucidation of the dual role of Mycobacterial MoeZR in molybdenum cofactor biosynthesis and cysteine biosynthesis

Abstract

The pathway of molybdenum cofactor biosynthesis has been studied in detail by using proteins from Mycobacterium species, which contain several homologs associated with the first steps of Moco biosynthesis. While all Mycobacteria species contain a MoeZR, only some strains have acquired an additional homolog, MoeBR, by horizontal gene transfer. The role of MoeBR and MoeZR was studied in detail for the interaction with the two MoaD-homologs involved in Moco biosynthesis, MoaD1 and MoaD2, in addition to the CysO protein involved in cysteine biosynthesis. We show that both proteins have a role in Moco biosynthesis, while only MoeZR, but not MoeBR, has an additional role in cysteine biosynthesis. MoeZR and MoeBR were able to complement an E. coli moeB mutant strain, but only in conjunction with the Mycobacterial MoaD1 or MoaD2 proteins. Both proteins were able to sulfurate MoaD1 and MoaD2 in vivo, while only MoeZR additionally transferred the sulfur to CysO. Our in vivo studies show that Mycobacteria have acquired several homologs to maintain Moco biosynthesis. MoeZR has a dual role in Moco- and cysteine biosynthesis and is involved in the sulfuration of MoaD and CysO, whereas MoeBR only has a role in Moco biosynthesis, which is not an essential function for Mycobacteria.

Figure 1. Purification of M. tuberculosis MoeZR and MoeBR after heterologous expression in E. coli.

5 µg of the purified proteins were separated by 15% SDS-PAGE and stained with Coomassie Blue. 1: MBP-MoeZR, 2: MBP-MoeBR. The band at 45 kDa in the MoeBR sample was shown to be MBP.

Figure 2. Circular Dichroism (CD) spectroscopy of MoeBR and MoeZR.

CD on purified MBP-MoeBR and MBP_MoeZR were recorded in a 0.1 cm path-length Suprasil quartz cell with a Jasco-J715 CD spectrometer. Far-UV CD was recorded from 260-198 nm using a step size of 1 nm with a signal averaging time of 4 seconds at each wavelength step. 200 µL of MoeBR and MoeZR at a concentration of 0.1 mg/mL was used.

Figure 3. Copurification of MoeBR or MoeZR with MoaD1, MoaD2 and CysO.

MBP-MoeBR and MBP-MoeZR were coexpressed in an E. coli moeB⁻ strain together with His₆-MoaD1, His₆-MoaD2 or His₆-CysO and purified by Ni-NTA chromatography afterwards. As control, His₆-MoaD1, His₆-MoaD2 or His₆-CysO were also expressed without MoeBR and MoeZR in the E. coli moeB⁻ strain. The proteins were separated by 15% SDS-PAGE and stained with Coomassie Blue or, in addition, the presence of MBP-MoeBR and MBP-MoeZR was determined by immunodetection using polyclonal MBP antisera (Sigma). 1: His₆-MoaD1, 2: His₆-MoaD2, 3: His₆-CysO, 4: His₆-MoaD1/MBP-MoeZR, 5: His₆-MoaD2/MBP-MoeZR, 6: His₆-CysO/MBP-MoeZR, 7: His₆-MoaD1/MBP-MoeBR, 8: His₆-MoaD2/MBP-MoeBR, 9: His₆-CysO/MBP-MoeBR.

Figure 4. Deconvoluted ESI mass spectra of MoaD1, MoaD2, and CysO.

The sulfuration level of His₆-MoaD1 (A–C), His₆-MoaD2 (D–F) and His₆-CysO (G–I) was analyzed after expression in E. coli moeB⁻ strain either alone or after coexpression with MBP-MoeZR or MBP-MoeBR. After coexpression, His₆-MoaD1, His₆-MoaD2 and His₆-CysO were purified by Ni-NTA chromatography and subjected to ESI-MS. The mass increase of +16 Da corresponds to the exchange of an oxygen versus a sulfur atom to generate thiocarboxylated proteins. (A) His₆-MoaD1, (B) His₆-MoaD1 after coexpression with MBP-MoeZ, (C) His₆-MoaD1 after coexpression with MBP-MoeBR. (D) His₆-MoaD2, (E) His₆-MoaD2 after coexpression with MBP-MoeZR, (F) His₆-MoaD2 after coexpression with MBP-MoeBR. (G) His₆-CysO, (H) His₆-CysO after coexpression with MBP-MoeZR, (I) His₆-CysO after coexpression with MBP-MoeBR. Additional peaks with mass increments of 22 Da are due to the corresponding Na-salts of proteins.

Figure 5. Amino acid sequence comparison of E. coli MoaD, M. tuberculosis MoaD1, M. tuberculosis MoaD2, and M. tuberculosis CysO.

Identical amino acids are boxed in black and homologous amino acids residues are shaded in grey.

Figure 6. Proposed model for the role of MoeZR and MoeBR in Mycobacteria.

Functional complementation studies of an E. coli moeB⁻ strain showed that both MoeZR and MoeBR are able to interact with either MoaD1 or MoaD2. In addition, MoeZR is able to sulfurate CysO for cysteine biosynthesis, while MoeBR is not able to interact with CysO.