The Role of the Nucleotides in the Insertion of the bis-Molybdopterin Guanine Dinucleotide Cofactor into apo-Molybdoenzymes

Abstract

The role of the GMP nucleotides of the bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor of the DMSO reductase family has long been a subject of discussion. The recent characterization of the bis-molybdopterin (bis-Mo-MPT) cofactor present in the E. coli YdhV protein, which differs from bis-MGD solely by the absence of the nucleotides, now enables studying the role of the nucleotides of bis-MGD and bis-MPT cofactors in Moco insertion and the activity of molybdoenzymes in direct comparison. Using the well-known E. coli TMAO reductase TorA as a model enzyme for cofactor insertion, we were able to show that the GMP nucleotides of bis-MGD are crucial for the insertion of the bis-MGD cofactor into apo-TorA.

Keywords: TMAO reductase; bis-MGD; chaperone; molybdenum cofactor.

Figure 1

Different structures of Moco in bacteria. The basic form of Moco (Mo-MPT) is a 5,6,7,8-tetrahydropyranopterin (MPT) with a dithiolene group coordinating the molybdenum atom in a trioxo form. Moco exists in different variants and is divided into three enzyme families according to the coordination at the molybdenum atom: the SO family, the XO family and the DMSO reductase family. The SO family is characterized by a molybdenum ligation with one oxo, one hydroxide and one cysteine ligand from the protein backbone. In E. coli, the XO family contains the sulfurated molybdopterin cytosine dinucleotide cofactor (MCD), while in other bacteria the sulfurated form of Mo-MPT without an additional nucleotide has also been identified. The DMSO reductase family contains two MPTs (bis-Mo-MPT) or two MGDs (bis-MGD) ligated to one molybdenum atom with additional ligands being an O/S and a sixth ligand which can be a serine, a cysteine, a selenocysteine, an aspartate or a hydroxide ligand.

Scheme 1

Overview of the different incubations for insertion of the different cofactor forms into apo-TorA or Apo-VdhV. Details are given in the text and Section 3.

Figure 2

Conversion efficiency over time for enzymatic conversion of cofactors. (A) Conversion of bis-MPT from 30 µM YdhV to bis-MGD catalyzed by 3 µM MobA in presence of 1 mM GTP and 1 mM MgCl₂. (B) Conversion of bis-MGD from 30 µM wtTorA to bis-MPT catalyzed by 10 U/mL phosphodiesterase I (PD). (C) Conversion of bis-MPT from 30 µM YdhV to dephospho-bis-MPT catalyzed by 10 U/mL alkaline phosphatase (AP). The reactions were terminated by the addition of acidic I₂/KI solution. The cofactors were quantified in form of their fluorescent oxidation products FormA-GMP and dephospho-FormA. On the right-hand side next to the panels, a scheme is shown of the enzymatic reaction that was performed to convert the cofactor into a different form.

Figure 3

Cofactor binding to apo-TorA, apo-YdhV and BSA. The bis-MGD content of 1.3 µM apo-TorA (A), apo-YdhV (C) and BSA (D) is shown after incubation with bis-MGD from different sources: bis-MGD isolated from wtTorA as donor (circles), bis-MGD formed in vitro out of bis-MPT in a MobA-catalyzed reaction (squares), residual bis-MGD after conversion into bis-MPT in a PD-catalyzed reaction (triangles). Bis-MGD was detected in form of its fluorescent oxidation product FormA-GMP. Panel (B) represents the specific TMAO reductase activity of the samples in panel A. The bis-Mo-MPT content of 1.3 µM apo-TorA (E), apo-YdhV (F) and BSA (G) is shown after incubation with bis-Mo-MPT from different sources: bis-Mo-MPT isolated from YdhV as donor (circles), residual bis-Mo-MPT after conversion into bis-MGD in a MobA-catalyzed reaction (squares), bis-Mo-MPT formed in vitro out of bis-MGD in a PD-catalyzed reaction (triangles). Bis-Mo-MPT was detected in form of its fluorescent oxidation product dephospho-FormA. No TMAO reductase activity was detected for these samples.

Figure 4

Effect of the presence of TorD and effect of free nucleotides in the reconstitution mixture on the insertion of bis-Mo-MPT into apo-TorA. Here, 1.3 µM apo-TorA, 2 µM TorD or both were incubated with bis-Mo-MPT isolated from 30 µM YdhV, and 50 mM GMP, GDP or GTP was added prior to the reconstitution mixture. Bis-Mo-MPT was detected in form of its fluorescent oxidation product dephospho-FormA.

Figure 5

Bis-Mo-MPT content of 1.3 µM apo-TorA (A), apo-YdhV (B) and BSA (C) incubated with bis-Mo-MPT or dephospho-bis-MPT. Bis-Mo-MPT (circles) was directly isolated from YdhV. Dephospho-bis-Mo-MPT (triangles) was obtained after in vitro hydrolysis of the terminal bis-Mo-MPT phosphate group by alkaline phosphatase (AP). Bis-MPT and dephospho-bis-MPT were detected in form of their fluorescent oxidation product dephospho-FormA.