The Molybdenum Cofactor Biosynthesis Network: In vivo Protein-Protein Interactions of an Actin Associated Multi-Protein Complex

Abstract

Survival of plants and nearly all organisms depends on the pterin based molybdenum cofactor (Moco) as well as its effective biosynthesis and insertion into apo-enzymes. To this end, both the central Moco biosynthesis enzymes are characterized and the conserved four-step reaction pathway for Moco biosynthesis is well-understood. However, protection mechanisms to prevent degradation during biosynthesis as well as transfer of the highly oxygen sensitive Moco and its intermediates are not fully enlightened. The formation of protein complexes involving transient protein-protein interactions is an efficient strategy for protected metabolic channelling of sensitive molecules. In this review, Moco biosynthesis and allocation network is presented and discussed. This network was intensively studied based on two in vivo interaction methods: bimolecular fluorescence complementation (BiFC) and split-luciferase. Whereas BiFC allows localisation of interacting partners, split-luciferase assay determines interaction strengths in vivo. Results demonstrate (i) interaction of Cnx2 and Cnx3 within the mitochondria and (ii) assembly of a biosynthesis complex including the cytosolic enzymes Cnx5, Cnx6, Cnx7, and Cnx1, which enables a protected transfer of intermediates. The whole complex is associated with actin filaments via Cnx1 as anchor protein. After biosynthesis, Moco needs to be handed over to the specific apo-enzymes. A potential pathway was discovered. Molybdenum-containing enzymes of the sulphite oxidase family interact directly with Cnx1. In contrast, the xanthine oxidoreductase family acquires Moco indirectly via a Moco binding protein (MoBP2) and Moco sulphurase ABA3. In summary, the uncovered interaction matrix enables an efficient transfer for intermediate and product protection via micro-compartmentation.

Keywords: bimolecular fluorescent complementation (BiFC); cytoskeleton; metabolic channelling; molybdenum cofactor; protein-protein interaction network; split-luciferase.

Figure 1

(A) The Moco enzyme pathway with the four-step reaction catalysed by Cnx2 and Cnx3 inside mitochondria as well as Cnx6, Cnx7, Cnx5, and Cnx1 in the cytosol. After biosynthesis, Moco is distributed to both families of Mo-enzymes and to Moco binding proteins. (B) Schematic presentation of an interaction approach and the necessary three controls for a split-protein assay. Depicted are the used constructs with the N-terminal (R-N) and the C-terminal (R-C) fragment of a reporter. (B1) The interaction approach needs two POI, while the negative control replace one POI for a non-interacting negative control protein (NC). (B2) In the abundance controls, a non-interacting abundance control protein (AC) is used. (C) FLuCI interaction study between ABA3 and MoBP2. Analysed was the full-length protein (FL) as well as the single N-terminal (NT) and C-terminal domain. Shown is the split-luciferase factor of the interaction approach in relation to the negative control. In addition, the split-luciferase factors of the two abundance controls are depicted, demonstrating the background factor without interaction. For interaction between the POI's, the split-luciferase factor has to be higher than the background factor.

Figure 2

Schematic presentation of the Moco biosynthesis interaction network. The cytosolic Moco biosynthesis enzymes Cnx5, Cnx6, Cnx7, and Cnx1 form a multi-enzyme complex on actin filaments. Molybdate as substrate is provided by molybdate transporters. An assembly of the Moco biosynthesis complex near these transporters at the cytoskeleton is hypothesised. After insertion of Mo from molybdate, di-oxo Moco is inserted into enzymes of the SO family via interaction with Cnx1. The enzymes of the XOR-family receive the mono-oxo form of Moco from ABA3, which generates this form of Moco from di-oxo Moco supplied by Cnx1 via MoBP2 as mediating protein.