Abstract
The molybdenum cofactor (Moco) exists in different variants in the cell and can be directly inserted into molybdoenzymes utilizing the molybdopterin (MPT) form of Moco. In bacteria such as Rhodobacter capsulatus and Escherichia coli, MPT is further modified by attachment of a GMP nucleotide, forming MPT guanine dinucleotide (MGD). In this work, we analyzed the distribution and targeting of different forms of Moco to their respective user enzymes by proteins that bind Moco and are involved in its further modification. The R. capsulatus proteins MogA, MoeA, MobA, and XdhC were purified, and their specific interactions were analyzed. Interactions between the protein pairs MogA-MoeA, MoeA-XdhC, MoeA-MobA, and XdhC-MobA were identified by surface plasmon resonance measurements. In addition, the transfer of Moco produced by the MogA-MoeA complex to XdhC was investigated. A direct competition of MobA and XdhC for Moco binding was determined. In vitro analyses showed that XdhC bound to MobA, prevented the binding of Moco to MobA, and thereby inhibited MGD biosynthesis. The data were confirmed by in vivo studies in R. capsulatus cells showing that overproduction of XdhC resulted in a 50% decrease in the activity of bis-MGD-containing Me(2)SO reductase. We propose that, in bacteria, the distribution of Moco in the cell and targeting to the respective user enzymes are accomplished by specific proteins involved in Moco binding and modification.
Highlights
The meta-stable intermediate Precursor Z [2, 3]
For R. capsulatus xanthine dehydrogenase (XDH), the XdhC protein has been identified as a chaperone that is involved in the maturation of XDH [21, 23, 36]
In contrast to proteins that bind the bis-MPT guanine dinucleotide (MGD) form of molybdenum cofactor (Moco) such as R. capsulatus Me2SO reductase, MobA is not essential for the generation of active XDH, which contains the MPT form of Moco [24]
Summary
Bacterial Strains, Plasmids, Media, and Growth Conditions— E. coli BL21(DE3) cells were used for heterologous expression of the R. capsulatus proteins MobA, MoeA, and MogA. 1 mM MgCl2 and 1 mM GTP were added, and the mixtures were incubated for 60 min at room temperature in a total volume of 400 l of 100 mM Tris (pH 7.2) before the protein was denatured and analyzed for the presence of Moco or MGD by conversion to Form A (as described below). Formation of MGD by MobA in the Presence of XdhC—To analyze the influence of XdhC on Moco binding to MobA, 10 M MobA was incubated with 0, 1, 10, and 100 M XdhC for 10 min at room temperature before the addition of 1 mM GTP, 1 mM MgCl2, 1 mM Na2MoO4, and 80 M Moco. Unbound Moco was removed by gel filtration, and 50 l of the Moco-loaded MobA fraction was incubated with 1 mM Na2MoO4, 1 mM GTP, and increasing amounts of XdhC (0, 1.16, 11.6, and 116 M) in a total volume of 400 l in 100 mM Tris (pH 7.2). Me2SO reductase activity (units/mg) is defined as the reduction of 1 mol of Me2SO/min/mg of protein
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