gene was identified as the third gene in the xdhABC
operon. The overlapping start codon of xdhC
with the 3′ end of xdhB
indicated translational coupling of these genes, ensuring the synthesis of equimolar amounts of the corresponding proteins. Downstream of xdhC
, another open reading frame (ORF1) was identified, which is probably also cotranscribed with xdhABC
. Interposon mutagenesis demonstrated that ORF1 is not required for XDH activity (21
). The N-terminal amino acid sequence of ORF1 shows high similarity to ATP binding proteins from ABC transport systems. However, it remains speculative whether ORF1 belongs to an ABC transport system involved in purine uptake. In all systems studied so far, purines are imported into the cell via permeases and not by ABC transport systems (7
). At least under the conditions of high xanthine or hypoxanthine concentrations used in this study, ORF1 is not essential for purine uptake in R. capsulatus
It was shown that XDHC is required for XDH activity, although the protein is not a subunit of the active protein. Furthermore, XDHC neither constitutes a transcriptional regulator of xdh gene expression nor influences XDH stability. Based on the finding that XDH purified from an xdhC mutant strain contained iron-sulfur clusters as well as the FAD cofactor, but not MPT, a possible function of XDHC in MPT insertion into XDH has to be assumed.
The crystal structures of several molybdoenzymes revealed (reviewed in references 16
) that Moco is deeply buried within the protein, at the end of a funnel-shaped passage giving access only to the substrate. Therefore, it seems unlikely that Moco is inserted into the protein after the assembly and correct folding of the protein. Thus, it has to be assumed that during Moco biosynthesis, until MPT is completed, the apo-XDHAB tetramer has to stay in a suitable open conformation which enables XDHB to bind mature MPT (Fig. ). This is in line with the observation that XDH isolated from Moco biosynthesis mutants exhibited a change in electrophoretic mobility in native polyacrylamide gels, indicating a different folding of XDH in these mutant strains. Additionally, XDH isolated from xdhC
mutants, which was shown to be devoid of MPT, exhibited the same electrophoretic mobility as XDH in crude extracts of Moco biosynthesis mutants. Thus, the role of XDHC could be as a molybdopterin carrier protein, an MPT insertase, or a chaperone involved in proper folding of XDH during or after the insertion of MPT.
FIG. 6 Model for the assembly of XDH in R. capsulatus. MPT for XDH is synthesized in a series of reactions catalyzed by the moa, mod, moe, and mog gene products (28). Finally, MPT might be inserted into XDH by the XDHC protein. The role of XDHC could either (more ...)
As mentioned above, R. capsulatus
XDHC is not a subunit of XDH but is essential for XDH activity. Similarly, E. coli
NarJ does not belong to the nitrate reductase complex but is also required for nitrate reductase activity (2
). E. coli
nitrate reductase A is a membrane-bound molybdoenzyme composed of three subunits, NarG, NarH, and NarI, harboring MGD. It has been suggested that NarJ functions as a chaperone in nitrate reductase maturation, being involved in MGD insertion into NarG (4
). After the insertion of MGD a conformational change in nitrate reductase results in dissociation of NarJ from the protein (4
). Taking into account similar functions in Moco insertion of XDHC for XDH to those of NarJ for nitrate reductase, it can be assumed that XDHC might also act as a specific chaperone for XDH. However, XDHC could not be copurified with XDHAB in the absence of MPT.
The presence of specific chaperones for molybdoenzymes in prokaryotes seems to be widespread, since a chaperone for trimethylamine N
-oxide reductase (TMAO reductase) in E. coli
has recently been identified (TorD) (27
). TorD homologous proteins were also identified for DMSO reductases of R. sphaeroides
and R. capsulatus
(DmsB and DorD, respectively), suggesting similar functions of these proteins for DMSO and TMAO reductases (27
). Additionally, an ORF with weak similarity to xdhC
could be identified in the E. coli
genome (AE000136-3) (5
), which is located immediately downstream of three ORFs with homology to XDH (AE000136-6, AE000136-5, and AE000136-4) (21
In conclusion, it seems likely that each prokaryotic molybdoenzyme has its own system-specific chaperone that plays a special role in Moco insertion and target protein folding, which cannot be replaced by another protein. This might explain why none of these proteins have structural features in common, which might imply general functional homologies.