Abstract
Pterin-4a-carbinolamine dehydratases (PCDs) recycle oxidized pterin cofactors generated by aromatic amino acid hydroxylases (AAHs). PCDs are known biochemically only from animals and one bacterium, but PCD-like proteins (COG2154 in the Clusters of Orthologous Groups [COGs] database) are encoded by many plant and microbial genomes. Because these genomes often encode no AAH homologs, the annotation of their COG2154 proteins as PCDs is questionable. Moreover, some COG2154 proteins lack canonical residues that are catalytically important in mammalian PCDs. Diverse COG2154 proteins of plant, fungal, protistan, and prokaryotic origin were therefore tested for PCD activity by functional complementation in Escherichia coli, and the plant proteins were localized using green fluorescent protein fusions. Higher and lower plants proved to have two COG2154 proteins, a mitochondrial one with PCD activity and a noncanonical, plastidial one without. Phylogenetic analysis indicated that the latter is unique to plants and arose from the former early in the plant lineage. All 10 microbial COG2154 proteins tested had PCD activity; six of these came from genomes with no AAH, and six were noncanonical. The results suggested the motif [EDKH]-x(3)-H-[HN]-[PCS]-x(5,6)-[YWF]-x(9)-[HW]-x(8,15)-D as a signature for PCD activity. Organisms having a functional PCD but no AAH partner include angiosperms, yeast, and various prokaryotes. In these cases, PCD presumably has another function. An ancillary role in molybdopterin cofactor metabolism, hypothesized from phylogenomic evidence, was supported by demonstrating significantly lowered activities of two molybdoenzymes in Arabidopsis thaliana PCD knockout mutants. Besides this role, we propose that partnerless PCDs support the function of as yet unrecognized pterin-dependent enzymes.
Highlights
Pterin-4a-carbinolamine dehydratases (PCDs) recycle oxidized pterin cofactors generated by aromatic amino acid hydroxylases (AAHs)
Given the strong probability of PCD activity that is implied by association evidence, it is noteworthy that microbial COG2154 proteins that are associated in these ways with AAH quite often lack a canonical catalytic motif (Fig. 3C)
The results of in vivo tests of COG2154 sequences from plants and diverse microorganisms (Fig. 4; Wang et al, 2006) allow definition of a new catalytic motif that can be used as a signature for PCD activity in any organism: [EDKH]-x(3)-H-[HN]-[PCS]-x(5,6)-[YWF]x(9)-[HW]-x(8,15)-D
Summary
We first systematically analyzed the distribution of COG2154 sequences in relation to that of AAH sequences in plants, fungi, protists, and prokaryotes. Given the strong probability of PCD activity that is implied by association evidence (i.e. co-occurrence and clustering), it is noteworthy that microbial COG2154 proteins that are associated in these ways with AAH quite often lack a canonical catalytic motif (Fig. 3C). This implies that the current motif (based only on PCDs from animals and one bacterium) is too narrowly defined. All type 1 sequences (whose catalytic motif is close to canonical) had PCD activity, whereas all the type 2 sequences lacked activity (Fig. 4, top four rows). COG2154 sequences in GenBank are occasionally annotated as ‘‘putative molybdopterin biosynthesis protein.’’
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