Abstract
The mimivirus genome contains many genes that lack homologs in the sequence database and are thus known as ORFans. In addition, mimivirus genes that encode proteins belonging to known fold families are in some cases fused to domain-sized segments that cannot be classified. One such ORFan region is present in the mimivirus enzyme R596, a member of the Erv family of sulfhydryl oxidases. We determined the structure of a variant of full-length R596 and observed that the carboxy-terminal region of R596 assumes a folded, compact domain, demonstrating that these ORFan segments can be stable structural units. Moreover, the R596 ORFan domain fold is novel, hinting at the potential wealth of protein structural innovation yet to be discovered in large double-stranded DNA viruses. In the context of the R596 dimer, the ORFan domain contributes to formation of a broad cleft enriched with exposed aromatic groups and basic side chains, which may function in binding target proteins or localization of the enzyme within the virus factory or virions. Finally, we find evidence for an intermolecular dithiol/disulfide relay within the mimivirus R596 dimer, the first such extended, intersubunit redox-active site identified in a viral sulfhydryl oxidase.
Highlights
More than half the approximately 900 predicted protein-coding genes in Acanthamoeba polyphaga mimivirus are ORFans [1], i.e., they have no detectable homologs in sequence databases and no predicted functions [2]
Mimivirus R596 is an enzyme of the Erv family of sulfhydryl oxidases, which catalyze the formation of disulfide bonds using an active-site di-cysteine motif juxtaposed to a flavin adenine dinucleotide (FAD) cofactor [4]
Comparing the R596 orthologs in Cafeteria roenbergensis virus (CroV) [11], Megavirus chilensis [12], and a few additional sequences likely to have arisen from uncharacterized viruses, we observed that the two cysteines immediately downstream of the Erv domain are the most highly conserved aside from the activesite disulfide (Figure 1)
Summary
More than half the approximately 900 predicted protein-coding genes in Acanthamoeba polyphaga mimivirus are ORFans [1], i.e., they have no detectable homologs in sequence databases and no predicted functions [2]. The origin of ORFan proteins and domains is poorly understood It remains to be determined how many ORFans correspond to known folds but fall below the threshold for detection on the basis of sequence homology or fold recognition, versus how many truly represent novel structural units. Data addressing this question may help determine whether mimivirus and other nucleocytoplasmic large DNA viruses (NCLDVs) exhibit so many apparent ORFans due to rapid sequence divergence or through a mechanism for generating novel folds. Found within these flanking regions is a second redox-active cysteine pair, which interacts with substrate and transfers electrons to the FADproximal disulfide [5,6]
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