Structure of a Baculovirus Sulfhydryl Oxidase, a Highly Divergent Member of the Erv Flavoenzyme Family

Abstract

Genomes of nucleocytoplasmic large DNA viruses (NCLDVs) encode enzymes that catalyze the formation of disulfide bonds between cysteine amino acid residues in proteins, a function essential for the proper assembly and propagation of NCLDV virions. Recently, a catalyst of disulfide formation was identified in baculoviruses, a group of large double-stranded DNA viruses considered phylogenetically distinct from NCLDVs. The NCLDV and baculovirus disulfide catalysts are flavin adenine dinucleotide (FAD)-binding sulfhydryl oxidases related to the cellular Erv enzyme family, but the baculovirus enzyme, the product of the Ac92 gene in Autographa californica multiple nucleopolyhedrovirus (AcMNPV), is highly divergent at the amino acid sequence level. The crystal structure of the Ac92 protein presented here shows a configuration of the active-site cysteine residues and bound cofactor similar to that observed in other Erv sulfhydryl oxidases. However, Ac92 has a complex quaternary structural arrangement not previously seen in cellular or viral enzymes of this family. This novel assembly comprises a dimer of pseudodimers with a striking 40-degree kink in the interface helix between subunits. The diversification of the Erv sulfhydryl oxidase enzymes in large double-stranded DNA viruses exemplifies the extreme degree to which these viruses can push the boundaries of protein family folds.In contrast to proteins that traverse the secretory pathway, cytosolic and nuclear proteins in mesophilic organisms rarely evolve to contain structural disulfide bonds. Some exceptions to this generalization are structural proteins encoded by nucleocytoplasmic large DNA viruses (NCLDVs), which do contain disulfides despite folding in an environment that typically is reducing (16). To promote disulfide formation in the cytosol or nucleus, NCLDVs (such as poxviruses, mimivirus, African swine fever virus [ASFV], iridoviruses, phycodnaviruses, and others) encode catalysts of disulfide formation (38, 40) similarly to cellular enzymes of the Erv (for essential for respiration and viability) family. The cellular Erv enzymes promote oxidative folding in the mitochondrial intermembrane space (30) and certain other biological settings (14, 41). Recently, an enzyme capable of catalyzing disulfide formation was identified in baculoviruses (28), a group of large double-stranded DNA viruses not classified as NCLDVs (21). The baculovirus sulfhydryl oxidase enzyme, one of a core group of conserved baculovirus gene products, is essential for virion assembly and thus for virus propagation (31, 48), similarly to its counterparts in NCLDVs (25, 39).The sulfhydryl oxidase encoded by the Ac92 gene in the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) contains amino acid sequence features shared by other cellular and viral sulfhydryl oxidases (28). In particular, a CXXC motif at the amino terminus of a predicted helix corresponds to the active-site disulfide. A motif consisting of a tryptophan, three histidine, and two asparagine amino acid...

Exploring ORFan Domains in Giant Viruses: Structure of Mimivirus Sulfhydryl Oxidase R596

“…We previously observed that the manner in which the FAD-binding fold of the viral sulfhydryl oxidases assembles into quaternary structures varies widely among NCLDVs and other large viruses [7], [10]. In the current study, we show that viral sulfhydryl oxidases exhibit tertiary as well as quaternary structural innovation.…”

“…Specifically, the African swine fever virus sulfhydryl oxidase, pB119L, and baculovirus Ac92 both use orthogonal protein surfaces for dimerization compared to cellular Erv sulfhydryl oxidases. In fact, the pB119L and Ac92 dimerization interfaces are orthogonal to one another as well [7], [10]. Here we present the structure of the intact mimivirus sulfhydryl oxidase R596, including both its sulfhydryl oxidase domain and its ORFan domain.…”

“…Other viral disulfide catalysts, however, have additional domain-sized segments fused to the Erv module. For example, the baculovirus sulfhydryl oxidase, Ac92, contains an amino-terminal fusion with a folded structure, but this domain does not seem to have a redox role in catalysis [7]. Mimivirus, ascovirus, and nudivirus sulfhydryl oxidases contain carboxy-terminal fusions, which bear no resemblance to any known protein or domain and thus may be considered ORFan regions [8].…”

The baculovirus sulfhydryl oxidase Ac92 (P33) interacts with the Spodoptera frugiperda P53 protein and oxidizes it in vitro

“…Viruses with a deletion in ac92 or a mutation in the sequence CXXC (Wu and Passarelli, 2010), a sulfhydryl oxidase motif important for oxidation in cellular enzymes (Fass, 2008), exhibited similar phenotypes, suggesting a requirement for disulfide bond formation in the proper assembly and propagation of AcMNPV virions. The structure of Ac92 revealed that the arrangement of active-site cysteine residues and bound flavin adenine dinucleotide cofactor is similar to that observed in other Erv family sulfhydryl oxidases (Hakim et al, 2011). Although Ac92 is a functional sulfhydryl oxidase and its enzymatic activity is essential for proper ODV formation and BV production, the target substrate(s) of Ac92 during baculovirus infection is unknown.…”

The Trichoplusia ni single nucleopolyhedrovirus tn79 gene encodes a functional sulfhydryl oxidase enzyme that is able to support the replication of Autographa californica multiple nucleopolyhedrovirus lacking the sulfhydryl oxidase ac92 gene

“…In addition, the structure of a sulfhydryl oxidase from the NCLDV mimivirus has been derived (Hakim et al, 2012). The sulfhydryl oxidases of the baculoviruses Autographa californica M nucleopolyhedrovirus (AcMNPV), Ac92 (P33), and Bombyx mori NPV, Bm75, have also been characterized structurally (Hakim et al, 2011; Hou et al, 2012) and functionally (Hou et al, 2012; Nie et al, 2011; Wu and Passarelli, 2010). …”