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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.

SUBMITTER: Hakim M

PROVIDER: S-EPMC3165737 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Structure of a baculovirus sulfhydryl oxidase, a highly divergent member of the erv flavoenzyme family.

Hakim Motti M Mandelbaum Amitai A Fass Deborah D

Journal of virology 20110713 18

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 (FA ...[more]

PMID: 21752922

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Project description:Typically, simple flavoprotein oxidases couple the oxidation of their substrates with the formation of hydrogen peroxide without release of significant levels of the superoxide ion. However, two evolutionarily related single-domain sulfhydryl oxidases (Erv2p; a yeast endoplasmic reticulum resident protein and augmenter of liver regeneration, ALR, an enzyme predominantly found in the mitochondrial intermembrane) release up to ~30% of the oxygen they reduce as the superoxide ion. Both enzymes oxidize dithiol substrates via a redox-active disulfide adjacent to the flavin cofactor within the helix-rich Erv domain. Subsequent reduction of the flavin is followed by transfer of reducing equivalents to molecular oxygen. Superoxide release was initially detected using tris(3-hydroxypropyl)phosphine (THP) as an alternative reducing substrate to dithiothreitol (DTT). THP, and other phosphines, showed anomalously high turnover numbers with Erv2p and ALR in the oxygen electrode, but oxygen consumption was drastically suppressed upon the addition of superoxide dismutase. The superoxide ion initiates a radical chain reaction promoting the aerobic oxidation of phosphines with the formation of hydrogen peroxide. Use of a known flux of superoxide generated by the xanthine/xanthine oxidase system showed that one superoxide ion stimulates the reduction of 27 and 4.5 molecules of oxygen using THP and tris(2-carboxyethyl)phosphine (TCEP), respectively. This superoxide-dependent amplification of oxygen consumption by phosphines provides a new kinetic method for the detection of superoxide. Superoxide release was also observed by a standard chemiluminescence method using a luciferin analogue (MCLA) when 2 mM DTT was employed as a substrate of Erv2p and ALR. The percentage of superoxide released from Erv2p increased to ~65% when monomeric mutants of the normally homodimeric enzyme were used. In contrast, monomeric multidomain quiescin sulfhydryl oxidase enzymes that also contain an Erv FAD-binding fold release only 1-5% of their total reduced oxygen species as the superoxide ion. Aspects of the mechanism and possible physiological significance of superoxide release from these Erv-domain flavoproteins are discussed.

Project description:Baculoviruses encode a conserved sulfhydryl oxidase, P33, which is necessary for budded virus (BV) production and multinucleocapsid occlusion-derived virus (ODV) formation. Here, the structural and functional relationship of P33 was revealed by X-ray crystallography, site-directed mutagenesis, and functional analysis. Based on crystallographic characterization and structural analysis, a series of P33 mutants within three conserved regions, i.e., the active site, the dimer interface, and the R127-E183 salt bridge, were constructed. In vitro experiments showed that mutations within the active site and dimer interface severely impaired the sulfhydryl oxidase activity of P33, while the mutations in the salt bridge had a relatively minor influence. Recombinant viruses containing mutated P33 were constructed and assayed in vivo Except for the active-site mutant AXXA, all other mutants produced infectious BVs, although certain mutants had a decreased BV production. The active-site mutant H114A, the dimer interface mutant H227D, and the salt bridge mutant R127A-E183A were further analyzed by electron microscopy and bioassays. The occlusion bodies (OBs) of mutants H114A and R127A-E183A had a ragged surface and contained mostly ODVs with a single nucleocapsid. The OBs of all three mutants contained lower numbers of ODVs and had a significantly reduced oral infectivity in comparison to control virus. Crystallographic analyses further revealed that all three regions may coordinate with one another to achieve optimal function of P33. Taken together, our data revealed that all the three conserved regions are involved in P33 activity and are crucial for virus morphogenesis and peroral infectivity.IMPORTANCE Sulfhydryl oxidase catalyzes disulfide bond formation of substrate proteins. P33, a baculovirus-encoded sulfhydryl oxidase, is different from other cellular and viral sulfhydryl oxidases, bearing unique features in tertiary and quaternary structure organizations. In this study, we found that three conserved regions, i.e., the active site, dimer interface, and the R127-E183 salt bridge, play important roles in the enzymatic activity and function of P33. Previous observations showed that deletion of p33 results in a total loss of budded virus (BV) production and in morphological changes in occlusion-derived virus (ODV). Our study revealed that certain P33 mutants lead to occlusion bodies (OBs) with a ragged surface, decreased embedded ODVs, and reduced oral infectivity. Interestingly, some P33 mutants with impaired ODV/OB still retained BV productivity, indicating that the impacts on BV and on ODV/OB are two distinctly different functions of P33, which are likely to be performed via different substrate proteins.

Project description:Quiescin sulfhydryl oxidase (QSOX) flavoenzymes catalyze the direct, facile, insertion of disulfide bonds into reduced unfolded proteins with the reduction of oxygen to hydrogen peroxide. To date, only QSOXs from vertebrates have been characterized enzymatically. These metazoan sulfhydryl oxidases have four recognizable domains: a redox-active thioredoxin (Trx) domain containing the first of three CxxC motifs (C(I)-C(II)), a second Trx domain with no obvious redox-active disulfide, a helix-rich domain, and then an Erv/ALR domain. This last domain contains the FAD moiety, a proximal C(III)-C(IV) disulfide, and a third CxxC of unknown function (C(V)-C(VI)). Plant and protist QSOXs lack the second Trx domain but otherwise appear to contain the same complement of redox centers. This work presents the first characterization of a single-Trx QSOX. Trypanosoma brucei QSOX was expressed in Escherichia coli using a synthetic gene and found to be a stable, monomeric, FAD-containing protein. Although evidently lacking an entire domain, TbQSOX shows catalytic activity and substrate specificity similar to the vertebrate QSOXs examined previously. Unfolded reduced proteins are more than 200-fold more effective substrates on a per thiol basis than glutathione and some 10-fold better than the parasite bisglutathione analogue, trypanothione. These data are consistent with a role for the protist QSOX in oxidative protein folding. Site-directed mutagenesis of each of the six cysteine residues (to serines) shows that the CxxC motif in the single-Trx domain is crucial for efficient catalysis of the oxidation of both reduced RNase and the model substrate dithiothreitol. As expected, the proximal disulfide C(III)-C(IV), which interacts with the flavin, is catalytically crucial. However, as observed with human QSOX1, the third CxxC motif shows no obvious catalytic role during the in vitro oxidation of reduced RNase or dithiothreitol. Pre-steady-state kinetics demonstrates that turnover in TbQSOX is limited by an internal redox step leading to 2-electron reduction of the FAD cofactor. In sum, the single-Trx domain QSOX studied here shows a striking similarity in enzymatic behavior to its double-Trx metazoan counterparts.

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Structure of a baculovirus sulfhydryl oxidase, a highly divergent member of the erv flavoenzyme family.

Publications

Structure of a baculovirus sulfhydryl oxidase, a highly divergent member of the erv flavoenzyme family.

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