Identification of the maturation factor for dual oxidase. Evolution of an eukaryotic operon equivalent

Dual oxidase 2 (DUOX2), an NADPH:O(2) oxidoreductase flavoprotein, is a component of the thyroid H(2)O(2) generator crucial for hormone synthesis at the apical membrane. Mutations in DUOX2 produce congenital hypothyroidism in humans. However, no functional DUOX-based NADPH oxidase has ever been reconstituted at the plasma membrane of transfected cells. It has been proposed that DUOX retention in the endoplasmatic reticulum (ER) of heterologous systems is due to the lack of an unidentified component required for functional maturation of the enzyme. By data mining of a massively parallel signature sequencing tissue expression data base, we identified an uncharacterized gene named DUOX maturation factor (DUOXA2) arranged head-to-head to and co-expressed with DUOX2. A paralog (DUOXA1) was similarly linked to DUOX1. The genomic rearrangement leading to linkage of ancient DUOX and DUOXA genes could be traced back before the divergence of echinoderms. We demonstrate that co-expression of DUOXA2, an ER-resident transmembrane protein, allows ER-to-Golgi transition, maturation, and translocation to the plasma membrane of functional DUOX2 in a heterologous system. The identification of DUOXA genes has important implications for studies of the molecular mechanisms controlling DUOX expression and the molecular genetics of congenital hypothyroidism.     

The type of DUOX-dependent ROS production is dictated by defined sequences in DUOXA

A deliberate generation of ROS is now recognized to be achieved by specific NADPH oxidases (NOX). Dual oxidases (DUOXs) are Ca(2+)-activated NOXs and operate as H(2)O(2)-generators in various tissues. A tight regulation is however required to avoid ROS overproduction that can rapidly be harmful to biological systems. DUOX activator (DUOXA) proteins act as organizing elements for surface expression and activity of the DUOX enzymes. To study DUOX activation by the maturation factors, chimeric DUOXA proteins were generated by replacing particular domains between DUOXA1 and DUOXA2. Their impact on DUOX function and membrane expression were explored in a reconstituted heterologous cell system composed of COS-7 cells. We have shown that the COOH-terminal end of DUOXA1 is responsible for DUOX1-dependent H(2)O(2) generation. The NH(2)-terminal tail of DUOXA2 is critical to specify the type of ROS released by DUOX2, hydrogen peroxide or superoxide. Native DUOXA2 would constrain DUOX2 to produce H(2)O(2). However, alterations of the DUOXA2 NH(2)-terminal domain modify DUOX2 activity triggering superoxide leaking. Our results demonstrate that specific domains of the DUOX maturation factors promote the activation of DUOXs as well as the type of ROS generated by the oxidases.     

Developmental regulation of DUOX1 expression and function in human fetal lung epithelial cells

The purpose of this study was to determine the expression and cellular functions of the epithelial NADPH oxidase DUOX1 during alveolar type II cell development. When human fetal lung cells (gestational age 11-22 wk) were cultured to confluency on permeable filters, exposure of cells to a hormone mixture (dexamethasone, 8-Br-cAMP, and IBMX, together referred to as DCI) resulted in differentiation of cells into a mature type II phenotype as assessed by expression of lamellar bodies, surfactant proteins, and transepithelial electrical parameters. After 6 days in culture in presence of DCI, transepithelial resistance (2,616 +/- 529 Omega.cm(2)) and potential (-8.5 +/- 0.6 mV) indicated epithelial polarization. At the same time, treatment with DCI significantly increased the mRNA expression of DUOX1 ( approximately 21-fold), its maturation factor DUOXA1 ( approximately 12-fold), as well as DUOX protein ( approximately 12-fold), which was localized near the apical cell pole in confluent cultures. For comparison, in fetal lung specimens, DUOX protein was not detectable at up to 27 wk of gestational age but was strongly upregulated after 32 wk. Function of DUOX1 was assessed by measuring H(2)O(2) and acid production. Rates of H(2)O(2) production were increased by DCI treatment and blocked by small interfering RNA directed against DUOX1 or by diphenylene iodonium. DCI-treated cultures also showed increased intracellular acid production and acid release into the mucosal medium, and acid production was largely blocked by knockdown of DUOX1 mRNA. These data establish the regulated expression of DUOX1 during alveolar maturation, and indicate DUOX1 in alveolar H(2)O(2) and acid secretion by differentiated type II cells.     

Mice deficient in dual oxidase maturation factors are severely hypothyroid

Dual oxidases (DUOX1 and DUOX2) are evolutionary conserved reduced nicotinamide adenine dinucleotide phosphate oxidases responsible for regulated hydrogen peroxide (H(2)O(2)) release of epithelial cells. Specific maturation factors (DUOXA1 and DUOXA2) are required for targeting of functional DUOX enzymes to the cell surface. Mutations in the single-copy Duox and Duoxa genes of invertebrates cause developmental defects with reduced survival, whereas knockdown in later life impairs intestinal epithelial immune homeostasis. In humans, mutations in both DUOX2 and DUOXA2 can cause congenital hypothyroidism with partial iodide organification defects compatible with a role of DUOX2-generated H(2)O(2) in driving thyroid peroxidase activity. The DUOX1/DUOXA1 system may account for residual iodide organification in patients with loss of DUOX2, but its physiological function is less clear. To provide a murine model recapitulating complete DUOX deficiency, we simultaneously targeted both Duoxa genes by homologous recombination. Knockout of Duoxa genes (Duoxa(-/-) mice) led to a maturation defect of DUOX proteins lacking Golgi processing of N-glycans and to loss of H(2)O(2) release from thyroid tissue. Postnatally, Duoxa(-/-) mice developed severe goitreous congenital hypothyroidism with undetectable serum T4 and maximally disinhibited TSH levels. Heterozygous mice had normal thyroid function parameters. (125)I uptake and discharge studies and probing of iodinated TG epitopes corroborated the iodide organification defect in Duoxa(-/-) mice. Duoxa(-/-) mice on continuous T4 replacement from P6 showed normal growth without an overt phenotype. Our results confirm in vivo the requirement of DUOXA for functional expression of DUOX-based reduced nicotinamide adenine dinucleotide phosphate oxidases and the role of DUOX isoenzymes as sole source of hormonogenic H(2)O(2).     

Conserved Cysteine Residues Provide a Protein-Protein Interaction Surface in Dual Oxidase (DUOX) Proteins

Intramolecular disulfide bond formation is promoted in oxidizing extracellular and endoplasmic reticulum compartments and often contributes to protein stability and function. DUOX1 and DUOX2 are distinguished from other members of the NOX protein family by the presence of a unique extracellular N-terminal region. These peroxidase-like domains lack the conserved cysteines that confer structural stability to mammalian peroxidases. Sequence-based structure predictions suggest that the thiol groups present are solvent-exposed on a single protein surface and are too distant to support intramolecular disulfide bond formation. To investigate the role of these thiol residues, we introduced four individual cysteine to glycine mutations in the peroxidase-like domains of both human DUOXs and purified the recombinant proteins. The mutations caused little change in the stabilities of the monomeric proteins, supporting the hypothesis that the thiol residues are solvent-exposed and not involved in disulfide bonds that are critical for structural integrity. However, the ability of the isolated hDUOX1 peroxidase-like domain to dimerize was altered, suggesting a role for these cysteines in protein-protein interactions that could facilitate homodimerization of the peroxidase-like domain or, in the full-length protein, heterodimeric interactions with a maturation protein. When full-length hDUOX1 was expressed in HEK293 cells, the mutations resulted in decreased H₂O₂ production that correlated with a decreased amount of the enzyme localized to the membrane surface rather than with a loss of activity or with a failure to synthesize the mutant proteins. These results support a role for the cysteine residues in intermolecular disulfide bond formation with the DUOX maturation factor DUOXA1.     

A novel small-molecule MRCK inhibitor blocks cancer cell invasion

Background

Dual oxidase maturation factor 1 (DUOXA1) has been associated with the maturation of the reactive oxygen species (ROS) producing enzyme, dual oxidase 1 (DUOX1) in the adult thyroid. However, ROS have also been implicated in the development of several tissues. We found that activated muscle satellite cells and primary myoblasts isolated from mice express robust levels of DUOXA1 and that its levels are altered as cells differentiate.

Results

To determine whether DUOXA1 levels affect muscle differentiation, we used an adenoviral construct (pCMV5-DUOXA1-GFP) to drive constitutive overexpression of this protein in primary myoblasts. High levels of DUOXA1 throughout myogenesis resulted in enhanced H₂O₂ production, fusion defects, reduced expression of early (myogenin) and late (myosin heavy chain) markers of differentiation, and elevated levels of apoptosis compared to control cells infected with an empty adenoviral vector (pCMV5-GFP). DUOXA1 knockdown (using a DUOXA1 shRNA construct) resulted in enhanced differentiation compared to cells subjected to a control shRNA, and subjecting DUOXA1 overexpressing cells to siRNAs targeting DUOX1 or apoptosis signal-regulating kinase 1 (ASK1) rescued the phenotype.

Conclusions

This study represents the first to demonstrate the importance of DUOXA1 in skeletal muscle myoblasts and that DUOXA1 overexpression in muscle stem cells induces apoptosis and inhibits differentiation through DUOX1 and ASK1.     

Perturbed heme binding is responsible for the blistering phenotype associated with mutations in the Caenorhabditis elegans dual oxidase 1 (DUOX1) peroxidase domain

Dual oxidase (DUOX) enzymes support a wide variety of essential reactions, from cellular signaling to thyroid hormone biosynthesis. In Caenorhabditis elegans, the DUOX system (CeDUOX1/2) plays a crucial role in innate immunity and in stabilizing the cuticle by forming tyrosine cross-links. The current model suggests that superoxide generated by CeDUOX1 at the C-terminal NADPH oxidase domain is rapidly converted to H(2)O(2). The H(2)O(2) is then utilized by the N-terminal peroxidase-like domain to cross-link tyrosines. We have now created a series of mutations in the isolated peroxidase domain, CeDUOX1(1-589). One set of mutations investigate the roles of a putative distal tyrosine (Tyr(105)) and Glu(238), a proposed covalent heme-binding residue. The results confirm that Glu(238) covalently binds to the heme group. A second set of mutations (G246D and D392N) responsible for a C. elegans blistering cuticle phenotype was also investigated. Surprisingly, although not among the catalytic residues, both mutations affected heme co-factor binding. The G246D mutant bound less total heme than the wild type, but a higher fraction of it was covalently bound. In contrast, the D392N mutant appears to fold normally but does not bind heme. Molecular dynamics simulations of a CeDUOX1(1-589) homology model implicate displacements of the proximal histidine residue as the likely cause. Both enzymes are structurally stable and through altered heme interactions exhibit partial or complete loss of tyrosine cross-linking activity, explaining the blistering phenotype. This result argues that the CeDUOX peroxidase domain is primarily responsible for tyrosine cross-linking.     

Maturation processing and characterization of streptopain

Streptopain is a cysteine protease expressed by Streptococcus pyogenes. To study the maturation mechanism of streptopain, wild-type and Q186N, C192S, H340R, N356D and W357A mutant proteins were expressed in Escherichia coli and purified to homogeneity. Proteolytic analyses showed that the maturation of prostreptococcal pyrogenic exotoxin B zymogen (pro-SPE B) involves eight intermediates with a combination of cis- and trans-processing. Based on the sequences of these intermediates, the substrate specificity of streptopain favors a hydrophobic residue at the P2 site. The relative autocatalytic rates of these mutants exhibited the order Q186N > W357A > N356D, C192S, H340R. Interestingly, the N356D mutant containing protease activity could not be converted into the 28-kDa form by autoprocessing. This observation suggested that Asn(356) might involve the cis-processing of the propeptide. In addition, the maturation rates of pro-SPE B with trypsin and plasmin were 10- and 60-fold slower than that with active mature streptopain. These findings indicate that active mature streptopain likely plays the most important role in the maturation of pro-SPE B under physiological conditions.     

Dual oxidase-2 has an intrinsic Ca2+-dependent H2O2-generating activity

Duox2 (and probably Duox1) is a glycoflavoprotein involved in thyroid hormone biosynthesis, as the thyroid H2O2 generator functionally associated with Tpo (thyroperoxidase). So far, because of the impairment of maturation and of the targeting process, transfecting DUOX into nonthyroid cell lines has not led to the expression of a functional H2O2-generating system at the plasma membrane. For the first time, we investigated the H2O2-generating activity in the particulate fractions from DUOX2- and DUOX1-transfected HEK293 and Chinese hamster ovary cells. The particulate fractions of these cells stably or transiently transfected with human or porcine DUOX cDNA demonstrate a functional NADPH/Ca2+-dependent H2O2-generating activity. The immature Duox proteins had less activity than pig thyrocyte particulate fractions, and their activity depended on their primary structures. Human Duox2 seemed to be more active than human Duox1 but only half as active as its porcine counterpart. TPO co-transfection produced a slight increase in the enzymatic activity, whereas p22(phox), the 22-kDa subunit of the leukocyte NADPH oxidase, had no effect. In previous studies on the mechanism of H2O2 formation, it was shown that mature thyroid NADPH oxidase does not release O2*- but H2O2. Using a spin-trapping technique combined with electron paramagnetic resonance spectroscopy, we confirmed this result but also demonstrated that the partially glycosylated form of Duox2, located in the endoplasmic reticulum, generates superoxide in a calcium-dependent manner. These results suggest that post-translational modifications during the maturation process of Duox2 could be implicated in the mechanism of H2O2 formation by favoring intramolecular superoxide dismutation.     

The chaperone protein BiP binds to a mutant prion protein and mediates its degradation by the proteasome

Familial prion diseases are thought to result from a change in structure of the mutant prion protein (PrP), which takes a pathogenic conformation. We have examined the role of molecular chaperones in the folding of normal and mutant PrP Q217R (PrP(217)) in transfected neuroblastoma cells. In a previous report we showed that, although most of the PrP(217) forms escape the endoplasmic reticulum quality control system and aggregate in post-Golgi compartments, a significant proportion of PrP(217) retains the C-terminal glycosylphosphatidyl inositol signal peptide (PrP32), and does not exit the endoplasmic reticulum (Singh, N., Zanusso, G., Chen, S. G., Fujioka, H., Richardson, S., Gambetti, P., and Petersen, R. B. (1997) J. Biol. Chem. 272, 28461-28470). We have now studied the folding and turnover of PrP32 to understand the mechanism by which abnormal PrP forms cause cellular toxicity in our cell culture model and in the human brain carrying the Gerstmann-Str?ussler-Scheinker disease Q217R mutation. In this report, we show that PrP32 remains associated with the chaperone BiP for an abnormally prolonged period of time and is degraded by the proteasomal pathway. This study is the first demonstration that BiP is chaperoning the folding of PrP and plays a role in maintaining the quality control in the PrP maturation pathway. Our data provide new insight into the diverse pathways of mutant PrP metabolism and neurotoxicity.     

IFNγ mediates DUOX2 expression via a STAT-independent signaling pathway

The biological roles of the dual oxidases, DUOX1 and DUOX2, are dependent upon the tissue in which they are expressed. However, the mechanisms that control DUOX expression in these tissues are largely unexplored. Given the known role of DUOX for host defense in the gut and respiratory tract, we characterized potential mechanisms that control DUOX2 expression in response to interferon gamma (IFNγ) in respiratory tract epithelium. We discovered that IFNγ-mediated DUOX2 expression was regulated by a STAT-independent, JAK-independent pathway. These data provide insights into a novel IFNγ signaling pathway with potential importance for regulation of host defense responses.     

Exploring the role of putative active site amino acids and pro-region motif of recombinant falcipain-2: a principal hemoglobinase of Plasmodium falciparum

Falcipain-2 is one of the principal hemoglobinases of Plasmodium falciparum, a human malaria parasite. It has a typical papain family cysteine protease structural organization, a large pro-domain, a mature domain with conserved active site amino acids. Pro-domain of falcipain-2 also contains two important conserved motifs, "GNFD" and "ERFNIN." The "GNFD" motif has been shown to be responsible for correct folding and stability in case of many papain family proteases. In the present study, we carried out site-directed mutagenesis to assess the roles of active site residues and pro-domain residues for the activity of falcipain-2. Our results showed that substitutions of putative active site residues; Q36, C42, H174, and N204 resulted in complete loss of falcipain-2 activity, while W206 and D155 mutants retained partial/complete activity in comparison to the wild type falcipain-2. Homology modeling data also corroborate the results of mutagenesis; Q36, C42, H174, N204, and W206 residues form the active site loop of the enzyme and D155 lie outside the active pocket. Substitutions in the pro-region did not affect the activity of falcipain-2. This implies that falcipain-2 shares active site residues with other members of papain family, however pro-region of falcipain-2 does not play any role in the activity of enzyme.     

Structural basis for type I and type II deficiencies of antithrombotic plasma protein C: Patterns revealed by three-dimensional molecular modelling of mutations of the protease domain

Familial deficiency of protein C is associated with inherited thrombophilia. To explore how specific missense mutations might cause observed clinical phenotypes, known protein C missense mutations were mapped onto three-dimensional homology models of the protein C protease domain, and the implications for domain folding and structure were evaluated. Most Type I missense mutations either replaced internal hydrophobic residues (I201T, L223F, A259V, A267T, A346T, A346V, G376D) or nearby interacting residues (I403M, T298M, Q184H), thus disrupting the packing of internal hydrophobic side chains, or changed hydrophilic residues, thus disrupting ion pairs (N256D, R178W). Mutations (P168L, R169W) at the activation site destabilized the region containing the activation peptide structure. Most Type II mutations involved solvent-exposed residues and were clustered either in a positively charged region (R147W, R157Q, R229Q, R352W) or were located in or near the active site region (S252N, D359N, G381S, G391S, H211Q). The cluster of arginines 147, 157, 229, and 352 may identify a functionally important exosite. Identification of the spatial relationships of natural mutations in the protein C model is helpful for understanding manifestations of protein C deficiency and for identification of novel, functionally important molecular features and exosites. © 1994 John Wiley & Sons, Inc.     

Photosystem I is indispensable for photoautotrophic growth, CO2 fixation, and H2 photoproduction in Chlamydomonas reinhardtii

Certain Chlamydomonas reinhardtii mutants deficient in photosystem I due to defects in psaA mRNA maturation have been reported to be capable of CO2 fixation, H2 photoevolution, and photoautotrophic growth (Greenbaum, E., Lee, J. W., Tevault, C. V., Blankinship, S. L. , and Mets, L. J. (1995) Nature 376, 438-441 and Lee, J. W., Tevault, C. V., Owens, T. G.; Greenbaum, E. (1996) Science 273, 364-367). We have generated deletions of photosystem I core subunits in both wild type and these mutant strains and have analyzed their abilities to grow photoautotrophically, to fix CO2, and to photoevolve O2 or H2 (using mass spectrometry) as well as their photosystem I content (using immunological and spectroscopic analyses). We find no instance of a strain that can perform photosynthesis in the absence of photosystem I. The F8 strain harbored a small amount of photosystem I, and it could fix CO2 and grow slowly, but it lost these abilities after deletion of either psaA or psaC; these activities could be restored to the F8-psaADelta mutant by reintroduction of psaA. We observed limited O2 photoevolution in mutants lacking photosystem I; use of 18O2 indicated that this O2 evolution is coupled to O2 uptake (i.e. respiration) rather than CO2 fixation or H2 evolution. We conclude that the reported instances of CO2 fixation, H2 photoevolution, and photoautotrophic growth of photosystem I-deficient mutants result from the presence of unrecognized photosystem I.     

Calcium is required for folding of newly made subunits of the asialoglycoprotein receptor within the endoplasmic reticulum.

By resolving immunoprecipitates on nonreducing sodium dodecyl sulfate gels, we have detected several disulfide-bonded intermediates in folding within the endoplasmic reticulum of newly made H1 subunits of the asialoglycoprotein receptor. H1 in the endoplasmic reticulum (ER) can be partially unfolded by treatment of cells with dithiothreitol, but H1 in Golgi or post-Golgi organelles is resistant to such unfolding. This defines a late step in H1 folding that occurs just prior to exit from the ER. Depletion of calcium from the endoplasmic reticulum, either by treatment with A23187 or thapsigargin, has no effect on folding or secretion of newly made albumin, but totally blocks H1 maturation from the ER. No ER intermediates in H1 folding are formed in cells treated with A23187 or thapsigargin, indicating that at least an early step in H1 folding requires a high Ca2+ concentration in the ER lumen. As judged by cross-linking experiments, formation of H1 dimers and trimers occurs immediately after biosynthesis of the peptide chain, before monomer folding, and occurs normally in cells in which ER Ca2+ is reduced and where the monomer never folds properly. Calcium is essential for the asialoglycoprotein receptor to bind galactose, and our results suggest that Ca2+ is also essential for the receptor polypeptides to fold in the ER.     

Different effects of calnexin deletion in Saccharomyces cerevisiae on the secretion of two glycosylated amyloidogenic lysozymes

Both glycosylated amyloidogenic lysozymes I55T/G49N and D66H/G49N were expressed in wild-type and calnexin-disrupted Saccharomyces cerevisiae. The secretion amounts of mutant I55T/G49N were almost similar in both wild-type and calnexin-disrupted S. cerevisiae. In contrast, the secretion of mutant D66H/G49N greatly increased in calnexin-disrupted S. cerevisiae, while the secretion was very low in the wild-type strain. In parallel, the induction level of the molecular chaperones BiP and PDI located in the endoplasmic reticulum (ER) was investigated when these glycosylated amyloidogenic lysozymes were expressed in wild-type and calnexin-disrupted S. cerevisiae. The mRNA concentrations of BiP and PDI were evidently increased when mutant lysozyme D66H/G49N was expressed in calnexin-disrupted S. cerevisiae, while they were not so increased when I55T/G49N mutant was expressed. This observation indicates that the conformation of mutant lysozyme D66H/G49N was less stable in the ER, thus leading to the higher-level expression of ER molecular chaperones via the unfolded protein response pathway. This suggests that glycosylated amyloidogenic lysozyme I55T/G49N may have a relatively stable conformation in the ER, thus releasing it from the quality control of calnexin compared with mutant lysozyme D66H/G49N.     

The ER glycoprotein quality control system

The endoplasmic reticulum (ER) is the major site for folding and sorting of newly synthesized secretory cargo proteins. One central regulator of this process is the quality control machinery, which retains and ultimately disposes of misfolded secretory proteins before they can exit the ER. The ER quality control process is highly effective and mutations in cargo molecules are linked to a variety of diseases. In mammalian cells, a large number of secretory proteins, whether membrane bound or soluble, are asparagine (N)-glycosylated. Recent attention has focused on a sugar transferase, UDP-Glucose: glycoprotein glucosyl transferase (UGGT), which is now recognized as a constituent of the ER quality control machinery. UGGT is capable of sensing the folding state of glycoproteins and attaches a single glucose residue to the Man9GlcNAc2 glycan of incompletely folded or misfolded glycoproteins. This enables misfolded glycoproteins to rebind calnexin and reenter productive folding cycles. Prolonging the time of glucose addition on misfolded glycoproteins ultimately results in either the proper folding of the glycoprotein or its presentation to an ER associated degradation machinery.     

Characterization of rat cathepsin E and mutants with changed active-site residues and lacking propeptides and N-glycosylation, expressed in human embryonic kidney 293T cells

To study the roles of the catalytic activity, propeptide, and N-glycosylation of the intracellular aspartic proteinase cathepsin E in biosynthesis, processing, and intracellular trafficking, we constructed various rat cathepsin E mutants in which active-site Asp residues were changed to Ala or which lacked propeptides and N-glycosylation. Wild-type cathepsin E expressed in human embryonic kidney 293T cells was mainly found in the LAMP-1-positive endosomal organelles, as determined by immunofluorescence microscopy. Consistently, pulse-chase analysis revealed that the initially synthesized pro-cathepsin E was processed to the mature enzyme within a 24 h chase. This process was completely inhibited by brefeldin A and bafilomycin A, indicating its transport from the endoplasmic reticulum (ER) to the endosomal acidic compartment. Mutants with Asp residues in the two active-site consensus motifs changed to Ala and lacking the propeptide (Leu23-Phe58) and the putative ER-retention sequence (Ser59-Asp98) were neither processed nor transported to the endosomal compartment. The mutant lacking the ER-retention sequence was rapidly degraded in the ER, indicating the importance of this sequence in correct folding. The single (N92Q or N324D) and double (N92Q/N324D) N-glycosylation-deficient mutants were neither processed into a mature form nor transported to the endosomal compartment, but were stably retained in the ER without degradation. These data indicate that the catalytic activity, propeptides, and N-glycosylation of this protein are all essential for its processing, maturation, and trafficking.     

Chromophore aspartate oxidation-decarboxylation in the green-to-red conversion of a fluorescent protein from Zoanthus sp. 2

The red fluorescence of a Discosoma coral protein is the result of an additional autocatalytic oxidation of a green fluorescent protein (GFP)-like chromophore. This reaction creates an extra pi-electron conjugation by forming a C=N-C=O substituent. Here we show that the red fluorescence of a protein from Zoanthus sp. 2 (z2FP574) arises from a coupled oxidation-decarboxylation of Asp-66, the first amino acid of the chromophore-precursory DYG sequence. Comparative mutagenesis of highly homologous green (zFP506) and red (z2FP574) fluorescent proteins from Zoanthus species reveals that an aspartate at position 66 is critical for the development of red fluorescence. The maturation kinetics of wild-type z2FP574 and the zFP506 N66D mutant indicates that the "green" GFP-like form is the actual intermediate in producing the red species. Furthermore, via maturation kinetics analysis of zFP506 N66D, combined with mass spectrometry, we determined that the oxidation-decarboxylation of Asp-66 occurs without detectable intermediate products. According to mass spectral data, the minor "red" chromophore of the z2FP574 D66E mutant appears to be oxidized and completely decarboxylation deficient, indicating that the side chain length of acidic amino acid 66 is critical in controlling efficient oxidation-decarboxylation. Substitutions with aspartate at the equivalent positions of a Condylactis gigantea purple chromoprotein and Dendronephthya sp. green fluorescent protein imply that additional oxidation of a GFP-like structure is a prerequisite for chromophore decarboxylation. In summary, these results lead to a mechanism that is related to the chemistry of beta-keto acid decarboxylation.