Unusual Post-translational Modification Research Articles

Genome Mining for Radical SAM Protein Determinants Reveals Multiple Sactibiotic-Like Gene Clusters

Thuricin CD is a two-component bacteriocin produced by Bacillus thuringiensis that kills a wide range of clinically significant Clostridium difficile. This bacteriocin has recently been characterized and consists of two distinct peptides, Trnβ and Trnα, which both possess 3 intrapeptide sulphur to α-carbon bridges and act synergistically. Indeed, thuricin CD and subtilosin A are the only antimicrobials known to possess these unusual structures and are known as the sactibiotics (sulplur to alpha carbon-containing antibiotics). Analysis of the thuricin CD-associated gene cluster revealed the presence of genes encoding two highly unusual SAM proteins (TrnC and TrnD) which are proposed to be responsible for these unusual post-translational modifications. On the basis of the frequently high conservation among enzymes responsible for the post-translational modification of specific antimicrobials, we performed an in silico screen for novel thuricin CD–like gene clusters using the TrnC and TrnD radical SAM proteins as driver sequences to perform an initial homology search against the complete non-redundant database. Fifteen novel thuricin CD–like gene clusters were identified, based on the presence of TrnC and TrnD homologues in the context of neighbouring genes encoding potential bacteriocin structural peptides. Moreover, metagenomic analysis revealed that TrnC or TrnD homologs are present in a variety of metagenomic environments, suggesting a widespread distribution of thuricin-like operons in a variety of environments. In-silico analysis of radical SAM proteins is sufficient to identify novel putative sactibiotic clusters.

Giardia Duodenalis 14-3-3 Protein Is Polyglycylated by a Tubulin Tyrosine Ligase-like Member and Deglycylated by Two Metallocarboxypeptidases

The flagellated protozoan Giardia duodenalis is a parasite of the upper part of the small intestine of mammals, including humans, and an interesting biological model. Giardia harbors a single 14-3-3 isoform, a multifunctional protein family, that is modified at the C terminus by polyglycylation, an unusual post-translational modification consisting of the covalent addition of one or multiple glycines on the γ-carboxyl groups of specific glutamic acids. Polyglycylation affects the intracellular localization of g14-3-3, as the shortening of the polyglycine chain is correlated with a partial relocalization of 14-3-3 inside the nuclei during encystation. In this work we demonstrate that the gTTLL3, a member of the tubulin tyrosine ligase-like family, is the enzyme responsible for the 14-3-3 polyglycylation. We also identify two metallopeptidases of the M20 family, here termed gDIP1 (giardial dipeptidase 1) and gDIP2, as enzymes able to shorten the g14-3-3 polyglycine tail both in vivo and in vitro. Finally, we show that the ectopic expression of gDIP2 alters the g14-3-3 localization and strongly hampers the cyst formation. In conclusion, we have identified a polyglycylase and two deglycylases that act in concert to modulate the stage-dependent glycylation status of the multifunctional regulatory g14-3-3 protein in G. duodenalis.

Inherited balanced translocation t(9;17)(q33.2;q25.3) concomitant with a 16p13.1 duplication in a patient with schizophrenia

We report two rare genetic aberrations in a schizophrenia patient that may act together to confer disease susceptibility. A previously unreported balanced t(9;17)(q33.2;q25.3) translocation was observed in two schizophrenia-affected members of a small family with diverse psychiatric disorders. The proband also carried a 1.5 Mbp microduplication at 16p13.1 that could not be investigated in other family members. The duplication has been reported to predispose to schizophrenia, autism and mental retardation, with incomplete penetrance and variable expressivity. The t(9;17) (q33.2;q25.3) translocation breakpoint occurs within the open reading frames of KIAA1618 on 17q25.3, and TTLL11 (tyrosine tubulin ligase like 11) on 9q33.2, causing no change in the expression level of KIAA1618 but leading to loss of expression of one TTLL11 allele. TTLL11 belongs to a family of enzymes catalyzing polyglutamylation, an unusual neuron-specific post-translational modification of microtubule proteins, which modulates microtubule development and dynamics. The 16p13.1 duplication resulted in increased expression of NDE1, encoding a DISC1 protein partner mediating DISC1 functions in microtubule dynamics. We hypothesize that concomitant TTLL11-NDE1 deregulation may increase mutation load, among others, also on the DISC1 pathway, which could contribute to disease pathogenesis through multiple effects on neuronal development, synaptic plasticity, and neurotransmission. Our data illustrate the difficulties in interpreting the contribution of multiple potentially pathogenic changes likely to emerge in future next-generation sequencing studies, where access to extended families will be increasingly important.

Glucose and weight control in mice with a designed ghrelin O-acyltransferase inhibitor.

Ghrelin is a gastric peptide hormone that stimulates weight gain in vertebrates. The biological activities of ghrelin require octanoylation of the peptide on Ser(3), an unusual posttranslational modification that is catalyzed by the enzyme ghrelin O-acyltransferase (GOAT). Here, we describe the design, synthesis, and characterization of GO-CoA-Tat, a peptide-based bisubstrate analog that antagonizes GOAT. GO-CoA-Tat potently inhibits GOAT in vitro, in cultured cells, and in mice. Intraperitoneal administration of GO-CoA-Tat improves glucose tolerance and reduces weight gain in wild-type mice but not in ghrelin-deficient mice, supporting the concept that its beneficial metabolic effects are due specifically to GOAT inhibition. In addition to serving as a research tool for mapping ghrelin actions, GO-CoA-Tat may help pave the way for clinical targeting of GOAT in metabolic diseases.

ChemInform Abstract: The 1,5-Disubstituted Tetrazole Ring as a cis-Amide Bond Surrogate

Proline occupies a special role among those amino acids incorporated into peptides and proteins by the normal ribosomal pathways, since it is the only residue that leads to an N-alkyl amide bond. In peptide natural products that often have special biosynthetic pathways or unusual posttranslational modifications, N-methyl amino acids are common and may play a special role because of their conformational properties, including their proclivity for cis-trans isomerism of the amide bond. Numerous peptides with important biological activities, such as cyclosporin and didemnin, contain N-methyl amino acids. Cis-trans isomerism of the N-alkyl amide bond involving the amino group can readily be observed (1) in the NMR of proline and N-methyl amino acid-containing peptides. In the case of angiotensin and thyroliberin (TRH) analogs, the quantity of cis-isomer in aqueous solution was correlated (2) with the biological activity. This suggested that the cis-isomer might be the one bound to the receptor and responsible for the observed biological activity. Bairaktari et al. (3) have reported that the normal amide bond between an He and Lys residues in the linear peptide, bombolitin, has the cis-conformation when bound to phospholipid micelles. In protei0n crystal structures, cis-amide bond conformations are occasionally observed for the normal, nonalkylated amide bond. A cis-amide bond predisposes the peptide for a reverse turn, a so-called Type VI β-turn. Brandl and Deber (4) have proposed that cis-trans isomerism of proline residue might play a role in transduction of transmembrane proteins.

Thuricin CD, a posttranslationally modified bacteriocin with a narrow spectrum of activity against Clostridium difficile

The last decade has seen numerous outbreaks of Clostridium difficile-associated disease (CDAD), which presented significant challenges for healthcare facilities worldwide. We have identified and purified thuricin CD, a two-component antimicrobial that shows activity against C. difficile in the nanomolar range. Thuricin CD is produced by Bacillus thuringiensis DPC 6431, a bacterial strain isolated from a human fecal sample, and it consists of two distinct peptides, Trn-alpha and Trn-beta, that act synergistically to kill a wide range of clinical C. difficile isolates, including ribotypes commonly associated with CDAD (e.g., ribotype 027). However, this bacteriocin thuricin CD has little impact on most other genera, including many gastrointestinal commensals. Complete amino acid sequencing using infusion tandem mass spectrometry indicated that each peptide is posttranslationally modified at its respective 21st, 25th, and 28th residues. Solution NMR studies on [(13)C,(15)N] Trn-alpha and [(13)C,(15)N]Trn-beta were used to characterize these modifications. Analysis of multidimensional NOESY data shows that specific cysteines are linked to the alpha-carbons of the modified residues, forming three sulfur to alpha-carbon bridges. Complete sequencing of the thuricin CD gene cluster revealed genes capable of encoding two S'-adenosylmethionine proteins that are characteristically associated with unusual posttranslational modifications. Thuricin CD is a two-component antimicrobial peptide system with sulfur to alpha-carbon linkages, and it may have potential as a targeted therapy in the treatment of CDAD while also reducing collateral impact on the commensal flora.

Posttranslational Modifications of the Bovine Lens Beaded Filament Proteins Filensin and CP49

The lens beaded filament proteins filensin and CP49 are phosphorylated proteins that undergo proteolytic degradation with fiber cell age; however, the specific sites of modifications remain largely unknown. The purpose of this study was to identify posttranslational modifications (PTMs) in bovine lens beaded filament proteins. Filensin and CP49 were enriched by urea extraction of lens fiber cell homogenates after the water-soluble fraction was removed. The urea-soluble fraction was separated by SDS-PAGE, and the corresponding filensin and CP49 bands were digested by trypsin, Lys C, or Glu C. The enzymatic digests were analyzed by HPLC mass spectrometry. The sequences of lens beaded filament proteins were systematically mapped, and putative database sequence errors of filensin were identified. The data also indicated that Met-1 of CP49 was removed and Ser2 was acetylated. Nine phosphorylation sites on filensin and seven phosphorylation sites on CP49 were identified. Filensin was found to be truncated at D431 and L39, and the resulting new N termini were N-myristoylated and N-acetylated, respectively. Truncation of CP49 occurred at D37. Aspartic acid isomerization to isoaspartic acid occurs at the major truncation sites of filensin (D431) and of CP49 (D37). This study identified sites of phosphorylation and truncation in filensin and CP49 and revealed two unusual PTMs: postproteolytic N-acetylation and N-myristoylation of filensin. The detailed knowledge about these PTMs provides important information for further study of their functional consequences-for example protein redistribution during lens fiber cell differentiation and aging.

TTLL10 can perform tubulin glycylation when co-expressed with TTLL8

Tubulin can undergo unusual post-translational modifications, glycylation and glutamylation. We previously failed to find glycylase (glycine ligase) for tubulin while identifying TTLL10 as a polyglycylase for nucleosome assembly protein 1. We here examine whether TTLL10 performs tubulin glycylation. We used a polyclonal antibody (R-polygly) raised against a poly(glycine) chain, which does not recognize monoglycylated protein. R-polygly strongly reacted with mouse tracheal cilia and axonemal tubulins. R-polygly detected many proteins in cell lysates co-expressing TTLL10 with TTLL8. Two-dimensional electrophoresis revealed that the R-polygly-reactive proteins included α- and β-tubulin. R-polygly labeling signals overlapped with microtubules. These results indicate that TTLL10 can strongly glycylate tubulin in a TTLL8-dependent manner. Furthermore, these two TTLL proteins can glycylate unidentified 170-, 110-, 75-, 40-, 35-, and 30-kDa acidic proteins.

Solution structure of Hyp10Pro variant of conomarphin, a cysteine-free and d-amino-acid containing conopeptide

Conotoxins are mainly disulfide-rich short peptides active on different ion channels, neurotransmitter receptors or transporters in nervous system, exhibiting highly diversified composition, structures and biological functions. Besides these kinds of conopeptides, some novel cysteine-free conopeptides have also been reported. Conomarphin, a cystine-free 15-residue conopeptide from Conus marmoreus, has been purified and classified into M-superfamily. In addition to its unique characteristic of a D-type phenylalanine at the third residue from the C-terminus, conomarphin has an unusual hydroxyproline residue at position 10. To make an effort to understand the role of hydroxyproline post-translational modification in conomarphin, 1H NMR solution structure of Hyp10Pro variant of conomarphin was resolved and compared with the native conomarphin in the present work. The Hyp10Pro conomarphin has a type II-β-turn near the C-terminus instead of a 3 10 helix in native conomarphin. The compact loop region in native conomarphin becomes more open when hydroxyproline is displaced by proline. This reveals that hydroxyproline residue is essential for the structure of conomarphin just like D-Phe13. The unusual post-translational modification of conomarphin implies a unique selectivity of hydroxylation in toxin sequence.

Post‐translational modifications of tubulin in the nervous system

Many studies have shown that microtubules (MTs) interact with MT-associated proteins and motor proteins. These interactions are essential for the formation and maintenance of the polarized morphology of neurons and have been proposed to be regulated in part by highly diverse, unusual post-translational modifications (PTMs) of tubulin, including acetylation, tyrosination, detyrosination, Delta2 modification, polyglutamylation, polyglycylation, palmitoylation, and phosphorylation. However, the precise mechanisms of PTM generation and the properties of modified MTs have been poorly understood until recently. Recent PTM research has uncovered the enzymes mediating tubulin PTMs and provided new insights into the regulation of MT-based functions. The identification of tubulin deacetylase and discovery of its specific inhibitors have paved the way to understand the roles of acetylated MTs in kinesin-mediated axonal transport and neurodegenerative diseases such as Huntington's disease. Studies with tubulin tyrosine ligase (TTL)-null mice have shown that tyrosinated MTs are essential in normal brain development. The discovery of TTL-like genes encoding polyglutamylase has led to the finding that polyglutamylated MTs which accumulate during brain development are involved in synapse vesicle transport or neurite outgrowth through interactions with motor proteins or MT-associated proteins, respectively. Here we review current exciting topics that are expected to advance MT research in the nervous system.

Cupryphans, metal‐binding, redox‐active, redesigned conopeptides

Contryphans are bioactive peptides, isolated from the venom of marine snails of the genus Conus, which are characterized by the short length of the polypeptide chain and the high degree of unusual post-translational modifications. The cyclization of the polypeptide chain through a single disulphide bond, the presence of two conserved Pro residues, and the epimerization of a Trp/Leu residue confer to Contryphans a stable and well-defined structure in solution, conserved in all members of the family, and tolerant to multiple substitutions. The potential of Contryphans as scaffolds for the design of redox-active (macro)molecules was tested by engineering a copper-binding site on two different variants of the natural peptide Contryphan-Vn. The binding site was designed by computational modeling, and the redesigned peptides were synthesized and characterized by optical, fluorescence, electron spin resonance, and nuclear magnetic resonance spectroscopy. The novel peptides, named Cupryphan and Arg-Cupryphan, bind Cu(2+) ions with a 1:1 stoichiometry and a K(d) in the 100 nM range. Other divalent metals (e.g., Zn(2+) and Mg(2+)) are bound with much lower affinity. In addition, Cupryphans catalyze the dismutation of superoxide anions with an activity comparable to other nonpeptidic superoxide dismutase mimics. We conclude that the Contryphan motif represents a natural robust scaffold which can be engineered to perform different functions, providing additional means for the design of catalytically active mini metalloproteins.

Inhibition of Neuronal Nicotinic Acetylcholine Receptor Subtypes by α-Conotoxin GID and Analogues

alpha-Conotoxins are small disulfide-rich peptides from the venom of the Conus species that target the nicotinic acetylcholine receptor (nAChR). They are valuable pharmacological tools and also have potential therapeutic applications particularly for the treatment of chronic pain. alpha-Conotoxin GID is isolated from the venom of Conus geographus and has an unusual N-terminal tail sequence that has been shown to be important for binding to the alpha4beta2 subtype of the nAChR. To date, only four conotoxins that inhibit the alpha4beta2 subtype have been characterized, but they are of considerable interest as it is the most abundant nAChR subtype in the mammalian brain and has been implicated in a range of diseases. In this study, analysis of alanine-scan and truncation mutants of GID reveals that a conserved proline in alpha-conotoxins is important for activity at the alpha7, alpha3beta2, and alpha4beta2 subtypes. Although the proline residue was the most critical residue for activity at the alpha3beta2 subtype, Asp(3), Arg(12), and Asn(14) are also critical at the alpha7 subtype. Interestingly, very few of the mutations tested retained activity at the alpha4beta2 subtype indicating a tightly defined binding site. This lack of tolerance to sequence variation may explain the lack of selective ligands discovered for the alpha4beta2 subtype to date. Overall, our findings contribute to the understanding of the structure-activity relationships of alpha-conotoxins and may be beneficial for the ongoing attempts to exploit modulators of the neuronal nAChRs as therapeutic agents.

Unusual Chromophore and Cross‐Links in Ranasmurfin: A Blue Protein from the Foam Nests of a Tropical Frog

A smurfin' good structure: The 1.16 Å resolution crystal structure of ranasmurfin, a blue protein extracted from the foam nests of the Malaysian tree frog Polypedates leucomystax, reveals unusual posttranslational chemical modifications. The chromophore involves an extended Zn-coordinated four-residue (Lys-Tyr-Tyr-Lys) inter-subunit cross-link of a kind not previously reported.

Characterization of Recombinant Human Prolyl 3-Hydroxylase Isoenzyme 2, an Enzyme Modifying the Basement Membrane Collagen IV

The single 3-hydroxyproline residue in the collagen I polypeptides is essential for proper fibril formation and bone development as its deficiency leads to recessive osteogenesis imperfecta. The vertebrate prolyl 3-hydroxylase (P3H) family consists of three members, P3H1 being responsible for the hydroxylation of collagen I. We expressed human P3H2 as an active recombinant protein in insect cells. Most of the recombinant polypeptide was insoluble, but small amounts were also present in the soluble fraction. P3H1 forms a complex with the cartilage-associated protein (CRTAP) that is required for prolyl 3-hydroxylation of fibrillar collagens. However, coexpression with CRTAP did not enhance the solubility or activity of the recombinant P3H2. A novel assay for P3H activity was developed based on that used for collagen prolyl 4-hydroxylases (C-P4H) and lysyl hydroxylases (LH). A large amount of P3H activity was found in the P3H2 samples with (Gly-Pro-4Hyp)5 as a substrate. The Km and Ki values of P3H2 for 2-oxoglutarate and its certain analogues resembled those of the LHs rather than the C-P4Hs. Unlike P3H1, P3H2 was strongly expressed in tissues rich in basement membranes, such as the kidney. P3H2 hydroxylated more effectively two synthetic peptides corresponding to sequences that are hydroxylated in collagen IV than a peptide corresponding to the 3-hydroxylation site in collagen I. These findings suggest that P3H2 is responsible for the hydroxylation of collagen IV, which has the highest 3-hydroxyproline content of all collagens. It is thus possible that P3H2 mutations may lead to a disease with changes in basement membranes.

Sulfated Tyrosine and HIV Entry

In order for human immunodeficiency virus type 1 (HIV-1) to enter host cells, its envelope glycoprotein gp120 must bind to the host-cell surface receptor CD4 and to a co-receptor. An unusual posttranslational modification, tyrosine sulfation, is important to the co-receptor interaction. Huang et al . have investigated HIV-1 gp120 interactions with a sulfated N-terminal peptide from the co-receptor CCR5 and determined the crystal structure of a tyrosine-sulfated antibody in complex with gp120 and CD4. A conserved site in gp120 recognizes sulfo-tyrosine and might be a target for design of therapeutics. C.-c. Huang, S. N. Lam, P. Acharya, M. Tang, S.-H. Xiang, S. Shahzad-ul Hussan, R. L. Stanfield, J. Robinson, J. Sodroski, I. A. Wilson, R. Wyatt, C. A. Bewley, P. D. Kwong, Structures of the CCR5 N terminus and of a tyrosine-sulfated antibody with HIV-1 gp120 and CD4. Science 317 , 1930-1934 (2007). [Abstract] [Full Text]

Characterization of the biochemical and biophysical properties of the phosphatidylserine receptor (PS-R) gene product

The PS-R gene product was originally described as a cell surface receptor that interacts with externalized phosphatidylserine (PS) on apoptotic cells, but more recent studies have shown that it plays a critical role in organ development and terminal differentiation of many cell types during embryogenesis. Despite these important developmental functions, the biochemical and molecular properties of PS-R are poorly understood. Here we have used several approaches to show that PS-R undergoes processive post-translational protein cross-linking to form covalent multimers within the nuclear compartment. Although PS-R has a potential Glu-Glu (QQ) duet that is often targeted by transglutaminase TG-2, the oligomerization of PS-R was not effected by QQ-->AA mutation, or when PS-R gene product was expressed in TG-2 (-/-) fibroblasts. Pulse-chase experiments with (35) S-methionine indicates that the PS-R undergoes an initial proteolytic cleavage, followed by progressive multimerization of the monomeric subunits over time. In summary, we report here that PS-R is modified by an unusual post-translational modification, and we speculate that homomultimer of PS-R might be playing an important function as a scaffolding protein in the nucleus.

Anchoring RCC1 by the tail

Generation of GTP-bound Ran on chromatin by its guanine nucleotide-exchange factor RCC1 provides a spatial signal that controls nuclear-envelope formation, nucleo-cytoplasmic transport and mitotic spindle assembly. A study now identifies an unusual post-translational modification, α-N-methylation of the amino-terminal tail of RCC1, which anchors the protein on chromosomes.

De Novo Sequencing and Disulfide Mapping of a Bromotryptophan-Containing Conotoxin by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

T-1-family conotoxins belong to the T-superfamily and are composed of 10-17 amino acids. They share a common cysteine framework and disulfide connectivity and exhibit unusual posttranslational modifications, such as tryptophan bromination, glutamic acid carboxylation, and threonine glycosylation. We have isolated and characterized a novel peptide, Mo1274, containing 11 amino acids, that shows the same cysteine pattern, -CC-CC, and disulfide linkage as those of the T-1-family members. The complete sequence, GNWCCSARVCC, in which W denotes bromotryptophan, was derived from MS-based de novo sequencing. The FT-ICR MS/MS techniques of electron capture dissociation (ECD), infrared multiphoton dissociation, and collision-induced dissociation served to detect and localize the tryptophan bromination. The bromine contributes a distinctive isotopic distribution in all fragments that contain bromotryptophan. ECD fragmentation results in the loss of bromine and return to the normal isotopic distribution. Disulfide connectivity of Mo1274, between cysteine pairs 1-3 and 2-4, was determined by mass spectrometry in combination with chemical derivatization employing tris(2-carboxyethyl)phosphine, followed by differential alkylation with N-ethylmaleimide and iodoacetamide. The ECD spectra of the native and partially modified peptide reveal a loss of bromine in a process that requires the presence of a disulfide bond.

The Giardia duodenalis 14-3-3 Protein Is Post-translationally Modified by Phosphorylation and Polyglycylation of the C-terminal Tail

The flagellated protozoan Giardia duodenalis (syn. lamblia or intestinalis) has been chosen as a model parasite to further investigate the multifunctional 14-3-3s, a family of highly conserved eukaryotic proteins involved in many cellular processes, such as cell cycle, differentiation, apoptosis, and signal transduction pathways. We confirmed the presence of a single 14-3-3 homolog gene (g14-3-3) by an in silico screening of the complete genome of Giardia, and we demonstrated its constitutive transcription throughout the life stages of the parasite. We cloned and expressed the g14-3-3 in bacteria, and by protein-protein interaction assays we demonstrated that it is a functional 14-3-3. Using an anti-peptide antibody raised against a unique 18-amino acid sequence at the N terminus, we observed variations both in the intracellular localization and in the molecular size of the native g14-3-3 during the conversion of Giardia from trophozoites to the cyst stage. An affinity chromatography, based on the 14-3-3 binding to the polypeptide difopein, was set to purify the native g14-3-3. By matrix-assisted laser desorption ionization mass spectroscopy analysis, we showed that polyglycylation, an unusual post-translational modification described only for tubulin, occurred at the extreme C terminus of the native g14-3-3 on Glu246, Glu247, or both and that the Thr214, located in the loop between helices 8 and 9, is phosphorylated. We propose that the addition of the polyglycine chain can promote the binding of g14-3-3 to alternative ligands and that the differential rate of polyglycylation/deglycylation during the encystation process can act as a novel mechanism to regulate the intracellular localization of g14-3-3.

Helix Switching of a Key Active-Site Residue in the Cytochromecbb3Oxidases

In the respiratory chains of mitochondria and many aerobic prokaryotes, heme-copper oxidases are the terminal enzymes that couple the reduction of molecular oxygen to proton pumping, contributing to the protonmotive force. The cbb(3) oxidases belong to the superfamily of enzymes that includes all of the heme-copper oxidases. Sequence analysis indicates that the cbb(3) oxidases are missing an active-site tyrosine residue that is absolutely conserved in all other known heme-copper oxidases. In the other heme-copper oxidases, this tyrosine is known to be subject to an unusual post-translational modification and to play a critical role in the catalytic mechanism. The absence of this tyrosine in the cbb(3) oxidases raises the possibility that the cbb(3) oxidases utilize a different catalytic mechanism from that of the other members of the superfamily. Using homology modeling, quantum chemistry, and molecular dynamics, a model of the structure of subunit I of a cbb(3) oxidase (Vibrio cholerae) was constructed. The model predicts that a tyrosine residue structurally analogous to the active-site tyrosine in other oxidases is present in the cbb(3) oxidases but that the tyrosine originates from a different transmembrane helix within the protein. The predicted active-site tyrosine is conserved in the sequences of all of the known cbb(3) oxidases. Mutagenesis of the tyrosine to phenylalanine in the V. cholerae oxidase resulted in a fully assembled enzyme with nativelike structure but lacking catalytic activity. These findings strongly suggest that all of the heme-copper oxidases utilize the same catalytic mechanism and provide an unusual example in which a critical active-site residue originates from different places within the primary sequence for different members of the same superfamily.