A new regime of heme-dependent aromatic oxygenase superfamily.

Two histidine-ligated heme-dependent monooxygenase proteins, TyrH and SfmD, have recently been found to resemble enzymes from the dioxygenase superfamily currently named after tryptophan 2,3-dioxygenase (TDO), that is, the TDO superfamily. These latest findings prompted us to revisit the structure and function of the superfamily. The enzymes in this superfamily share a similar core architecture and a histidine-ligated heme. Their primary functions are to promote O-atom transfer to an aromatic metabolite. TDO and indoleamine 2,3-dioxygenase (IDO), the founding members, promote dioxygenation through a two-step monooxygenation pathway. However, the new members of the superfamily, including PrnB, SfmD, TyrH, and MarE, expand its boundaries and mediate monooxygenation on a broader set of aromatic substrates. We found that the enlarged superfamily contains eight clades of proteins. Overall, this protein group is a more sizeable, structure-based, histidine-ligated heme-dependent, and functionally diverse superfamily for aromatics oxidation. The concept of TDO superfamily or heme-dependent dioxygenase superfamily is no longer appropriate for defining this growing superfamily. Hence, there is a pressing need to redefine it as a heme-dependent aromatic oxygenase (HDAO) superfamily. The revised concept puts HDAO in the context of thiol-ligated heme-based enzymes alongside cytochrome P450 and peroxygenase. It will update what we understand about the choice of heme axial ligand. Hemoproteins may not be as stringent about the type of axial ligand for oxygenation, although thiolate-ligated hemes (P450s and peroxygenases) more frequently catalyze oxygenation reactions. Histidine-ligated hemes found in HDAO enzymes can likewise mediate oxygenation when confronted with a proper substrate.

Occurrence and sequence of Sphaeroides Heme Protein and diheme cytochrome C in purple photosynthetic bacteria in the family Rhodobacteraceae.

BACKGROUND: Sphaeroides Heme Protein (SHP) was discovered in the purple photosynthetic bacterium, Rhodobacter sphaeroides, and is the only known c-type heme protein that binds oxygen. Although initially not believed to be widespread among the photosynthetic bacteria, the gene has now been found in more than 40 species of proteobacteria and generally appears to be regulated. Rb. sphaeroides is exceptional in not having regulatory genes associated with the operon. We have thus analyzed additional purple bacteria for the SHP gene and examined the genetic context to obtain new insights into the operon, its distribution, and possible function. RESULTS: We found SHP in 9 out of 10 strains of Rb. sphaeroides and in 5 out of 10 purple photosynthetic bacterial species in the family Rhodobacteraceae. We found a remarkable similarity within the family including the lack of regulatory genes. Within the proteobacteria as a whole, SHP is part of a 3-6 gene operon that includes a membrane-spanning diheme cytochrome b and one or two diheme cytochromes c. Other genes in the operon include one of three distinct sensor kinase - response regulators, depending on species, that are likely to regulate SHP. CONCLUSIONS: SHP is not as rare as generally believed and has a role to play in the photosynthetic bacteria. Furthermore, the two companion cytochromes along with SHP are likely to function as an electron transfer pathway that results in the reduction of SHP by quinol and formation of the oxygen complex, which may function as an oxygenase. The three distinct sensors suggest at least as many separate functional roles for SHP. Two of the sensors are not well characterized, but the third is homologous to the QseC quorum sensor, which is present in a number of pathogens and typically appears to regulate genes involved in virulence.

The cbb3 oxidases are an ancient innovation of the domain bacteria.

A survey of genomes for the presence of gene clusters related to cbb(3) oxidases detected bona fide members of the family in almost all phyla of the domain Bacteria. No archaeal representatives were found. The subunit composition was seen to vary substantially between clades observed on the phylogenetic tree of the catalytic subunit CcoN. The protein diade formed by CcoN and the monoheme cytochrome CcoO appears to constitute the functionally essential "core" of the enzyme conserved in all sampled cbb(3) gene clusters. The topology of the phylogenetic tree contradicts the scenario of a recent origin of cbb(3) oxidases and substantiates the status of this family as a phylogenetic entity on the same level as the other subgroups of the heme-copper superfamily (including nitric oxide reductase). This finding resuscitates and exacerbates the conundrum of the evolutionary origin of heme-copper oxidases.

Oxypred: prediction and classification of oxygen-binding proteins.

This study describes a method for predicting and classifying oxygen-binding proteins. Firstly, support vector machine (SVM) modules were developed using amino acid composition and dipeptide composition for predicting oxygen-binding proteins, and achieved maximum accuracy of 85.5% and 87.8%, respectively. Secondly, an SVM module was developed based on amino acid composition, classifying the predicted oxygen-binding proteins into six classes with accuracy of 95.8%, 97.5%, 97.5%, 96.9%, 99.4%, and 96.0% for erythrocruorin, hemerythrin, hemocyanin, hemoglobin, leghemoglobin, and myoglobin proteins, respectively. Finally, an SVM module was developed using dipeptide composition for classifying the oxygen-binding proteins, and achieved maximum accuracy of 96.1%, 98.7%, 98.7%, 85.6%, 99.6%, and 93.3% for the above six classes, respectively. All modules were trained and tested by five-fold cross validation. Based on the above approach, a web server Oxypred was developed for predicting and classifying oxygen-binding proteins (available from http://www.imtech.res.in/raghava/oxypred/).

A variant System I for cytochrome c biogenesis in archaea and some bacteria has a novel CcmE and no CcmH.

C-type cytochromes are characterized by post-translational covalent attachment of heme to thiols that occur in a Cys-Xxx-Xxx-Cys-His motif. Three distinct biogenesis systems are known for this heme attachment. Archaea are now shown to contain a significantly modified form of cytochrome c maturation System I (the Ccm system). The most notable adaptation relative to the well-studied apparatus from proteobacteria and plants is a novel form of the heme chaperone CcmE, lacking the highly conserved histidine that covalently binds heme and is essential for function in Escherichia coli. In most archaeal CcmEs this histidine, normally found in a His-Xxx-Xxx-Xxx-Tyr motif, is replaced by a cysteine residue that occurs in a Cys-Xxx-Xxx-Xxx-Tyr motif. The CcmEs from two halobacteria contain yet another form of CcmE, having HxxxHxxxH approximately corresponding in alignment to the H/CxxxY motif. The CxxxY-type of CcmE is, surprisingly, also found in some bacterial genomes (including Desulfovibrio species). All of the modified CcmEs cluster together in a phylogenetic tree, as do other Ccm proteins from the same organisms. Significantly, CcmH is absent from all of the complete archaeal genomes we have studied, and also from most of the bacterial genomes that have CxxxY-type CcmE.

Recombinant PAS-heme domains of oxygen sensing proteins: high level production and physical characterization.

Details of a high-level recombinant production method for the heme-PAS domains of heme oxygen sensing proteins from Sinorhizobium meliloti (Sm) (formerly Rhizobium meliloti, Rm), Bradyrhizobium japonicum (Bj), and Escherichia coli (Ec) are described. Using a newly proposed, concise, and unambiguous naming system (also described here) these proteins are: SmFixLH(128-264), BjFixLH(140-270), and EcDosH(1-147). In addition, high-level production of BjFixL(140-505), the soluble full-length protein containing both heme (oxygen sensing) and kinase (catalytic) domains is described. Using an IPTG-inducible pET/BL21 expression system and a rapid, two-column purification has resulted in increased yields of 3- to 17-fold over literature values. The recombinant proteins are highly pure as judged by SDS-PAGE, MALDI-TOF mass spectrometry, and a UV-visible purity index. To our knowledge, this work includes the first mass spectrometry analysis of any PAS-heme protein and provides high-resolution confirmation of each protein's identity. These production and characterization improvements make possible future spectroscopic and dynamics studies designed to elucidate the intramolecular/interdomain signal that follows heme-domain oxygen dissociation.

Heme-based sensors: defining characteristics, recent developments, and regulatory hypotheses.

In a great variety of organisms throughout all kingdoms of life, the heme-based-sensor proteins are the key regulators of adaptive responses to fluctuating oxygen, carbon monoxide, and nitric oxide levels. These signal transducers achieve their responses by coupling a regulatory heme-binding domain to a neighboring transmitter. The past decade has witnessed an explosion in the numbers of these modular sensory proteins known, from just two recognized members, FixL and soluble guanylyl cyclase (sGC), to four broad families comprising more than 50 sensors. Heme-based sensors so far feature four different types of heme-binding modules: the heme-binding PAS domain, globin-coupled sensor (GCS), CooA, and heme-NO-binding (HNOB). The transmitters for coupling to such heme-binding domains include histidine protein kinases, cyclic nucleotide phosphodiesterases, chemotaxis methyl-carrier protein receptors, and transcription factors of the basic helix-loop-helix and helix-turn-helix classes. Some well-studied sensors are the FixL, EcDos, AxPDEA1, NPAS2, HemAT-Bs, HemAT-Hs, CooA, and sGC proteins. This review elaborates the defining characteristics of heme-based sensors, examines recent developments on those proteins, and discusses the regulatory hypotheses proposed for those sensors. A general, "helix-swap", model is also proposed here for signal transduction by PAS domains.

Globin-coupled sensors, protoglobins, and the last universal common ancestor.

The strategy for detecting oxygen, carbon monoxide, nitric oxide, and sulfides is predominantly through heme-based sensors utilizing either a globin domain or a PAS domain. Whereas PAS domains bind various cofactors, globins bind only heme. Globin-coupled sensors (GCSs) were first described as regulators of the aerotactic responses in Bacillus subtilis and Halobacterium salinarum. GCSs were also identified in diverse microorganisms that appear to have roles in regulating gene expression. Functional and evolutionary analyses of the GCSs, their protoglobin ancestor, and their relationship to the last universal common ancestor (LUCA) are discussed in the context of globin-based signal transduction.

Neuroglobin and cytoglobin in search of their role in the vertebrate globin family.

Neuroglobin and cytoglobin are two recent additions to the family of heme-containing respiratory proteins of man and other vertebrates. Here, we review the present state of knowledge of the structures, ligand binding kinetics, evolution and expression patterns of these two proteins. These data provide a first glimpse into the possible physiological roles of these globins in the animal's metabolism. Both, neuroglobin and cytoglobin are structurally similar to myoglobin, although they contain distinct cavities that may be instrumental in ligand binding. Kinetic and structural studies show that neuroglobin and cytoglobin belong to the class of hexa-coordinated globins with a biphasic ligand-binding kinetics. Nevertheless, their oxygen affinities resemble that of myoglobin. While neuroglobin is evolutionarily related to the invertebrate nerve-globins, cytoglobin shares a more recent common ancestry with myoglobin. Neuroglobin expression is confined mainly to brain and a few other tissues, with the highest expression observed in the retina. Present evidence points to an important role of neuroglobin in neuronal oxygen homeostasis and hypoxia protection, though other functions are still conceivable. Cytoglobin is predominantly expressed in fibroblasts and related cell types, but also in distinct nerve cell populations. Much less is known about its function, although in fibroblasts it might be involved in collagen synthesis.

Global self-organization of all known protein sequences reveals inherent biological signatures.

A global classification of all currently known protein sequences is performed. Every protein sequence is partitioned into segments of 50 amino acid residues and a dynamic programming distance is calculated between each pair of segments. This space of segments is initially embedded into Euclidean space. The algorithm that we apply embeds every finite metric space into Euclidean space so that (1) the dimension of the host space is small, (2) the metric distortion is small. A novel self-organized, cross-validated clustering algorithm is then applied to the embedded space with Euclidean distances. We monitor the validity of our clustering by randomly splitting the data into two parts and performing an hierarchical clustering algorithm independently on each part. At every level of the hierarchy we cross-validate the clusters in one part with the clusters in the other. The resulting hierarchical tree of clusters offers a new representation of protein sequences and families, which compares favorably with the most updated classifications based on functional and structural data about proteins. Some of the known families clustered into well distinct clusters. Motifs and domains such as the zinc finger, EF hand, homeobox, EGF-like and others are automatically correctly identified, and relations between protein families are revealed by examining the splits along the tree. This clustering leads to a novel representation of protein families, from which functional biological kinship of protein families can be deduced, as demonstrated for the transporter family. Finally, we introduce a new concise representation for complete proteins that is very useful in presenting multiple alignments, and in searching for close relatives in the database. The self-organization method presented is very general and applies to any data with a consistent and computable measure of similarity between data items.

The crystal structure of chloroperoxidase: a heme peroxidase--cytochrome P450 functional hybrid.

BACKGROUND: Chloroperoxidase (CPO) is a versatile heme-containing enzyme that exhibits peroxidase, catalase and cytochrome P450-like activities in addition to catalyzing halogenation reactions. The structure determination of CPO was undertaken to help elucidate those structural features that enable the enzyme to exhibit these multiple activities. RESULTS: Despite functional similarities with other heme enzymes, CPO folds into a novel tertiary structure dominated by eight helical segments. The catalytic base, required to cleave the peroxide O-O bond, is glutamic acid rather than histidine as in other peroxidases. CPO contains a hydrophobic patch above the heme that could be the binding site for substrates that undergo P450-like reactions. The crystal structure also shows extensive glycosylation with both N- and O-linked glycosyl chains. CONCLUSIONS: The proximal side of the heme in CPO resembles cytochrome P450 because a cysteine residue serves as an axial heme ligand, whereas the distal side of the heme is 'peroxidase-like' in that polar residues form the peroxide-binding site. Access to the heme pocket is restricted to the distal face such that small organic substrates can interact with the iron-linked oxygen atom which accounts for the P450-like reactions catalyzed by chloroperoxidase.

The heme-copper oxidase family consists of three distinct types of terminal oxidases and is related to nitric oxide reductase.

Among aerobic prokaryotes, many different terminal oxidase complexes have been described. Sequence comparison has revealed that the aa3-type cytochrome c oxidase and the bo3-type quinol oxidase are variations on the same theme: the heme-copper oxidase. A third member of this family has recently been recognized: the cbb3-type cytochrome c oxidase. Here we give an overview, and report that nitric oxide (NO) reductase, a bc-type cytochrome involved in denitrification, shares important features with these terminal oxidases as well. Tentative structural, functional and evolutionary implications are discussed.

Heme-based sensors, exemplified by the kinase FixL, are a new class of heme protein with distinctive ligand binding and autoxidation.

FixL's are chimeric heme protein kinases from symbiotic nitrogen-fixing Rhizobia. We have overexpressed three FixL variants in Escherichia coli. Bradyrhizobium japonicum FixL, a soluble dimeric protein, is the first full-length FixL to be purified. The other two proteins are soluble truncations of Rhizobium meliloti FixL, which is a membrane protein. One contains both heme and kinase domains and is dimeric; the other has only the heme domain and is monomeric. We find that all the FixL's bind oxygen and carbon monoxide non-cooperatively, with very low affinities due entirely to slow association rates. FixL P50's for oxygen are 17-76 mmHg. FixL's may sense nitric oxide and carbon monoxide in addition to oxygen, especially at the low oxygen pressures encountered in vivo. Autoxidation rates are about 50 times faster than that of sperm whale myoglobin. The carbon monoxide affinity of FixL's is about 300 times lower than that of myoglobin, resulting in the unusually low values of 7.5-17 for the partition constant, M = P50(O2)/P50(CO), between carbon monoxide and oxygen. Met-FixL's have their Soret absorption maximum at 395 nm instead of the typical 408 nm and a steep hydroxymet transition at pH > or = 9.3; these properties indicate a pentacoordinated high-spin ferric heme and suggest a sterically hindered hydrophobic heme pocket lacking a distal (E7) histidine. FixL is the first member of a new class of heme proteins, the heme-based sensors, distinct from the oxygen carriers and electron transporters. We expect that some of the novel properties of FixL will be characteristic of the class.