Structure-Based Phylogenetic Analysis of the Lipocalin Superfamily

Lipocalins constitute a superfamily of extracellular proteins that are found in all three kingdoms of life. Although very divergent in their sequences and functions, they show remarkable similarity in 3-D structures. Lipocalins bind and transport small hydrophobic molecules. Earlier sequence-based phylogenetic studies of lipocalins highlighted that they have a long evolutionary history. However the molecular and structural basis of their functional diversity is not completely understood. The main objective of the present study is to understand functional diversity of the lipocalins using a structure-based phylogenetic approach. The present study with 39 protein domains from the lipocalin superfamily suggests that the clusters of lipocalins obtained by structure-based phylogeny correspond well with the functional diversity. The detailed analysis on each of the clusters and sub-clusters reveals that the 39 lipocalin domains cluster based on their mode of ligand binding though the clustering was performed on the basis of gross domain structure. The outliers in the phylogenetic tree are often from single member families. Also structure-based phylogenetic approach has provided pointers to assign putative function for the domains of unknown function in lipocalin family. The approach employed in the present study can be used in the future for the functional identification of new lipocalin proteins and may be extended to other protein families where members show poor sequence similarity but high structural similarity.     

Functional divergence outlines the evolution of novel protein function in NifH/BchL protein family

Biological nitrogen fixation is accomplished by prokaryotes through the catalytic action of complex metalloenzyme, nitrogenase. Nitrogenase is a two-protein component system comprising MoFe protein (NifD&K) and Fe protein (NifH). NifH shares structural and mechanistic similarities as well as evolutionary relationships with light-independent protochlorophyllide reductase (BchL), a photosynthesis-related metalloenzyme belonging to the same protein family. We performed a comprehensive bioinformatics analysis of the NifH/BchL family in order to elucidate the intrinsic functional diversity and the underlying evolutionary mechanism among the members. To analyse functional divergence in the NifH/BchL family, we have conducted pair-wise estimation in altered evolutionary rates between the member proteins. We identified a number of vital amino acid sites which contribute to predicted functional diversity. We have also made use of the maximum likelihood tests for detection of positive selection at the amino acid level followed by the structure-based phylogenetic approach to draw conclusion on the ancient lineage and novel characterization of the NifH/BchL protein family. Our investigation provides ample support to the fact that NifH protein and BchL share robust structural similarities and have probably deviated from a common ancestor followed by divergence in functional properties possibly due to gene duplication     

Genetic manipulation of 11beta-hydroxysteroid dehydrogenases in mice

11beta-hydroxysteroid dehydrogenases (HSDs) interconvert active 11-hydroxy glucocorticoids (cortisol, corticosterone) and their inert 11-keto derivatives (cortisone, 11-dehydrocorticosterone). 11beta-HSD type 1 is a predominant reductase that regenerates active glucocorticoids in expressing cells, thus amplifying local glucocorticoid action, whereas 11beta-HSD type 2 catalyzes rapid dehydrogenation, potently inactivating intracellular glucocorticoids. Both isozymes thus regulate receptor activation by substrate availability. Spatial and temporal regulation of expression are important determinants of the physiological roles of 11beta-HSDs, with each isozyme exhibiting a distinct, tissue-restricted pattern together with dynamic regulation during development and in response to environmental challenges, including diet and stress. Transgenic approaches in the mouse have contributed significantly toward an understanding of the importance of these prereceptor regulatory mechanisms on corticosteroid receptor activity and have highlighted its potential relevance to human health and disease. Here we discuss current ideas of the physiological roles of 11beta-HSDs, with emphasis on the key contributions made by studies of 11beta-HSD gene manipulation in vivo.     

Evolutionary History of <Emphasis Type="SmallCaps">d</Emphasis>-Lactate Dehydrogenases: A Phylogenomic Perspective on Functional Diversity in the FAD Binding Oxidoreductase/Transferase Type 4 Family

Lactate dehydrogenases which convert lactate to pyruvate are found in almost every organism and comprise a group of highly divergent proteins in amino acid sequence, catalytic properties, and substrate specificity. While the l-lactate dehydrogenases are among the most studied enzymes, very little is known about the structure and function of d-lactate dehydrogenases (d-LDHs) which include two discrete classes of enzymes that are classified based on their ability to transfer electrons and/or protons to NAD in NAD-dependent lactate dehydrogenases (nLDHs), and FAD in NAD-independent lactate dehydrogenases (iLDHs). In this study, we used a combination of structural and phylogenomic approaches to reveal the likely evolutionary events in the history of the recently described FAD binding oxidoreductase/transferase type 4 family that led to the evolution of d-iLDHs (commonly referred as DLD). Our phylogenetic reconstructions reveal that DLD genes from eukaryotes form a paraphyletic group with respect to d-2-hydroxyglutarate dehydrogenase (D2HGDH). All phylogenetic reconstructions recovered two divergent yeast DLD phylogroups. While the first group (DLD1) showed close phylogenetic relationships with the animal and plant DLDs, the second yeast group (DLD2) revealed strong phylogenetic and structural similarities to the plant and animal D2HGDH group. Our data strongly suggest that the functional assignment of the yeast DLD2 group should be carefully revisited. The present study demonstrates that structural phylogenomic approach can be used to resolve important evolutionary events in functionally diverse superfamilies and to provide reliable functional predictions to poorly characterized genes.     

Flavonoids and cinnamic acid esters as inhibitors of fungal 17beta-hydroxysteroid dehydrogenase: a synthesis, QSAR and modelling study

The 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) modulate the biological potency of estrogens and androgens by interconversion of inactive 17-keto-steroids and their active 17beta-hydroxy- counterparts. We have shown previously that flavonoids are potentially useful lead compounds for developing inhibitors of 17beta-HSDs. In this paper, we describe the synthesis and biochemical evaluation of structurally analogous inhibitors, the trans-cinnamic acid esters and related compounds. Additionally, quantitative structure-activity relationship (QSAR) and modelling studies were performed to rationalize the results and to suggest further optimization. The results stress the importance of a hydrogen bond with Asn154 and hydrophobic interactions with the aromatic side chain of Tyr212 for optimal molecular recognition.     

Structure and function of retinol dehydrogenases of the short chain dehydrogenase/reductase family

All-trans-retinol is the common precursor of the active retinoids 11-cis-retinal, all-trans-retinoic acid (atRA) and 9-cis-retinoic acid (9cRA). Genetic and biochemical data supports an important role of the microsomal members of the short chain dehydrogenases/reductases (SDRs) in the first oxidative conversion of retinol into retinal. Several retinol dehydrogenases of this family have been reported in recent years. However, the structural and functional data on these enzymes is limited. The prototypic enzyme RDH5 and the related enzyme CRAD1 have been shown to face the lumen of the endoplasmic reticulum (ER), suggesting a compartmentalized synthesis of retinal. This is a matter of debate as a related enzyme has been proposed to have the opposite membrane topology. Recent data indicates that RDH5, and presumably other members of the SDRs, occur as functional homodimers, and need to interact with other proteins for proper intracellular localization and catalytic activity. Further analyses on the compartmentalization, membrane topology, and functional properties of microsomal retinol dehydrogenases, will give important clues about how retinoids are processed.     

Hydroxysteroid dehydrogenases (HSDs) in bacteria: a bioinformatic perspective

Steroidal compounds including cholesterol, bile acids and steroid hormones play a central role in various physiological processes such as cell signaling, growth, reproduction, and energy homeostasis. Hydroxysteroid dehydrogenases (HSDs), which belong to the superfamily of short-chain dehydrogenases/reductases (SDR) or aldo-keto reductases (AKR), are important enzymes involved in the steroid hormone metabolism. HSDs function as an enzymatic switch that controls the access of receptor-active steroids to nuclear hormone receptors and thereby mediate a fine-tuning of the steroid response. The aim of this study was the identification of classified functional HSDs and the bioinformatic annotation of these proteins in all complete sequenced bacterial genomes followed by a phylogenetic analysis. For the bioinformatic annotation we constructed specific hidden Markov models in an iterative approach to provide a reliable identification for the specific catalytic groups of HSDs. Here, we show a detailed phylogenetic analysis of 3α-, 7α-, 12α-HSDs and two further functional related enzymes (3-ketosteroid-Δ(1)-dehydrogenase, 3-ketosteroid-Δ(4)(5α)-dehydrogenase) from the superfamily of SDRs. For some bacteria that have been previously reported to posses a specific HSD activity, we could annotate the corresponding HSD protein. The dominating phyla that were identified to express HSDs were that of Actinobacteria, Proteobacteria, and Firmicutes. Moreover, some evolutionarily more ancient microorganisms (e.g., Cyanobacteria and Euryachaeota) were found as well. A large number of HSD-expressing bacteria constitute the normal human gastro-intestinal flora. Another group of bacteria were originally isolated from natural habitats like seawater, soil, marine and permafrost sediments. These bacteria include polycyclic aromatic hydrocarbons-degrading species such as Pseudomonas, Burkholderia and Rhodococcus. In conclusion, HSDs are found in a wide variety of microorganisms including bacteria and archaea, suggesting that steroid metabolism is an evolutionarily conserved mechanism that might serve different functions such as nutrient supply and signaling. Article from a special issue on steroids and microorganisms.     

Evolutionary Descent of Prion Genes from the ZIP Family of Metal Ion Transporters

In the more than twenty years since its discovery, both the phylogenetic origin and cellular function of the prion protein (PrP) have remained enigmatic. Insights into a possible function of PrP may be obtained through the characterization of its molecular neighborhood in cells. Quantitative interactome data demonstrated the spatial proximity of two metal ion transporters of the ZIP family, ZIP6 and ZIP10, to mammalian prion proteins in vivo. A subsequent bioinformatic analysis revealed the unexpected presence of a PrP-like amino acid sequence within the N-terminal, extracellular domain of a distinct sub-branch of the ZIP protein family that includes ZIP5, ZIP6 and ZIP10. Additional structural threading and orthologous sequence alignment analyses argued that the prion gene family is phylogenetically derived from a ZIP-like ancestral molecule. The level of sequence homology and the presence of prion protein genes in most chordate species place the split from the ZIP-like ancestor gene at the base of the chordate lineage. This relationship explains structural and functional features found within mammalian prion proteins as elements of an ancient involvement in the transmembrane transport of divalent cations. The phylogenetic and spatial connection to ZIP proteins is expected to open new avenues of research to elucidate the biology of the prion protein in health and disease.     

New Functions for the Ancient DedA Membrane Protein Family

The DedA protein family is a highly conserved and ancient family of membrane proteins with representatives in most sequenced genomes, including those of bacteria, archaea, and eukarya. The functions of the DedA family proteins remain obscure. However, recent genetic approaches have revealed important roles for certain bacterial DedA family members in membrane homeostasis. Bacterial DedA family mutants display such intriguing phenotypes as cell division defects, temperature sensitivity, altered membrane lipid composition, elevated envelope-related stress responses, and loss of proton motive force. The DedA family is also essential in at least two species of bacteria: Borrelia burgdorferi and Escherichia coli. Here, we describe the phylogenetic distribution of the family and summarize recent progress toward understanding the functions of the DedA membrane protein family.     

Characterization of fungal 17beta-hydroxysteroid dehydrogenases

To promote understanding of the evolution of the steroid hormone signalling and hydroxysteroid dehydrogenases (HSDs), comparative characterization of fungal 17beta-HSDs was performed. Constitutive 17beta-HSD activity was determined in cytosols of the fungi: Cochliobolus lunatus, Pleospora herbarum, Fusarium lini, Trichoderma viride, Mucor spinosus, Rhizopus nigricans and Pleurotus ostreatus. The reaction equilibrium in all species except P. ostreatus was shifted towards reduction. The preferential coenzyme for reduction of androstenedione was NADPH, while for oxidation of testosterone, NAD4 was preferred. The highest enzyme activities were found in the Ascomycete C. lunatus (152.4 nmol mg(-1) h(-1)) and in the Basidiomycete P. ostreatus (69.1 nmol mg(-1) h(-1)). No similarities on the protein and mRNA level between fungal 17beta-HSDs and the purified enzyme from C. lunatus were observed. To investigate the nature of these enzymes, 17beta-HSD was purified from P. ostreatus using ammonium sulphate precipitation, hydrophobic interaction chromatography, and affinity chromatography. The purified enzyme has an apparent molecular mass of approximately 35 kDa and is probably a dimer as determined by gel filtration. Chemical modifications exposed Lys, His and Tyr as important for enzyme activity. Additionally, no similarities of C. lunatus and P. ostreatus enzymes were found to bacterial 3alpha,20beta-HSD from Streptomyces hydrogenans, 3beta,17beta-HSD from Comamonas testosteroni and mammalian 17beta-HSD types 1 and 4. The results thus suggest that there are most probably different enzymes responsible for 17beta-HSD activity in filamentous fungi.     

The repertoire of solute carriers of family 6: identification of new human and rodent genes

Tremendous amount of primary sequence information has been made available from the genome sequencing projects, although a complete annotation and identification of all genes is still far from being complete. Here, we present the identification of two new human genes from the pharmacologically important family of transporter proteins, solute carriers family 6 (SLC6). These were named SLC6A17 and SLC6A18 by HUGO. The human repertoire of SLC6 proteins now consists of 19 functional members and four pseudogenes. We also identified the corresponding orthologues and additional genes from mouse and rat genomes. Detailed phylogenetic analysis of the entire family of SLC6 proteins in mammals shows that this family can be divided into four subgroups. We used Hidden Markov Models for these subgroups and identified in total 430 unique SLC6 proteins from 10 animal, one plant, two fungi, and 196 bacterial genomes. It is evident that SLC6 proteins are present in both animals and bacteria, and that three of the four subfamilies of mammalian SLC6 proteins are present in Caenorhabditis elegans, showing that these subfamilies are evolutionary very ancient. Moreover, we performed tissue localization studies on the entire family of SLC6 proteins on a panel of 15 rat tissues and further, the expression of three of the new genes was studied using quantitative real-time PCR showing expression in multiple central and peripheral tissues. This paper presents an overall overview of the gene repertoire of the SLC6 gene family and its expression profile in rats.     

Pseudomonas 3 beta-hydroxysteroid dehydrogenase. Primary structure and relationships to other steroid dehydrogenases

The 3 beta-hydroxysteroid dehydrogenase of Pseudomonas testosteroni commercially available was purified by an FPLC step and submitted to sequence determination by peptide analysis. The structure obtained reveals a 253-residue polypeptide chain, with an N-terminal, free alpha-amino group, and a low cysteine content. Comparisons with other hydroxysteroid dehydrogenases recently characterized reveal distant similarities with prokaryotic and, to some extent, also eukaryotic forms of separate specificities. Residue identities with a Streptomyces 20 beta-hydroxysteroid dehydrogenase are 35% and distributed over the entire molecule, whereas residue identities with the mammalian 17 beta-hydroxysteroid dehydrogenase only constitute 20%, and are essentially limited to the N-terminal and central parts, Nevertheless, all these enzymes exhibit a conserved tyrosine residue (position 151 in the present enzyme) noted as possibly having a functional role in some members of this protein family. Combined, the results establish the prokaryotic 3 beta-hydroxysteroid dehydrogenase as belonging to the family of short-chain alcohol dehydrogenases, reveal that the hydroxysteroid dehydrogenases are no more closely related than dehydrogenases with other enzyme activities within the family (e.g. glucose, ribitol, hydroxyprostaglandin dehydrogenases), show several of the mammalian hydroxysteroid dehydrogenases to have subunits of longer size with different patterns of similarity than those of the prokaryotic family members characterized, and define important segments of the coenzyme-binding region for this enzyme group.     

Novel inhibitors of 17beta-hydroxysteroid dehydrogenase type 1: templates for design

The 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) catalyze the interconversion between the oxidized and reduced forms of androgens and estrogens at the 17 position. The 17beta-HSD type 1 enzyme (17beta-HSD1) catalyzes the reduction of estrone (E1) to estradiol and is expressed in malignant breast cells. Inhibitors of this enzyme thus have potential as treatments for hormone dependent breast cancer. Syntheses and biological evaluation of novel non-steroidal inhibitors designed to mimic the E1 template are reported using information from potent steroidal inhibitors. Of the templates investigated biphenyl ethanone was promising and led to inhibitors with IC(50) values in the low micromolar range.     

Comparison of sequence-based and structure-based phylogenetic trees of homologous proteins: Inferences on protein evolution

Several studies based on the known three-dimensional (3-D) structures of proteins show that two homologous proteins with insignificant sequence similarity could adopt a common fold and may perform same or similar biochemical functions. Hence, it is appropriate to use similarities in 3-D structure of proteins rather than the amino acid sequence similarities in modelling evolution of distantly related proteins. Here we present an assessment of using 3-D structures in modelling evolution of homologous proteins. Using a dataset of 108 protein domain families of known structures with at least 10 members per family we present a comparison of extent of structural and sequence dissimilarities among pairs of proteins which are inputs into the construction of phylogenetic trees. We find that correlation between the structure-based dissimilarity measures and the sequence-based dissimilarity measures is usually good if the sequence similarity among the homologues is about 30% or more. For protein families with low sequence similarity among the members, the correlation coefficient between the sequence-based and the structure-based dissimilarities are poor. In these cases the structure-based dendrogram clusters proteins with most similar biochemical functional properties better than the sequence-similarity based dendrogram. In multi-domain protein families and disulphide-rich protein families the correlation coefficient for the match of sequence-based and structure-based dissimilarity (SDM) measures can be poor though the sequence identity could be higher than 30%. Hence it is suggested that protein evolution is best modelled using 3-D structures if the sequence similarities (SSM) of the homologues are very low.     

PALI: a database of alignments and phylogeny of homologous protein structures

PALI is a database of structure-based sequence alignments and phylogenetic relationships derived on the basis of three-dimensional structures of homologous proteins. This database enables grouping of pairs of homologous protein structures on the basis of their sequence identity calculated from the structure-based alignment and PALI also enables association of a new sequence to a family and automatic generation of a dendrogram combining the query sequence and homologous protein structures.     

Unusual evolution of 11β- and 17β-hydroxysteroid and retinol dehydrogenases

11β-hydroxysteroid dehydrogenases regulate glucocorticoid concentrations and 17β-hydroxysteroid dehydrogenases regulate estrogen and androgen concentrations in mammals. Phylogenetic analysis of the sequences from two 11β-hydroxysteroid dehydrogenases and four mammalian 17β-hydroxysteroid dehydrogenases indicates unusual evolution in these enzymes. Type 1 11β- and 17β-hydroxysteroid dehydrogenases are on the same branch; Type 2 enzymes cluster on another branch with β-hydroxybutyrate dehydrogenase, 11-cis retinol dehydrogenase and retinol dehydrogenase; Type 3 17β-hydroxysteroid dehydrogenase is on a third branch; while the pig dehydrogenase clusters with a yeast multifunctional enzyme on a fourth branch. Pig 17β-hydroxysteroid dehydrogenase appears to have evolved independently from the other three 17β-hydroxysteroid dehydrogenase dehydrogenases; in which case, the evolution of 17β-hydroxysteroid dehydrogenase activity is an example of functional convergence. The phylogeny also suggests that independent evolution of specificity toward C11 substituents on glucocorticoids and C17 substituents on androgens and estrogens has occurred in Types 1 and 2 11β- and 17β-hydroxysteroid dehydrogenases.     

SNARE complex-mediated degranulation in mast cells

Mast cell function and dysregulation is important in the development and progression of allergic and autoimmune disease. Identifying novel proteins involved in mast cell function and disease progression is the first step in the design of new therapeutic strategies. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are a family of proteins demonstrated to mediate the transport and fusion of secretory vesicles to the membrane in mast cells, leading to the subsequent release of the vesicle cargo through an exocytotic mechanism. The functional role[s] of specific SNARE family member complexes in mast cell degranulation has not been fully elucidated. Here, we review recent and historical data on the expression, formation and localization of various SNARE proteins and their complexes in murine and human mast cells. We summarize the functional data identifying the key SNARE family members that appear to participate in mast cell degranulation. Furthermore, we discuss the utilization of RNA interference (RNAi) methods to validate SNARE function and the use of siRNA as a therapeutic approach to the treatment of inflammatory disease. These studies provide an overview of the specific SNARE proteins and complexes that serve as novel targets for the development of new therapies to treat allergic and autoimmune disease.     

Expression of 17beta-hydroxysteroid dehydrogenases in mesophilic and extremophilic yeast

17beta-hydroxysteroid dehydrogenases (17beta-HSDs) are enzymes responsible for reversible interconversions of biologically active 17-hydroxy and inactive 17-keto steroids. We have performed a survey of 17beta-HSD activity in yeast. Constitutive 17beta-HSD activity was found in three mesophilic yeast species: Candida tropicalis, Cryptococcus tsukubaensis, and Saccharomyces cerevisiae as well as in three extremophilic black yeast species: Hortaea werneckii, Trimmatostroma salinum, and Phaeotheca triangularis, indicating that 17beta-HSD activity is widely distributed among yeast. In extremophilic black yeast, NaCl modulated enzyme activity. Enzymes resembling 17beta-HSD from the filamentous fungus Cochliobolus lunatus were detected in Trimmatostroma salinum and Phaeotheca triangularis. Sequences with identity to the Saccharomyces cerevisiae YBR159w gene were not observed in other yeast species possessing a similar enzyme activity. The results suggest the existence of at least three different types of 17beta-HSD in yeast.     

Functional aspects of 17beta-hydroxysteroid dehydrogenase 1 determined by comparison to a closely related retinol dehydrogenase

Determining the functional aspects of a gene or protein is a difficult and time-consuming process. De novo analysis is surely the hardest and so it is often quite useful to start with a comparison to functionally or structurally related proteins. Although 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD 1) can hardly be called a new protein but rather the best characterized among the family of 17beta-HSDs some aspects of structure–function relationships remain unclear. We have sought new aspects of 17beta-HSD 1 function through a comparison with its closest homolog, a photoreceptor-associated retinol dehydrogenase (prRDH). Overall amino acid identity and size of the proteins are highly conserved, but major differences occur in the C-termini, where prRDH, but not 17beta-HSD 1, harbors motifs indicative of membrane localization. To gain insight into substrate discrimination by prRDH and 17beta-HSD 1, we constructed 3D-structure models of the corresponding zebrafish enzymes. Investigation of the substrate binding site revealed a few identical amino acids, and suggested a role for G143 in zebrafish 17beta-HSD 1 and M146 and M147 in the two zebrafish paralogs prRDH 1 and prRDH 2, respectively, in substrate specificity. Activity measurements of modified proteins in transiently transfected intact HEK 293 cells hint at a putative role of these amino acids in discrimination between steroid and retinoid substrates.     

Comparative analysis of two members of the metal ion-containing group III-alcohol dehydrogenases from Dickeya zeae

Purpose of work

A pair of NAD⁺- and NADP⁺-dependent group III-alcohol dehydrogenases was characterized from the enterobacterium, Dickeya zeae, to expand our understanding of the distribution and biochemical properties of this interesting group of enzymes. Two putative group III-alcohol dehydrogenases (ADHs) were identified in the genome of Dickeya zeae. Amino acid alignments and phylogenetic analysis revealed that Adh3.1 and Adh3.2 are only distantly related (~25 % identity at the protein level). Both proteins were purified to homogeneity after heterologous expression in E. coli. A specific activity of 1.8 U/mg was measured for the NAD⁺-dependent enzyme Adh3.1 with ethanol used as substrate, while NADPH-dependent Adh3.2 preferred butanal (29.1 U/mg) as substrate. Maximum activity for Adh3.1 was at 50 °C and pH 10 and for Adh3.2 at 70 °C and pH 6. Cell viability assays were used to confirm activity towards butanal and glyoxals. Biochemical characterization and phylogenetic analyses led to the hypothesis that Adh3.1 and Adh3.2 are probably the result of an ancient gene duplication event followed by functional diversification.