Crystal structures of the tryptophan repressor binding protein WrbA and complexes with flavin mononucleotide

The tryptophan repressor binding protein WrbA binds to the tryptophan repressor protein TrpR. Although the biological role of WrbA remains unclear, it has been proposed to function in enhancing the stability of TrpR-DNA complexes. Sequence database analysis has identified WrbA as a founding member of a flavodoxin-like family of proteins. Here we present crystal structures of WrbA from Deinococcus radiodurans and Pseudomonas aeruginosa and their complexes with flavin mononucleotide. The protomer structure is similar to that of previously determined long-chain flavodoxins; however, each contains a conserved inserted region unique to the WrbA family. Interestingly, each WrbA protein forms a homotetramer with 222 symmetry, unique among flavodoxin-like proteins, in which each protomer binds one flavin mononucleotide cofactor molecule.     

WrbA from Escherichia coli and Archaeoglobus fulgidus is an NAD(P)H:quinone oxidoreductase

WrbA (tryptophan [W] repressor-binding protein) was discovered in Escherichia coli, where it was proposed to play a role in regulation of the tryptophan operon; however, this has been put in question, leaving the function unknown. Here we report a phylogenetic analysis of 30 sequences which indicated that WrbA is the prototype of a distinct family of flavoproteins which exists in a diversity of cell types across all three domains of life and includes documented NAD(P)H:quinone oxidoreductases (NQOs) from the Fungi and Viridiplantae kingdoms. Biochemical characterization of the prototypic WrbA protein from E. coli and WrbA from Archaeoglobus fulgidus, a hyperthermophilic species from the Archaea domain, shows that these enzymes have NQO activity, suggesting that this activity is a defining characteristic of the WrbA family that we designate a new type of NQO (type IV). For E. coli WrbA, the K(m)(NADH) was 14 +/- 0.43 microM and the K(m)(benzoquinone) was 5.8 +/- 0.12 microM. For A. fulgidus WrbA, the K(m)(NADH) was 19 +/- 1.7 microM and the K(m)(benzoquinone) was 37 +/- 3.6 microM. Both enzymes were found to be homodimeric by gel filtration chromatography and homotetrameric by dynamic light scattering and to contain one flavin mononucleotide molecule per monomer. The NQO activity of each enzyme is retained over a broad pH range, and apparent initial velocities indicate that maximal activities are comparable to the optimum growth temperature for the respective organisms. The results are discussed and implicate WrbA in the two-electron reduction of quinones, protecting against oxidative stress.     

WrbA bridges bacterial flavodoxins and eukaryotic NAD(P)H:quinone oxidoreductases

The crystal structure of the flavodoxin-like protein WrbA with oxidized FMN bound reveals a close relationship to mammalian NAD(P)H:quinone oxidoreductase, Nqo1. Structural comparison of WrbA, flavodoxin, and Nqo1 indicates how the twisted open-sheet fold of flavodoxins is elaborated to form multimers that extend catalytic function from one-electron transfer between protein partners using FMN to two-electron reduction of xenobiotics using FAD. The structure suggests a novel physiological role for WrbA and Nqo1.     

Mutations in Elongation Factor 1β, a Guanine Nucleotide Exchange Factor, Enhance Translational Fidelity

Translation elongation factor 1beta (EF-1beta) is a member of the family of guanine nucleotide exchange factors, proteins whose activities are important for the regulation of G proteins critical to many cellular processes. EF-1beta is a highly conserved protein that catalyzes the exchange of bound GDP for GTP on EF-1alpha, a required step to ensure continued protein synthesis. In this work, we demonstrate that the highly conserved C-terminal region of Saccharomyces cerevisiae EF-1beta is sufficient for normal cell growth. This region of yeast and metazoan EF-1beta and the metazoan EF-1beta-like protein EF-1delta is highly conserved. Human EF-1beta, but not human EF-1delta, is functional in place of yeast EF-1beta, even though both EF-1beta and EF-1delta have previously been shown to have guanine nucleotide exchange activity in vitro. Based on the sequence and functional homology, mutagenesis of two C-terminal residues identical in all EF-1beta protein sequences was performed, resulting in mutants with growth defects and sensitivity to translation inhibitors. These mutants also enhance translational fidelity at nonsense codons, which correlates with a reduction in total protein synthesis. These results indicate the critical function of EF-1beta in regulating EF-1alpha activity, cell growth, translation rates, and translational fidelity.     

Spermidine biosynthesis in Saccharomyces cerevisiae. Biosynthesis and processing of a proenzyme form of S-adenosylmethionine decarboxylase

We have cloned and sequenced the Saccharomyces cerevisiae gene for S-adenosylmethionine decarboxylase. This enzyme contains covalently bound pyruvate which is essential for enzymatic activity. We have shown that this enzyme is synthesized as a Mr 46,000 proenzyme which is then cleaved post-translationally to form two polypeptide chains: a beta subunit (Mr 10,000) from the amino-terminal portion and an alpha subunit (Mr 36,000) from the carboxyl-terminal portion. The protein was overexpressed in Escherichia coli and purified to homogeneity. The purified enzyme contains both the alpha and beta subunits. About half of the alpha subunits have pyruvate blocking the amino-terminal end; the remaining alpha subunits have alanine in this position. From a comparison of the amino acid sequence deduced from the nucleotide sequence with the amino acid sequence of the amino-terminal portion of each subunit (determined by Edman degradation), we have identified the cleavage site of the proenzyme as the peptide bond between glutamic acid 87 and serine 88. The pyruvate moiety, which is essential for activity, is generated from serine 88 during the cleavage. The amino acid sequence of the yeast enzyme has essentially no homology with S-adenosylmethionine decarboxylase of E. coli (Tabor, C. W., and Tabor, H. (1987) J. Biol. Chem. 262, 16037-16040) and only a moderate degree of homology with the human and rat enzymes (Pajunen, A., Crozat, A., J?nne, O. A., Ihalainen, R., Laitinen, P. H., Stanley, B., Madhubala, R., and Pegg, A. E. (1988) J. Biol. Chem. 263, 17040-17049); all of these enzymes are pyruvoyl-containing proteins. Despite this limited overall homology the cleavage site of the yeast proenzyme is identical to the cleavage sites in the human and rat proenzymes, and seven of the eight amino acids adjacent to the cleavage site are identical in the three eukaryote enzymes.     

Both helical propensity and side-chain hydrophobicity at a partially exposed site in alpha-helix contribute to the thermodynamic stability of ubiquitin

Pfam family DUF1023 consists entirely of uncharacterized proteins generated by sequencing the genomes of Actinobacteria (Bateman A., et al., Nucleic Acids Res. 2004;32 Database issue:D138-141.) Utilizing sequence similarity detection methods, we infer homology between DUF1023 and alpha/beta hydrolases. DUF1023 proteins conserve the core secondary structures in alpha/beta hydrolase fold, and share similar catalytic machinery as that of alpha/beta hydrolases. We predict DUF1023 spatial structure and deduce that they function as hydrolases utilizing catalytic Ser-His-Asp triad with the serine as a nucleophile.     

Anabaena apoflavodoxin hydrogen exchange: on the stable exchange core of the alpha/beta(21345) flavodoxin-like family

An important issue in modern protein biophysics is whether structurally homologous proteins share common stability and/or folding features. Flavodoxin is an archetypal alpha/beta protein organized in three layers: a central beta-sheet (strand order 21345) flanked by helices 1 and 5 on one side and helices 2, 3, and 4 on the opposite side. The backbone internal dynamics of the apoflavodoxin from Anabaena is analyzed here by the hydrogen exchange method. The hydrogen exchange rates indicate that 46 amide protons, distributed throughout the structure of apoflavodoxin, exchange relatively slowly at pH 7.0 (k(ex) < 10(-1) min(-1)). According to their distribution in the structure, protein stability is highest on the beta-sheet, helix 4, and on the layer formed by helices 1 and 5. The exchange kinetics of Anabaena apoflavodoxin was compared with those of the apoflavodoxin from Azotobacter, with which it shares a 48% sequence identity, and with Che Y and cutinase, two other alpha/beta (21345) proteins with no significant sequence homology with flavodoxins. Both similarities and differences are observed in the cores of these proteins. It is of interest that a cluster of a few structurally equivalent residues in the central beta-strands and in helix 5 is common to the cores.     

Isolation, cloning, mapping, and nucleotide sequencing of the gene encoding flavodoxin in Escherichia coli.

The flavodoxins constitute a highly conserved family of small, acidic electron transfer proteins with flavin mononucleotide prosthetic groups. They are found in prokaryotes and in red and green algae, where they provide electrons at low potentials for the reduction of nitrogen by nitrogenase, for the light-dependent reduction of NADP+ in photosynthesis, and for the reduction of sulfite. Proteins with the physical characteristics of flavodoxins have been implicated in the reductive activation of pyruvate formate-lyase and cobalamin-dependent methionine synthase in Escherichia coli. We have purified flavodoxin to homogeneity from E. coli, determined its N-terminal amino acid sequence, and used this sequence to construct a 64-fold degenerate oligonucleotide probe for the flavodoxin gene. Because the phenotype of a flavodoxin mutant is not known, we used this degenerate probe to screen the phages of the Kohara library and identified two phages, with inserts mapping at approximately 16 min, that hybridized to the probe. The flavodoxin gene, designated fldA, was subcloned from the DNA in the overlap region of these two clones. The deduced amino acid sequence, determined by nucleotide sequencing of the flavodoxin gene, shows strong homology with flavodoxins from nitrogen-fixing bacteria and cyanobacteria. The fldA gene maps at 15.9 min on the E. coli chromosome and is transcribed in a counterclockwise direction.     

Biophysical characterization of cyclic nucleotide phosphodiesterases

We have compared selected biophysical properties of three phosphodiesterases, from Arabidopsis thaliana, Saccharomyces cerevisiae, and Escherichia coli. All of them belong to a recently identified family of cyclic nucleotide phosphodiesterases. Experiments elucidating folding stability, protein fluorescence, oligomerization behavior, and the effects of substrates were conducted, revealing differences between the plant and the yeast protein. According to CD spectroscopy, the latter protein exhibits an (alpha + beta) fold rather than an (alpha/beta) fold as found with CPDase (A. thaliana). The redox-dependent structural reorganization recently found for the plant protein by X-ray crystallography could not be detected by CD spectroscopy due to its only marginal effect on the total percentage of helical content. However, in the present study a redox-dependent effect was also observed for the yeast CPDase. The enzymatic activity of wild type CPDase (A. thaliana) as well as of four mutants were characterized by isothermal titration calorimetry and the results prove the requirement of all four residues of the previously identified tandem signature motif for the catalytic function. Within the comparison of the three proteins in this study, the PDase Homolog/RNA ligase (E. coli) shares more similarities with the plant than with the yeast protein.     

Molecular cloning of a Chinese hamster mitochondrial protein related to the "chaperonin" family of bacterial and plant proteins

The complete cDNA sequence of a mitochondrial protein from Chinese hamster ovary cells, designated P1, which was originally identified as a microtubule-related protein (Gupta, R.S., Ho, T.K.W., Moffat, M.R.K., and Gupta, R. (1982) J. Biol. Chem. 257, 1071-1078), has been determined. The P1 cDNA encodes a protein of 60,983 Da including a 26-amino acid putative mitochondrial targeting sequence at its N-terminal end. The deduced amino acid sequence of Chinese hamster P1 shows 97% identity to the human P1 protein. Most interestingly, the amino acid sequences of mammalian P1 proteins show extensive sequence homology (42-60% identical residues and an additional 15-25% conservative replacements) to the "chaperonin" family of bacterial, yeast, and plant proteins (viz. groEL protein of Escherichia coli, hsp 60 protein of yeast, and ribulose-1,5-bisphosphate carboxylase subunit binding protein of plant chloroplasts) and to the 60-65-kDa major antigenic protein of mycobacteria and Coxiella burnetii. The homology between mammalian P1 and other proteins begins after the putative mitochondrial presequence and extends up to the C-terminal end. Furthermore, similar to the chaperonin family of proteins, P1 appears to exist in cells as a homooligomeric complex of seven subunits and shows ATPase activity. These observations strongly indicate that P1 protein is a member of the chaperonin family and that it may be involved in a similar function in mammalian cells.     

Cloning, expression, and functional characterization of the beta regulatory subunit of human methionine adenosyltransferase (MAT II)

MAT II, the extrahepatic form of methionine adenosyltransferase (MAT), consists of catalytic alpha(2)/alpha(2') subunits and a noncatalytic beta subunit, believed to have a regulatory function. The full-length cDNA that encodes the beta subunit of human MAT II was cloned and found to encode for a 334-amino acid protein with a calculated molecular weight of 37,552. Analysis of sequence homology showed similarity with bacterial enzymes that catalyze the reduction of TDP-linked sugars. The beta subunit cDNA was cloned into the pQE-30 expression vector, and the recombinant His tagged protein, which was expressed in Escherichia coli, was recognized by antibodies to the human MAT II, to synthetic peptides copying the sequence of native beta subunit protein, and to the rbeta protein. There is no cross-reactivity between the MAT II alpha(2) or beta subunits. None of the anti-beta subunit antibodies reacted with protein extracts of E. coli host cells, suggesting that these bacteria have no beta subunit protein. Interestingly, the rbeta subunit associated with E. coli as well as human MAT alpha subunits. This association changed the kinetic properties of both enzymes and lowered the K(m) of MAT for L-methionine. Together, the data show that we have cloned and expressed the human MAT II beta subunit and confirmed its long suspected regulatory function. This knowledge affords a molecular means by which MAT activity and consequently the levels of AdoMet may be modulated in mammalian cells.     

The primary structure and the functional domains of an elongation factor-1 alpha from Mucor racemosus

We have determined the complete nucleotide sequence for TEF-1, one of three genes coding for elongation factor (EF)-1 alpha in Mucor racemosus. The deduced EF-1 alpha protein contains 458 amino acids encoded by two exons. The presence of an intervening sequence located near the 3' end of the gene was predicted by the nucleotide sequence data and confirmed by alkaline S1 nuclease mapping. The amino acid sequence of EF-1 alpha was compared to the published amino acid sequences of EF-1 alpha proteins from Saccharomyces cerevisiae and Artemia salina. These proteins shared nearly 85% homology. A similar comparison to the functionally analogous EF-Tu from Escherichia coli revealed several regions of amino acid homology suggesting that the functional domains are conserved in elongation factors from these diverse organisms. Secondary structure predictions indicated that alpha helix and beta sheet conformations associated with the functional domains in EF-Tu are present in the same relative location in EF-1 alpha from M. racemosus. Through this comparative structural analysis we have predicted the general location of functional domains in EF-1 alpha which interact with GTP and tRNA.     

Isolation and characterization of the yeast gene coding for the alpha subunit of mitochondrial phenylalanyl-tRNA synthetase

The respiratory defect of pet mutants of Saccharomyces cerevisiae assigned to complementation group G120 has been ascribed to their inability to acylate the mitochondrial phenylalanyl tRNA. A fragment of wild type yeast genomic DNA capable of complementing the genetic lesion of G120 mutants has been cloned by transformation with a yeast genomic recombinant library of a representative mutant from this complementation group. The gene designated as MSF1 has been subcloned on a 2.2-kilobase pair fragment and its nucleotide sequence determined. The predicted protein product of MSF1 has a molecular weight of 55,314 and has several domains of high primary sequence homology to the alpha subunit of the Escherichia coli phenylalanyl-tRNA synthetase. Based on the phenotype of G120 mutants and the homology to the bacterial protein, MSF1 is proposed to code for the alpha subunit of yeast mitochondrial phenylalanyl-tRNA synthetase. Disruption of the chromosomal copy of MSF1 in the respiratory-competent haploid strain W303-1B induces a phenotype similar to G120 mutants but does not affect cell viability, indicating that the cytoplasmic phenylalanyl-tRNA synthetase of yeast is encoded by a separate gene. Although the E. coli and yeast mitochondrial aminoacyl-tRNA synthetases are sufficiently similar in their primary sequences to suggest a common evolutionary origin, they have undergone significant changes as evidenced by the low homology in some regions of the polypeptide chains and the presence in the mitochondrial enzyme of two domains that are lacking in the bacterial phenylalanyl-tRNA synthetase.     

Isolation and sequencing of cDNA clones encoding alpha and beta subunits of Drosophila melanogaster casein kinase II.

Cloned cDNAs encoding both subunits of Drosophila melanogaster casein kinase II have been isolated by immunological screening of lambda gt11 expression libraries, and the complete amino acid sequence of both polypeptides has been deduced by DNA sequencing. The alpha cDNA contained an open reading frame of 336 amino acid residues, yielding a predicted molecular weight for the alpha polypeptide of 39,833. The alpha sequence contained the expected semi-invariant residues present in the catalytic domain of previously sequenced protein kinases, confirming that it is the catalytic subunit of the enzyme. Pairwise homology comparisons between the alpha sequence and the sequences of a variety of vertebrate protein kinase suggested that casein kinase II is a distantly related member of the protein kinase family. The beta subunit was derived from an open reading frame of 215 amino acid residues and was predicted to have a molecular weight of 24,700. The beta subunit exhibited no extensive homology to other proteins whose sequences are currently known.     

The PUF family of RNA-binding proteins: does evolutionarily conserved structure equal conserved function?

Drosophila Pumilio (Pum) protein is a founder member of a novel family of RNA-binding proteins, known as the PUF family. The PUF proteins constitute an evolutionarily highly conserved family of proteins present from yeast to humans and plants, and are characterized by a highly conserved C-terminal RNA-binding domain, composed of eight tandem repeats. The conserved biochemical features and genetic function of PUF family members have emerged from studies of model organisms. PUF proteins bind to related sequence motifs in the 3' untranslated region (3'UTR) of specific target mRNAs and repress their translation. Frequently, PUF proteins function asymmetrically to create protein gradients, thus causing asymmetric cell division and regulating cell fate specification. Thus, it was recently proposed that the primordial role of PUF proteins is to sustain mitotic proliferation of stem cells. Here we review the evolution, conserved genetic and biochemical properties of PUF family of proteins, and discuss protein interactions, upstream regulators and downstream targets of PUF proteins. We also suggest that a conserved mechanism of PUF function extends to the newly described mammalian members of the PUF family (human PUM1 and PUM2, and mouse Pum1 and Pum2), that show extensive homology to Drosophila Pum, and could have an important role in cell development, fate specification and differentiation.     

Functional divergence prediction from evolutionary analysis: a case study of vertebrate hemoglobin

It is a central assumption of evolution that gene duplications provide the genetic raw material from which to create proteins with new functions. The increasing availability in multigene family sequences that has resulted from genome projects has inspired the creation of novel in silico approaches to predict details of protein function. The underlying principle of all such approaches is to compare the evolutionary properties of homologous sequence positions in paralogous proteins. It has been proposed that the positions that show switches in substitution rate over time-i.e., "heterotachous sites," are good indicators of functional divergence. Here, we analyzed the alpha and beta paralogous subunits of hemoglobin in search for such signatures. We found as many heterotachous sites in comparisons between groups of paralogous subunits (alpha/beta) as between orthologous ones (alpha/alpha, beta/beta). Thus, the importance of substitution rate shifts as predictors of specialization between protein subfamilies might be reconsidered. Instead, such shifts may reflect a more general process of protein evolution, consistent with the fact that they can be compatible with function conservation. As an alternative, we focused on those residues showing highly constrained states in two sequence groups, but different in each group, and we named them CBD (for "constant but different"). As opposed to heterotachous positions, CBD sites were markedly overrepresented in paralogous (alpha/beta) comparisons, as opposed to orthologous ones (alpha/alpha, beta/beta), identifying them as likely signatures of functional specialization between the two subunits. When superimposed onto the three-dimensional structure of hemoglobin, CBD positions consistently appeared to cluster preferentially on inter-subunit surfaces, two contact areas crucial to function in vertebrate tetrameric hemoglobin. The identification and analysis of CBD sites by complementing structural information with evolutionary data may represent a promising direction for future studies dealing with the functional characterization of a growing number of multigene families identified by complete genome analyses.