Α2β2 Heterotetramer Research Articles

Structural and Functional Characterization of a Gene Cluster Responsible for Deglycosylation of C-glucosyl Flavonoids and Xanthonoids by Deinococcus aerius

Plant C-glycosylated aromatic polyketides are important for plant and animal health. These are specialized metabolites that perform functions both within the plant, and in interaction with soil or intestinal microbes. Despite the importance of these plant compounds, there is still limited knowledge of how they are metabolized. The Gram-positive aerobic soil bacterium Deinococcus aerius strain TR0125 and other Deinococcus species thrive in a wide range of harsh environments. In this work, we identified a C-glycoside deglycosylation gene cluster in the genome of D. aerius. The cluster includes three genes coding for a GMC-type oxidoreductase (DaCGO1) that oxidizes the glucosyl C3 position in aromatic C-glucosyl compounds, which in turn provides the substrate for the C-glycoside deglycosidase (DaCGD; composed of α+β subunits) that cleaves the glucosyl-aglycone C–C bond. Our results from size-exclusion chromatography, single particle cryo-electron microscopy and X-ray crystallography show that DaCGD is an α2β2 heterotetramer, which represents a novel oligomeric state among bacterial CGDs. Importantly, the high-resolution X-ray structure of DaCGD provides valuable insights into the activation of the catalytic hydroxide ion by Lys261. DaCGO1 is specific for the 6-C-glucosyl flavones isovitexin, isoorientin and the 2-C-glucosyl xanthonoid mangiferin, and the subsequent C–C-bond cleavage by DaCGD generated apigenin, luteolin and norathyriol, respectively. Of the substrates tested, isovitexin was the preferred substrate (DaCGO1, Km 0.047 mM, kcat 51 min−1; DaCGO1/DaCGD, Km 0.083 mM, kcat 0.42 min−1).

Examination of the Catalytic Role of the Axial Cystine Ligand in the Co-Type Nitrile Hydratase from Pseudonocardia thermophila JCM 3095

The strictly conserved αSer162 residue in the Co-type nitrile hydratase from Pseudonocardia thermophila JCM 3095 (PtNHase), which forms a hydrogen bond to the axial αCys108-S atom, was mutated into an Ala residue. The αSer162Ala yielded two different protein species: one was the apoform (αSerA) that exhibited no observable activity, and the second (αSerB) contained its full complement of cobalt ions and was active with a kcat value of 63 ± 3 s−1 towards acrylonitrile at pH 7.5. The X-ray crystal structure of αSerA was determined at 1.85 Å resolution and contained no detectable cobalt per α2β2 heterotetramer. The axial αCys108 ligand itself was also mutated into Ser, Met, and His ligands. All three of these αCys108 mutant enzymes contained only half of the cobalt complement of wild-type PtNHase, but were able to hydrate acrylonitrile with kcat values of 120 ± 6, 29 ± 3, and 14 ± 1 s−1 for the αCys108His, Ser, and Met mutant enzymes, respectively. As all three of these mutant enzymes are catalytically competent, these data provide the first experimental evidence that transient disulfide bond formation is not catalytically essential for NHases.

Substrate recognition induces sequential electron transfer across subunits in the nitrogenase-like DPOR complex

A key step in bacteriochlorophyll biosynthesis is the reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), catalyzed by dark-operative protochlorophyllide oxidoreductase (DPOR). DPOR is made of electron donor (BchL) and acceptor (BchNB) component proteins. BchNB is further composed of two subunits each of BchN and BchB arranged as an α2β2 heterotetramer with two active sites for substrate reduction. Such oligomeric architectures are found in several other electron transfer (ET) complexes, but how this architecture influences activity is unclear. Here, we describe allosteric communication between the two identical active sites in Rhodobacter sphaeroides BchNB that drives sequential and asymmetric ET. Pchlide binding to one BchNB active site initiates ET from the pre-reduced [4Fe-4S] cluster of BchNB, a process similar to the deficit spending mechanism observed in the structurally related nitrogenase complex. Pchlide binding in one active site is recognized in trans by an Asp-274 from the opposing half, which is positioned to serve as the initial proton donor. A D274A variant DPOR binds to two Pchlide molecules in the BchNB complex, but only one is bound productively, stalling Pchlide reduction in both active sites. A half-active complex combining one WT and one D274A monomer also stalled after one electron was transferred in the WT half. We propose that such sequential electron transfer in oligomeric enzymes serves as a regulatory mechanism to ensure binding and recognition of the correct substrate. The findings shed light on the functional advantages imparted by the oligomeric architecture found in many electron transfer enzymes.

Protein dynamics and molecular motions study in relation to molecular interaction between proteins from sulfur oxidizing proteobacteria Allochromatium vinosum

The gamma-proteobacteria Allochromatium vinosum DSM 180T (A. vinosum) encodes the sulfur oxidizing dsr operon comprising of 15 genes. Dsr proteins are involved in oxidation of sulfur globules produced as an obligatory intermediate during the sulfur oxidation process. The dsrA and dsrB gene products are known to function as a α2β2 hetero-tetramer and the protein complex plays the catalytic role in sulfur oxidation process. DsrC has a highly conserved C-terminal domain that forms a flexible arm, where two strictly conserved cysteines were found to act as a substrate donating residue for DsrAB instead of being a subunit of this redox enzyme. Therefore, to elucidate the molecular mechanism of the sulfur oxidation process here an attempt was made to study the dynamics, stability and binding mechanisms of DsrAB and DsrC proteins through computational docking and molecular dynamics (MD) simulations. This structure function relationship investigation revealed that the C-terminal domain of DsrC interacts with DsrA of DsrAB protein complex for catalytic functions. Some basic amino acid residues of DsrC are found to form the catalytic pockets along with DsrAB protein complex where the sulfur anions bind to get oxidized. Structural dynamics and fluctuations as well as the secondary structural alterations study revealed the possible regions responsible for protein-protein interactions. Principal Component Analysis (PCA) of protein motions displayed that the collective motions of DsrAB-DsrC complex was higher and more anti-correlated than the unbound DsrAB form. The present molecular insight study would therefore help researchers to predict the plausible biochemical mechanism of sulfur oxidation process in sulfur metabolic pathways in near future. Communicated by Ramaswamy H. Sarma

IpdE1-IpdE2 Is a Heterotetrameric Acyl Coenzyme A Dehydrogenase That Is Widely Distributed in Steroid-Degrading Bacteria.

Steroid-degrading bacteria, including Mycobacterium tuberculosis (Mtb), utilize an architecturally distinct subfamily of acyl coenzyme A dehydrogenases (ACADs) for steroid catabolism. These ACADs are α2β2 heterotetramers that are usually encoded by adjacent fadE-like genes. In mycobacteria, ipdE1 and ipdE2 (formerly fadE30 and fadE33) occur in divergently transcribed operons associated with the catabolism of 3aα-H-4α(3′-propanoate)-7aβ-methylhexahydro-1,5-indanedione (HIP), a steroid metabolite. In Mycobacterium smegmatis, ΔipdE1 and ΔipdE2 mutants had similar phenotypes, showing impaired growth on cholesterol and accumulating 5-OH HIP in the culture supernatant. Bioinformatic analyses revealed that IpdE1 and IpdE2 share many of the features of the α- and β-subunits, respectively, of heterotetrameric ACADs that are encoded by adjacent genes in many steroid-degrading proteobacteria. When coproduced in a rhodococcal strain, IpdE1 and IpdE2 of Mtb formed a complex that catalyzed the dehydrogenation of 5OH-HIP coenzyme A (5OH-HIP-CoA) to 5OH-3aα-H-4α(3′-prop-1-enoate)-7aβ-methylhexa-hydro-1,5-indanedione coenzyme A ((E)-5OH-HIPE-CoA). This corresponds to the initial step in the pathway that leads to degradation of steroid C and D rings via β-oxidation. Small-angle X-ray scattering revealed that the IpdE1-IpdE2 complex was an α2β2 heterotetramer typical of other ACADs involved in steroid catabolism. These results provide insight into an important class of steroid catabolic enzymes and a potential virulence determinant in Mtb.

The NifZ accessory protein has an equivalent function in maturation of both nitrogenase MoFe protein P-clusters

The Mo-dependent nitrogenase comprises two interacting components called the Fe protein and the MoFe protein. The MoFe protein is an α2β2 heterotetramer that harbors two types of complex metalloclusters, both of which are necessary for N2 reduction. One type is a 7Fe-9S-Mo-C-homocitrate species designated FeMo-cofactor, which provides the N2-binding catalytic site, and the other is an 8Fe-7S species designated the P-cluster, involved in mediating intercomponent electron transfer to FeMo-cofactor. The MoFe protein's catalytic partner, Fe protein, is also required for both FeMo-cofactor formation and the conversion of an immature form of P-clusters to the mature species. This latter process involves several assembly factors, NafH, NifW, and NifZ, and precedes FeMo-cofactor insertion. Here, using various protein affinity–based purification methods as well as in vivo, EPR spectroscopy, and MALDI measurements, we show that several MoFe protein species accumulate in a NifZ-deficient background of the nitrogen-fixing microbe Azotobacter vinelandii. These included fully active MoFe protein replete with FeMo-cofactor and mature P-cluster, inactive MoFe protein having no FeMo-cofactor and only immature P-cluster, and partially active MoFe protein having one αβ-unit with a FeMo-cofactor and mature P-cluster and the other αβ-unit with no FeMo-cofactor and immature P-cluster. Also, NifW could associate with MoFe protein having immature P-clusters and became dissociated upon P-cluster maturation. Furthermore, both P-clusters could mature in vitro without NifZ. These findings indicate that NifZ has an equivalent, although not essential, function in the maturation of both P-clusters contained within the MoFe protein.

Analyzing the function of the insert region found between the α and β-subunits in the eukaryotic nitrile hydratase from Monosiga brevicollis

The functional roles of the (His)17 region and an insert region in the eukaryotic nitrile hydratase (NHase, EC 4.2.1.84) from Monosiga brevicollis (MbNHase), were examined. Two deletion mutants, MbNHaseΔ238−257 and MbNHaseΔ219−272, were prepared in which the (His)17 sequence and the entire insert region were removed. Each of these MbNHase enzymes provided an α2β2 heterotetramer, identical to that observed for prokaryotic NHases and contains their full complement of cobalt ions. Deletion of the (His)17 motif provides an MbNHase enzyme that is ∼55% as active as the WT enzyme when expressed in the absence of the Co-type activator (ε) protein from Pseudonocardia thermophila JCM 3095 (PtNHaseact) but ∼28% more active when expressed in the presence of PtNHaseact. MbNHaseΔ219−272 exhibits ∼55% and ∼89% of WT activity, respectively, when expressed in the absence or presence of PtNHaseact. Proteolytic cleavage of MbNHase provides an α2β2 heterotetramer that is modestly more active compared to WT MbNHase (kcat = 163 ± 4 vs 131 ± 3 s−1). Combination of these data establish that neither the (His)17 nor the insert region are required for metallocentre assembly and maturation, suggesting that Co-type eukaryotic NHases utilize a different mechanism for metal ion incorporation and post-translational activation compared to prokaryotic NHases.

An unexpected vestigial protein complex reveals the evolutionary origins of an s-triazine catabolic enzyme

Cyanuric acid is a metabolic intermediate of s-triazines, such as atrazine (a common herbicide) and melamine (used in resins and plastics). Cyanuric acid is mineralized to ammonia and carbon dioxide by the soil bacterium Pseudomonas sp. strain ADP via three hydrolytic enzymes (AtzD, AtzE, and AtzF). Here, we report the purification and biochemical and structural characterization of AtzE. Contrary to previous reports, we found that AtzE is not a biuret amidohydrolase, but instead it catalyzes the hydrolytic deamination of 1-carboxybiuret. X-ray crystal structures of apo AtzE and AtzE bound with the suicide inhibitor phenyl phosphorodiamidate revealed that the AtzE enzyme complex consists of two independent molecules in the asymmetric unit. We also show that AtzE forms an α2β2 heterotetramer with a previously unidentified 68-amino acid-long protein (AtzG) encoded in the cyanuric acid mineralization operon from Pseudomonas sp. strain ADP. Moreover, we observed that AtzG is essential for the production of soluble, active AtzE and that this obligate interaction is a vestige of their shared evolutionary origin. We propose that AtzEG was likely recruited into the cyanuric acid-mineralizing pathway from an ancestral glutamine transamidosome that required protein-protein interactions to enforce the exclusion of solvent from the transamidation reaction.

Mannose in Complex with Colocasia esculenta Tuber Agglutinin

The major tuber storage protein of Colocasia esculenta is a monocot mannose-binding, widely used, dietarylectin, for which a crystal structure was previously shown to consist of four β-prism II domains or two αβheterodimers, each forming a α2β2 heterotetramer with a symmetry related unit. In the present study, the same lectincrystallized in the presence of a 50 molar excess of free mannose, persists in forming such α2β2 heterotetramers.Lectin crystals apparently complexed to mannose were obtained by hanging-drop, vapor-diffusion method at roomtemperature and high-resolution X-ray diffraction data were collected using a home X-ray source. Using the crystalstructure of the Remusatia vivipara lectin, the structure of the complex has been solved by molecular replacementand subsequent crystallographic refinement. Five different mannose binding sites per heterodimer with partialoccupancy have been found, among which only one appears to bind close to a classical carbohydrate recognitionsite characterized earlier for these lectins. Mannose binding at these five sites is described.

Assembly of the elongated collagen prolyl 4-hydroxylase α2β2 heterotetramer around a central α2 dimer.

Collagen prolyl 4-hydroxylase (C-P4H), an α2β2 heterotetramer, is a crucial enzyme for collagen synthesis. The α-subunit consists of an N-terminal dimerization domain, a central peptide substrate-binding (PSB) domain, and a C-terminal catalytic (CAT) domain. The β-subunit [also known as protein disulfide isomerase (PDI)] acts as a chaperone, stabilizing the functional conformation of C-P4H. C-P4H has been studied for decades, but its structure has remained elusive. Here, we present a three-dimensional small-angle X-ray scattering model of the entire human C-P4H-I heterotetramer. C-P4H is an elongated, bilobal, symmetric molecule with a length of 290 Å. The dimerization domains from the two α-subunits form a protein-protein dimer interface, assembled around the central antiparallel coiled-coil interface of their N-terminal α-helices. This region forms a thin waist in the bilobal tetramer. The two PSB/CAT units, each complexed with a PDI/β-subunit, form two bulky lobes pointing outward from this waist region, such that the PDI/β-subunits locate at the far ends of the βααβ complex. The PDI/β-subunit interacts extensively with the CAT domain. The asymmetric shape of two truncated C-P4H-I variants, also characterized in the present study, agrees with this assembly. Furthermore, data from these truncated variants show that dimerization between the α-subunits has an important role in achieving the correct PSB-CAT assembly competent for catalytic activity. Kinetic assays with various proline-rich peptide substrates and inhibitors suggest that, in the competent assembly, the PSB domain binds to the procollagen substrate downstream from the CAT domain.

Crystal Structure of Colocasia esculenta Tuber Agglutinin at 1.74 Å Resolution and Its Quaternary Interactions

Many members of the monocot mannose-binding lectin family, characterized by specificity towards mannose have been characterized and cloned. A majority of these lectins molecules contain 1-4 polypeptides of about 110 residues each. From the previously solved crystal structures of a few such lectins, mostly from non-edible plants, these lectins are thought to possess a common β-prism II fold structure. The major tuber storage protein of Colocasia esculenta is a monocot mannose-binding, widely used, dietary lectin. This tuber agglutinin contains two polypeptides of 12.0 and 12.4 kDa by matrix assisted laser desorption ionisation time-of-flight mass spectrometry. By gel filtration at pH 7.2, the purified lectin has a α2β2 form of apparent molecular mass of 48.2 kDa in solution but at pH 3, it has the heterodimeric αβ form. Lectin crystals were obtained by hanging-drop, vapor-diffusion method at room temperature and high-resolution X-ray diffraction data were collected using a home X-ray source. Among previously solved crystal structures of this family are garlic, Solomon’s seal, snowdrop, daffodil and Spanish blue-bell lectins, but the protein sequence of the Colocasia esculenta tuber agglutinin was found to be closest to that of the Remusatia vivipara lectin having no simple mannose-binding property. Using the previously solved 2.4A crystal structure of the Remusatia vivipara lectin, that of Colocasia esculenta has been solved by molecular replacement and subsequent crystallographic refinement and root mean square deviations between various lectins are tabulated and rationalized. The asymmetric unit in our lectin crystal structure contains four β-prism II domains or two αβ heterodimers, each forming a α2β2 heterotetramer with a symmetry related unit. The tetrameric interface obtained from our crystal structure is used to explain the conversion to dimers in acidic pH. Five ordered magnesium ions were located in the asymmetric unit and the presence of magnesium verified by atomic absorption spectroscopy.

Mycobacterium tuberculosis Utilizes a Unique Heterotetrameric Structure for Dehydrogenation of the Cholesterol Side Chain

Compounding evidence supports the important role in pathogenesis that the metabolism of cholesterol by Mycobacterium tuberculosis plays. Elucidating the pathway by which cholesterol is catabolized is necessary to understand the molecular mechanism by which this pathway contributes to infection. On the basis of early metabolite identification studies in multiple actinomycetes, it has been proposed that cholesterol side chain metabolism requires one or more acyl-CoA dehydrogenases (ACADs). There are 35 genes annotated as encoding ACADs in the M. tuberculosis genome. Here we characterize a heteromeric ACAD encoded by Rv3544c and Rv3543c, formerly named fadE28 and fadE29, respectively. We now refer to genes Rv3544c and Rv3543c as chsE1 and chsE2, respectively, in recognition of their validated activity in cholesterol side chain dehydrogenation. Analytical ultracentrifugation and liquid chromatography-ultraviolet experiments establish that ChsE1-ChsE2 forms an α(2)β(2) heterotetramer, a new architecture for an ACAD. Our bioinformatic analysis and mutagenesis studies reveal that heterotetrameric ChsE1-ChsE2 has only two active sites. E241 in ChsE2 is required for catalysis of dehydrogenation by ChsE1-ChsE2. Steady state kinetic analysis establishes the enzyme is specific for an intact steroid ring system versus hexahydroindanone substrates with specificity constants (k(cat)/K(M)) of (2.5 ± 0.5) × 10(5) s(-1) M(-1) versus 9.8 × 10(2) s(-1) M(-1), respectively, at pH 8.5. The characterization of a unique ACAD quaternary structure involved in sterol metabolism that is encoded by two distinct cistronic ACAD genes opens the way to identification of additional sterol-metabolizing ACADs in M. tuberculosis and other actinomycetes through bioinformatic analysis.

Trmt61B is a methyltransferase responsible for 1-methyladenosine at position 58 of human mitochondrial tRNAs

In human mitochondria, 1-methyladenosine (m¹A) occurs at position 58 of tRNA(Leu(UUR)). In addition, partial m¹A58 modifications have been found in human mitochondrial tRNA(Lys) and tRNA(Ser(UCN)). We identified human Trmt61B, which encodes a mitochondria-specific tRNA methyltransferase responsible for m¹A58 in these three tRNAs. Trmt61B is dominantly localized to the mitochondria. m¹A58 formation in human mitochondrial tRNA(Leu(UUR)) could be reconstituted in vitro using recombinant Trmt61B in the presence of Ado-Met as a methyl donor. Unlike the cytoplasmic tRNA m¹A58 methyltransferase that consists of an α2β2 heterotetramer formed by Trmt61A and Trmt6, Trmt61B formed a homo-oligomer (presumably a homotetramer) that resembled the bacterial homotetrameric m¹A58 methyltransferase. The bacterial origin of Trmt61B is supported by the results of the phylogenetic analysis.

The crystal structures of the α-subunit of the α2β2 tetrameric Glycyl-tRNA synthetase

Aminoacyl-tRNA synthetases (AARSs) are ligases (EC.6.1.1.-) that catalyze the acylation of amino acids to their cognate tRNAs in the process of translating genetic information from mRNA to protein. Their amino acid and tRNA specificity are crucial for correctly translating the genetic code. Glycine is the smallest amino acid and the glycyl-tRNA synthetase (GlyRS) belongs to Class II AARSs. The enzyme is unusual because it can assume different quaternary structures. In eukaryotes, archaebacteria and some bacteria, it forms an α(2) homodimer. In some bacteria, GlyRS is an α(2)β(2) heterotetramer and shows a distant similarity to α(2) GlyRSs. The human pathogen eubacterium Campylobacter jejuni GlyRS (CjGlyRS) is an α(2)β(2) heterotetramer and is similar to Escherichia coli GlyRS; both are members of Class IIc AARSs. The two-step aminoacylation reaction of tetrameric GlyRSs requires the involvement of both α- and β-subunits. At present, the structure of the GlyRS α(2)β(2) class and the details of the enzymatic mechanism of this enzyme remain unknown. Here we report the crystal structures of the catalytic α-subunit of CjGlyRS and its complexes with ATP, and ATP and glycine. These structures provide detailed information on substrate binding and show evidence for a proposed mechanism for amino acid activation and the formation of the glycyl-adenylate intermediate for Class II AARSs.

The role of symmetry in the regulation of bacterial carboxyltransferase.

Carboxyltransferase is one component of the multifunctional enzyme acetyl-CoA carboxylase which catalyzes the first committed step in fatty acid biosynthesis. Carboxyltransferase is an α2β2 heterotetramer and possesses two distinct but integrated functions. One function catalyzes the transfer of carbon dioxide from biotin to acetyl-CoA, whereas the other involves binding to the mRNA encoding both subunits. When carboxyltransferase binds to the mRNA both enzymatic activity and translation of the mRNA are inhibited. However, the substrate acetyl-CoA competes with mRNA for binding. Thus, mRNA binding by carboxyltransferase provides an effective mechanism for regulating enzymatic activity and gene expression. This conceptual review takes the position that regulation of enzymatic activity and gene expression of carboxyltransferase by binding to its own mRNA is at its most fundamental level the result of the symmetry in the chemical reaction catalyzed by the enzyme. The chemical reaction is symmetrical in that both substrates generate enolate anions during the course of catalysis. The chemical symmetry led to a structural symmetry in the enzyme where both the α and β subunits contain oxyanion holes that stabilize the enolate anions. Then the region of the mRNA that codes for the oxyanion holes provided the binding sites for carboxyltransferase. Thus, the symmetry of the chemical reaction formed the foundation for the evolution of the mechanism for regulation of carboxyltransferase.

Discovery of a putative acetoin dehydrogenase complex in the hyperthermophilic archaeon Sulfolobus solfataricus

Like many other aerobic archaea, the hyperthermophile Sulfolobus solfataricus possesses a gene cluster encoding components of a putative 2-oxoacid dehydrogenase complex. In the current paper, we have cloned and expressed the first two genes of this cluster and demonstrate that the protein products form an α2β2 hetero-tetramer possessing the catalytic activity characteristic of the first component enzyme of an acetoin dehydrogenase multienzyme complex. This represents the first report of an acetoin multienzyme complex in archaea, and contrasts with the branched-chain 2-oxoacid dehydrogenase complex activities characterised in two other archaea, Thermoplasma acidophilum and Haloferax volcanii.

Hydroquinone Dioxygenase from Pseudomonas fluorescens ACB: a Novel Member of the Family of Nonheme-Iron(II)-Dependent Dioxygenases

Hydroquinone 1,2-dioxygenase (HQDO), an enzyme involved in the catabolism of 4-hydroxyacetophenone in Pseudomonas fluorescens ACB, was purified to apparent homogeneity. Ligandation with 4-hydroxybenzoate prevented the enzyme from irreversible inactivation. HQDO was activated by iron(II) ions and catalyzed the ring fission of a wide range of hydroquinones to the corresponding 4-hydroxymuconic semialdehydes. HQDO was inactivated by 2,2'-dipyridyl, o-phenanthroline, and hydrogen peroxide and inhibited by phenolic compounds. The inhibition with 4-hydroxybenzoate (K(i) = 14 microM) was competitive with hydroquinone. Online size-exclusion chromatography-mass spectrometry revealed that HQDO is an alpha2beta2 heterotetramer of 112.4 kDa, which is composed of an alpha-subunit of 17.8 kDa and a beta-subunit of 38.3 kDa. Each beta-subunit binds one molecule of 4-hydroxybenzoate and one iron(II) ion. N-terminal sequencing and peptide mapping and sequencing based on matrix-assisted laser desorption ionization--two-stage time of flight analysis established that the HQDO subunits are encoded by neighboring open reading frames (hapC and hapD) of a gene cluster, implicated to be involved in 4-hydroxyacetophenone degradation. HQDO is a novel member of the family of nonheme-iron(II)-dependent dioxygenases. The enzyme shows insignificant sequence identity with known dioxygenases.

Evolutionary Analysis of the TPP-Dependent Enzyme Family

The evolutionary relationships of the thiamine pyrophosphate (TPP)-dependent family of enzymes was investigated by generation of a neighbor joining phylogenetic tree using sequences from the conserved pyrophosphate (PP) and pyrimidine (Pyr) binding domains of 17 TPP-dependent enzymes. This represents the most comprehensive analysis of TPP-dependent enzyme evolution to date. The phylogeny was shown to be robust by comparison with maximum likelihood trees generated for each individual enzyme and also broadly confirms the evolutionary history proposed recently from structural comparisons alone (Duggleby 2006). The phylogeny is most parsimonious with the TPP enzymes having arisen from a homotetramer which subsequently diverged into an alpha(2)beta(2) heterotetramer. The relationship between the PP- and Pyr-domains and the recruitment of additional protein domains was examined using the transketolase C-terminal (TKC)-domain as an example. This domain has been recruited by several members of the family and yet forms no part of the active site and has unknown function. Removal of the TKC-domain was found to increase activity toward beta-hydroxypyruvate and glycolaldehyde. Further truncations of the Pyr-domain yielded several variants with retained activity. This suggests that the influence of TKC-domain recruitment on the evolution of the mechanism and specificity of transketolase (TK) has been minor, and that the smallest functioning unit of TK comprises the PP- and Pyr-domains, whose evolutionary histories extend to all TPP-dependent enzymes.

Human Prolyl-4-hydroxylase α(I) Transcription Is Mediated by Upstream Stimulatory Factors

Prolyl-4-hydroxylase alpha(I) (P4Halpha(I)) is the rate-limiting subunit for P4H enzyme activity, which is essential for procollagen hydroxylation and secretion. In the current study, we have characterized the human P4Halpha(I) promoter for transcription factors and DNA elements regulating P4Halpha(I) expression. Using a progressive deletion cloning approach, we have constructed pGL3-P4Halpha(I) recombinant plasmids. We have identified a positive regulatory region at the positions of bp -184 to -97 responsible for approximately 80% of the P4Halpha(I) promoter efficiency. Three E-boxes were located within this region, and the E-box at position bp -135 explains most of the regulatory capacity. Upstream stimulatory factors (USF1/USF2) were shown to bind on the E-box using chromatin immunoprecipitation assay. Suppression of USF1 and/or USF2 using specific short interference RNA resulted in a significant reduction in P4Halpha(I) promoter activity, and overexpressed USF1 or USF2 increased P4Halpha(I) promoter activity significantly. Although transforming growth factor beta1 increased the USF1/USF2-E-box binding and P4Halpha(I) promoter activity, this up-regulatory effect can be largely prevented by USF1/USF2-specific short interference RNA. On the other hand, cigarette smoking extracts, which have been shown to suppress P4Halpha(I) expression, inhibited the binding between the USF1/USF2 and E-box, resulting in a reduced P4Halpha(I) promoter activity. Furthermore, the E-box on the P4Halpha(I) promoter appeared to indiscriminately bind with either USF1 or USF2, with a similar outcome on the promoter efficiency. In conclusion, our study shows that USF1/USF2 plays a critical role in basal P4Halpha(I) expression, and both positive (transforming growth factor beta1) and negative (cigarette smoking extract) regulators appear to influence the USF-E-box interaction and affect P4Halpha(I) expression.

Crystal Structure of Human Taspase1, a Crucial Protease Regulating the Function of MLL

Taspase1 catalyzes the proteolytic processing of the mixed lineage leukemia (MLL) nuclear protein, which is required for maintaining Hox gene expression patterns. Chromosomal translocations of the MLL gene are associated with leukemia in infants. Taspase1, a threonine aspartase, is a member of the type 2 asparaginase family, but is the only protease in this family. We report here the crystal structures of human activated Taspase1 and its proenzyme, as well as the characterization of the effects of mutations in the active site region using a newly developed fluorogenic assay. The structure of Taspase1 has significant differences from other asparaginases, especially near the active site. Mutation of the catalytic nucleophile, Thr234, abolishes autocatalytic processing in cis but does not completely block proteolysis in trans. The structure unexpectedly showed the binding of a chloride ion in the active site, and our kinetic studies confirm that chlorides ions are inhibitors of this enzyme at physiologically relevant concentrations.