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BBA - Proteins and Proteomics (v.1774, #5)

Editorial Board (pp. ii).

Sulfite oxidizing enzymes by Changjian Feng; Gordon Tollin; John H. Enemark (pp. 527-539).

Sulfite oxidizing enzymes are essential mononuclear molybdenum (Mo) proteins involved in sulfur metabolism of animals, plants and bacteria. There are three such enzymes presently known: (1) sulfite oxidase (SO) in animals, (2) SO in plants, and (3) sulfite dehydrogenase (SDH) in bacteria. X-ray crystal structures of enzymes from all three sources (chicken SO, Arabidopsis thaliana SO, and Starkeya novella SDH) show nearly identical square pyramidal coordination around the Mo atom, even though the overall structures of the proteins and the presence of additional cofactors vary. This structural information provides a molecular basis for studying the role of specific amino acids in catalysis. Animal SO catalyzes the final step in the degradation of sulfur-containing amino acids and is critical in detoxifying excess sulfite. Human SO deficiency is a fatal genetic disorder that leads to early death, and impaired SO activity is implicated in sulfite neurotoxicity. Animal SO and bacterial SDH contain both Mo and heme domains, whereas plant SO only has the Mo domain. Intraprotein electron transfer (IET) between the Mo and Fe centers in animal SO and bacterial SDH is a key step in the catalysis, which can be studied by laser flash photolysis in the presence of deazariboflavin. IET studies on animal SO and bacterial SDH clearly demonstrate the similarities and differences between these two types of sulfite oxidizing enzymes. Conformational change is involved in the IET of animal SO, in which electrostatic interactions may play a major role in guiding the docking of the heme domain to the Mo domain prior to electron transfer. In contrast, IET measurements for SDH demonstrate that IET occurs directly through the protein medium, which is distinctly different from that in animal SO. Point mutations in human SO can result in significantly impaired IET or no IET, thus rationalizing their fatal effects. The recent developments in our understanding of sulfite oxidizing enzyme mechanisms that are driven by a combination of molecular biology, rapid kinetics, pulsed electron paramagnetic resonance (EPR), and computational techniques are the subject of this review.

Keywords: Abbreviations; Mo; molybdenum; SO; sulfite oxidase; SDH; sulfite dehydrogenases; nitrate reductase; NR; cytochrome; cyt; (cyt; c; ); _ox; and (cyt; c; ); _red; : ferricytochrome; c; and ferrocytochrome; c; respectively; IET; intraprotein electron transfer; k; _et; rate constant for IET; EPR; electron paramagnetic resonance; CW; continuous wave; CD; circular dichroism; ^{dRF and dRFH}; : 5-deazariboflavin and 5-deazariboflavin semiquinone; respectivelySulfite oxidase; Sulfite dehydrogenase; Electron transfer; Laser flash photolysis; Sulfite oxidase deficiency; EPR

Temperature modulates binding specificity and affinity of thed-trehalose/d-maltose-binding protein from the hyperthermophilic archaeon Thermococcus litoralis by Petr Herman; Ivan Barvik Jr.; Maria Staiano; Annalisa Vitale; Jaroslav Vecer; Mose' Rossi; Sabato D'Auria (pp. 540-544).

We investigated the effect of temperature on the binding specificity of the recombinantd-trehalose/d-maltose-binding protein from the hyperthermophilic archaeon Thermococcus litoralis (TMBP). Importantly, we found that TMBP can bindd-glucose (Glc). The Glc binding was characterized by means of fluorescence spectroscopy in the temperature range of 25 °C–85 °C. Our results show that at 25 °C the binding of Glc to TMBP is well represented by a bimodal model with apparent K_d of 20 μM and approximately 3–8 mM for the first and the second binding step, respectively. At 60 °C the binding of Glc to TMBP is represented by a simple hyperbolic model with an apparent K_d value of about 40 μM. Finally, at 85 °C Glc did not bind to TMBP. Molecular dynamics (MD) simulations were used to shed light on the molecular mechanism of the Glc binding. Our results suggest that after proper fluorescent labeling TMBP can be used as a highly thermostable and non-consuming analyte biosensor for monitoring the level of glucose in fluids (e.g. human blood) where other sugars are not present.

Keywords: Glucose; TMBP; Thermostability; Biosensor; Fluorescence; Molecular dynamics

Low molecular weight protein tyrosine phosphatase (LMW-PTP) and its possible physiological functions of redox signaling in the eye lens by Kuiyi Xing; Ashraf Raza; Stefan Löfgren; M. Rohan Fernando; Ye-Shih Ho; Marjorie F. Lou (pp. 545-555).

Low molecular weight protein tyrosine phosphatase (LMW-PTP) was cloned from human lens epithelial B3 cells (HLE B3) and the recombinant enzyme was purified to homogeneity. The pure enzyme reacted positively with anti-LMW-PTP antibody, displayed tyrosine-specific phosphatase activity and was extremely sensitive to H₂O₂. The inactivated LMW-PTP could be regenerated by thioltransferase (TTase)/GSH system as demonstrated by both activity assay and by mass spectrometry (MS). The MS study also showed that an intramolecular disulfide bond was formed between C13 and C18 at the active site, and was reduced by the TTase/GSH system. The putative role of LMW-PTP in regulating platelet derived growth factor (PDGF)-stimulated cell signaling was demonstrated in wild type mouse lens epithelial cells (LEC) in which LMW-PTP was transiently inactivated, corroborated with the transient phosphorylation of Tyr857 at the active site of PDGF receptor and the downstream signaling components of Akt and ERK1/2. In contrast, LMW-PTP activity in PDGF-stimulated LEC from TTase^−/− mice was progressively lost, concomitant with the high basal and sustained high phosphorylation levels at Tyr857, Akt and ERK1/2. We conclude that the reversible LMW-PTP activity regulated by ROS-mediated oxidation and TTase/GSH reduction is the likely mechanism of redox signaling in lens epithelial cells.

Keywords: Low molecular weight protein tyrosine phosphatase; Signal transduction; Protein S-thiolation; Reactive oxygen species; Lens epithelial cells

The role of cofactor binding in tryptophan accessibility and conformational stability of His-taggedd-amino acid oxidase from Trigonopsis variabilis by Miguel Arroyo; Margarita Menéndez; José Luis García; Nuria Campillo; Daniel Hormigo; Isabel de la Mata; María Pilar Castillón; Carmen Acebal (pp. 556-565).

d-amino acid oxidase from Trigonopsis variabilis ( TvDAAO) is a flavoenzyme with high biotechnological and industrial interest. The overexpression and purification of the apoprotein form of a recombinant His-tagged TvDAAO allowed us to go deep into the structural differences between apoenzyme and holoenzyme, and on the cofactor binding and its contribution to enzyme stability. A significant decrease in intrinsic fluorescence emission took place upon FAD binding, associated to cofactor induced conformational transitions or subunit dimerization that could affect the local environment of protein tryptophan residues. Furthermore, acrylamide-quenching experiments indicated that one of the five tryptophan residues of TvDAAO became less accessible upon FAD binding. A K_d=1.5±0.1×10−⁷ M for the dissociation of FAD from TvDAAO was calculated from binding experiments based on both quenching of FAD fluorescence and activity titration curves. Secondary structure prediction indicated that TvDAAO is a mixed α/β protein with 8 α-helices and 14 β-sheets connected by loops. Prediction results were in good agreement with the estimates obtained by circular dichroism which indicated that both the apoenzyme and the holoenzyme had the same structural component ratios: 34% α-helix content, 20% β-structure content (14% antiparallel and 6% parallel β-sheet), 15% β-turns and 31% of random structure. Circular dichroism thermal-transition curves suggested single-step denaturation processes with apparent midpoint transition temperatures ( T_m) of 37.9 °C and 41.4 °C for the apoenzyme and the holoenzyme, respectively. A three-dimensional model of TvDAAO built by homology modelling and consistent with the spectroscopic studies is shown. Comparing our results with those reported for pig kidney ( pkDAAO) and Rhodotorula gracilis ( RgDAAO)d-amino acid oxidases, a “head-to-head” interaction between subunits in the TvDAAO dimer might be expected.

Keywords: Flavoprotein; d; -amino acid oxidase; Secondary structure; Fluorescence; Circular dichroism; Thermal stability; Dissociation constant; Cofactor binding

Interactions of rotor subunits in the chloroplast ATP synthase modulated by nucleotides and by Mg²⁺ by Melanie Gertz; Holger Seelert; Norbert A. Dencher; Ansgar Poetsch (pp. 566-574).

ATP synthases – rotary nano machines – consist of two major parts, F_O and F₁, connected by two stalks: the central and the peripheral stalk. In spinach chloroplasts, the central stalk (subunits γ, ε) forms with the cylinder of subunits III the rotor and transmits proton motive force from F_O to F₁, inducing conformational changes of the catalytic centers in F₁. The ε subunit is an important regulator affecting adjacent subunits as well as the activity of the whole protein complex. Using a combination of chemical cross-linking and mass spectrometry, we monitored interactions of subunit ε in spinach chloroplast ATP synthase with III and γ. Onto identification of interacting residues in subunits ε and III, one cross-link defined the distance between ε-Cys6 and III-Lys48 to be 9.4 Å at minimum. ε-Cys6 was competitively cross-linked with subunit γ. Altered cross-linking yields revealed the impact of nucleotides and Mg²⁺ on cross-linking of subunit ε. The presence of nucleotides apparently induced a displacement of the N-terminus of subunit ε, which separated ε-Cys6 from both, III-Lys48 and subunit γ, and thus decreasing the yield of the cross-linked subunits ε and γ as well as ε and III. However, increasing concentrations of the cofactor Mg²⁺ favoured cross-linking of ε-Cys6 with subunit γ instead of III-Lys48 indicating an approximation of subunits γ and ε and a separation from III-Lys48.

Keywords: Abbreviations; AMP-PNP; adenosine 5′-(β,γ-imido)triphosphate; BN; blue-native; β-DM; n-dodecyl β-; d; -maltoside; ESI; electrospray ionisation; F; _O; F; ₁; ATP synthase holoenzyme; F; _O; membrane integral portion of F; _O; F; F; ₁; hydrophilic portion of F; _O; F; ₁; MALDI; matrix-assisted laser-desorption ionisation; PMF; peptide mass fingerprint; TFA; trifluoroacetic acid; TOF; time-of-flightATP synthase; Cross-linking; F; _O; F; ₁; Central stalk; Mass spectrometry

Proteomic analysis of proteins regulated by TRPS1 transcription factor in DU145 prostate cancer cells by Glenn T.G. Chang; Simon C. Gamble; Mila Jhamai; Robin Wait; Charlotte L. Bevan; Albert O. Brinkmann (pp. 575-582).

The aim of the present study was to identify proteins differentially regulated by TRPS1 in human prostate cancer cells in order to better understand the role of TRPS1 in prostate cancer development. The proteomes of androgen-independent DU145 prostate cancer cells, that do not express TRPS1 and of genetically engineered DU145 cells that stable and inducible express recombinant TRPS1 protein, were compared. Using two-dimensional electrophoresis followed by mass spectrometric analysis, 13 proteins that were differentially expressed between these two cell lines were identified. These proteins represent a dominant reduction of expression of antioxidant proteins, including superoxide dismutase, protein disulfide isomerase A3 precursor, endoplasmin precursor and annexin A2. Furthermore, regulation was observed for mitochondrion-associated proteins, glycolytic enzymes, a cytoskeleton-associated protein, a nuclear protein and proteins involved in apoptosis. Our data indicate that overexpression of TRPS1 protein is correlated with reduced protein expression of certain antioxidants. This suggests a possible involvement of TRPS1 in oxidative stress, and possibly in apoptosis in androgen-independent DU145 prostate cancer cells.

Keywords: TRPS1; Proteomic analysis; DU145; Prostate cancer; Antioxidant

An EcoRI– RsrI chimeric restriction endonuclease retains parental sequence specificity by Tungalag Chuluunbaatar; Tetiana Ivanenko-Johnston; Mónika Fuxreiter; Ruslan Meleshko; Tamás Raskó; István Simon; Joseph Heitman; Antal Kiss (pp. 583-594).

To test their structural and functional similarity, hybrids were constructed between EcoRI and RsrI, two restriction endonucleases recognizing the same DNA sequence and sharing 50% amino acid sequence identity. One of the chimeric proteins (EERE), in which the EcoRI segment His147–Ala206 was replaced with the corresponding RsrI segment, showed EcoRI/ RsrI-specific endonuclease activity. EERE purified from inclusion bodies was found to have ∼100-fold weaker activity but higher specific DNA binding affinity, than EcoRI. Increased binding is consistent with results of molecular dynamics simulations, which indicate that the number of hydrogen bonds formed with the recognition sequence increased in the chimera as compared to EcoRI. The success of obtaining an EcoRI– RsrI hybrid endonuclease, which differs from EcoRI by 22 RsrI-specific amino acid substitutions and still preserves canonical cleavage specificity, is a sign of structural and functional similarity shared by the parental enzymes. This conclusion is also supported by computational studies, which indicate that construction of the EERE chimera did not induce substantial changes in the structure of EcoRI. Surprisingly, the chimeric endonuclease was more toxic to cells not protected by EcoRI methyltransferase, than the parental EcoRI mutant. Molecular modelling revealed structural alterations, which are likely to impede coupling between substrate recognition and cleavage and suggest a possible explanation for the toxic phenotype.

Keywords: Restriction endonuclease; Sequence-specific DNA – recognition; Protein refolding; Molecular dynamics simulations

Synaptonemal complex protein SYCP3: Conserved polymerization properties among vertebrates by Andrea Baier; Manfred Alsheimer; Ricardo Benavente (pp. 595-602).

Synaptonemal complexes (SCs) are meiosis-specific, nuclear structures that are critically involved in synapsis, recombination and segregation of homologous chromosomes. Although the SC structure is conserved in evolution this is not the case for its protein components. To provide information on SC proteins which would be important for our understanding of the conserved SC structure and function, here we compared ortholog SYCP3 proteins of two evolutionary distant vertebrate species, namely rat and medaka fish. To this end we have investigated the polymerization properties of both proteins by immunocytochemistry, electron microscopy and cell fractionation. We found that despite of the sequence differences that have accumulated over the last 450 million years mammalian and fish SYCP3 have similar properties that allow them to co-assemble higher order structures under experimental conditions. We also provide a likely explanation as to how heterozygous mutations in the SYCP3 gene can lead to a defective meiosis.

Keywords: Meiosis; Chromosomes; Synaptonemal complex; SYCP3; Vertebrates; Rodents; Medaka

Identification of a novel two component system in Thermotoga maritima. Complex stoichiometry and crystallization by Patricia Casino; Ana Fernández-Alvarez; Carlos Alfonso; Germán Rivas; Alberto Marina (pp. 603-609).

Two-component signal transduction systems, comprised of histidine kinase and its cognate response regulator, are the predominant mechanism by which microorganisms sense and respond to changes in many different environmental conditions. Different Thermotoga maritima histidine kinases have been used as prototypes; among them, the orphan TM0853 has been presented as a structural model of class I histidine kinases. We used phosphotransfer assays to identify TM0468 as the partner response regulator of TM0853. Since full-length TM0853 can be produced as a soluble protein in Escherichia coli, it was used to analyze the union stoichiometry in an intact two-component system for the first time. We demonstrate that TM0853, or its cytoplasmic catalytic portion, form a 1:1 complex with TM0468 with native PAGE. The complex band is unique, even in the presence of an excess of each individual protein, indicating that the union is cooperative. We corroborated these findings by using ultracentrifugation assays. Therefore, we propose that the general mode of interaction in an orthodox two-component system may be the stoichiometric and cooperative complex between a dimeric histidine kinase and two response regulators. Finally, we have been able to produce protein crystals of the complex between the cytoplasmic portion of TM0853 and TM0468 that diffract to 2.8 Å Bragg spacing.

Keywords: Two-component signal transduction; Histidine kinase; Phosphotransfer profiling

The conformation of acetylated virginiamycin M₁ and virginiamycin M₁ in explicit solvents by Chai Ann Ng; Wen Zhao; Jason Dang; Mikael Bergdahl; Frances Separovic; Robert T.C. Brownlee; Robert P. Metzger (pp. 610-618).

The three-dimensional structure of acetylated virginiamycin M₁ (acetylated VM1) in chloroform and in a water/acetonitrile mixture (83:17 v/v) have been established through 2D high resolution NMR experiments and molecular dynamics modeling and the results compared with the conformation of the antibiotic VM1 in the same and other solvents. The results indicated that acetylation of the C-14 OH group of VM1 caused it to rotate about 90° from the position it assumed in non-acetylated VM1. The conformation of both VM1 and acetylated VM1 appear to flatten in moving from a nonpolar to polar solvent. However, the acetylated form has a more hydrophobic nature. The acetylated VM1 in chloroform and in water/acetonitrile solution had a similar configuration to that of VM1 bound to 50S ribosomes and to the Vat(D) active sites as previously determined by X-ray crystallography. Docking studies of VM1 to the 50S ribosomal binding site and the Vat(D) gave conformations very similar to those derived from X-ray crystallographic studies. The docking studies with acetylated VM1 suggested the possibility of a hydrogen bond from the acetyl carbonyl group oxygen of acetylated VM1 to the 2′ hydroxyl group of ribose of adenosine 2538 at the ribosomal VM1 binding site. No hydrogen bonds between acetylated VM1 and the Vat(D) active sites were found; the loss of this binding interaction partly accounts for the release of the product from the active site.

Keywords: Virginiamycin M1 antibiotic; NMR structure; Solvent effects; Enzyme binding; ribosome binding; Molecular dynamics

Biochemical characterization of an aspartic protease from Vigna radiata: Kinetic interactions with the classical inhibitor pepstatin implicating a tight binding mechanism by Aarohi Kulkarni; Mala Rao (pp. 619-627).

Aspartic proteases are the focus of recent research interest in understanding the physiological importance of this class of enzymes in plants. This is the first report of an aspartic protease from the seeds of Vigna radiata. The aspartic protease was purified to homogeneity by fractional ammonium sulfate precipitation and pepstatin-A agarose affinity column. It was found to have a molecular weight of 67,406 Da by gel filtration chromatography. SDS-PAGE analysis revealed the presence of a heterodimer with subunits of molecular weights of 44,024 and 23,349 Da respectively. The enzyme was pH stable with the amino acid analysis confirming the molecular weight of the protein. The substrate cleavage site as analyzed by using the synthetic substrate was found to be the Phe–Tyr bond. The kinetic interactions of the enzyme were studied with the universal inhibitor, pepstatin A. This is the first report on the interactions of a plant aspartic protease with pepstatin-A, an inhibitor from a microbial source. A competitive one-step mechanism of binding is observed. The progress curves are time-dependent and consistent with tight binding inhibition. The K_i value of the reversible complex of pepstatin with the enzyme was 0.87 μM whereas the overall inhibition constant K_i* was 0.727 μM.

Keywords: Aspartic protease; Pepstatin; Tight binding; Competitive inhibition

Post-translational modifications of rat liver mitochondrial outer membrane proteins identified by mass spectrometry by Anne M. Distler; Janos Kerner; Charles L. Hoppel (pp. 628-636).

The identification of post-translational modifications is difficult especially for hydrophobic membrane proteins. Here we present the identification of several types of protein modifications on membrane proteins isolated from mitochondrial outer membranes. We show, in vivo, that the mature rat liver mitochondrial carnitine palmitoyltransferase-I enzyme is N-terminally acetylated, phosphorylated on two threonine residues, and nitrated on two tyrosine residues. We show that long chain acyl-CoA synthetase 1 is acetylated at both the N-terminal end and at a lysine residue and tyrosine residues are found to be phosphorylated and nitrated. For the three voltage-dependent anion channel isoforms present in the mitochondria, the N-terminal regions of the protein were determined and sites of phosphorylation were identified. These novel findings raise questions about regulatory aspects of carnitine palmitoyltransferase-I, long chain acyl-CoA synthetase and voltage dependent anion channel and further studies should advance our understanding about regulation of mitochondrial fatty acid oxidation in general and these three proteins in specific.

Keywords: Carnitine palmitoyltransferase-I; Long chain acyl-CoA synthetase; Voltage dependent anion channel; Post-translational modifications; Mitochondria

Structural features of the ligand binding site on human complement protein C8γ: A member of the lipocalin family by Brian Chiswell; Leslie L. Lovelace; Charity Brannen; Eric A. Ortlund; Lukasz Lebioda; James M. Sodetz (pp. 637-644).

Human C8 is one of five components of the cytolytic membrane attack complex of complement. It contains three subunits (C8α, C8β, C8γ) arranged as a disulfide-linked C8α-γ heterodimer that is noncovalently associated with C8β. C8γ has the distinction of being the only lipocalin in the complement system. Lipocalins have a core β-barrel structure forming a calyx with a binding site for a small hydrophobic ligand. A natural ligand for C8γ has not been identified; however previous structural studies indicate C8γ has a typical lipocalin fold that is suggestive of a ligand-binding capability. A distinctive feature of C8γ is the division of its putative ligand binding pocket into a hydrophilic upper portion and a large hydrophobic lower cavity. Access to the latter is restricted by the close proximity of two tyrosine side chains (Y83 and Y131). In the present study, binding experiments were performed using lauric acid as a pseudoligand to investigate the potential accessibility of the lower cavity. The crystal structure of a C8γ·laurate complex revealed that Y83 and Y131 can move to allow penetration of the hydrocarbon chain of laurate into the lower cavity. Introducing a Y83W mutation blocked access but had no effect on the ability of C8γ to enhance C8 cytolytic activity. Together, these results indicate that the lower cavity in C8γ could accommodate a ligand if such a ligand has a narrow hydrophobic moiety at one end. Entry of that moiety into the lower cavity would require movement of Y83 and Y131, which act as a gate at the cavity entrance.

Keywords: Abbreviations; MACPF; membrane attack complex/perforin domain; C8γ·laurate; a complex of C8γ and laurate ion; Y83W; recombinant human C8γ containing C40A and Y83W substitutions; C8α·C8β; a noncovalent complex of C8α and C8β; C8γ·C8α·C8β; a noncovalent complex of C8γ, C8α and C8β; EAC1–7; sensitized sheep erythrocytes carrying human complement C1–C7; 2-AS; 2-(9-anthroyloxy)stearic acid; OBP; odorant binding proteinComplement C8γ; Lipocalin; C8γ crystal structure; C8γ·laurate complex; Membrane attack complex

NMR solution structure of the angiostatic peptide anginex by Monica M. Arroyo; Kevin H. Mayo (pp. 645-651).

Anginex, a designed peptide 33mer, is known to function both as an antiangiogenic and bactericidal agent. Solving the NMR solution structure of the peptide is key to understand better its structure–activity relationships and to design more bioactive peptides and peptide mimetics. However, structure elucidation of anginex has been elusive due to subunit exchange-induced resonance broadening. Here, we found that performing NMR structural studies in a micellar environment abolishes exchange broadening and allows the structure of anginex to be determined. Anginex folds in an amphipathic, three-stranded antiparallel β-sheet conformation with functionally key hydrophobic residues lying on one face of the β-sheet and positively charged, mostly lysine residues, lying on the opposite face. Structural comparison is made with a homologous, yet relatively inactive peptide, βpep-28. These results contribute to the design of peptidomimetics of anginex for therapeutic use against angiogenically-related diseases like cancer, as well as infectious diseases.

Keywords: Abbreviations; NMR; nuclear magnetic resonance spectroscopy; NOE; nuclear Overhauser effect; NOESY; two-dimensional NOE spectroscopy; FID; free induction decay; HPLC; high performance liquid chromatography; DPC; dodecylphosphocoline; DMSO; dimethyl sulfoxideDrug design; Anti-angiogenic; Anti-bacterial; Conformation

Pressure effects on the heat-induced aggregation of equine serum albumin by FT-IR spectroscopic study: Secondary structure, kinetic and thermodynamic properties by Akira Okuno; Minoru Kato; Yoshihiro Taniguchi (pp. 652-660).

Pressure can restrain the heat-induced aggregation and dissociate the heat-induced aggregates. We investigated the aggregation-preventing pressure effect and the aggregates-dissociating pressure effect to characterize the heat-induced aggregation of equine serum albumin (ESA) by Fourier transform infrared spectroscopy. The results suggest that the α-helical structure collapses at the beginning of heat-induced aggregation, then the rearrangement of structure from partially unfolded structure to the intermolecular β-sheet takes place through the activated state. We determined the activation volume for the heat-induced aggregation (Δ V^≠=+92±8 ml mol−¹) and the partial molar volume difference between native state and heat-induced aggregates (Δ VN→_HA=+32 ml mol−¹). This positive partial molar volume difference suggests that the heat-induced aggregates have larger internal voids than the native structure. Moreover, the positive volume change implies that the formation of the intermolecular β-sheet is unfavorable under high pressure. We also determined the free energy profile of ESA. This energy profile explains the restriction of the formation of heat-induced aggregates by pressure. These results explain the structural differences between heat-induced aggregates with intermolecular β-sheet and pressure-induced aggregates without intermolecular β-sheet.

Keywords: Serum albumin; High pressure FT-IR; Heat-induced protein aggregation; Aggregation-preventing pressure effect; Aggregates-dissociating pressure effect

Molecular cloning and biochemical characterization of the first archaeal maltogenic amylase from the hyperthermophilic archaeon Thermoplasma volcanium GSS1 by Jung-Woo Kim; Yung-Hee Kim; Hee-Seob Lee; Sung-Jae Yang; Young-Wan Kim; Myoung-Hee Lee; Jung-Wan Kim; Nam-Seok Seo; Cheon-Seok Park; Kwan-Hwa Park (pp. 661-669).

Maltogenic amylases (MAases), a subclass of cyclodextrin (CD)-hydrolyzing enzymes belonging to glycoside hydrolase family 13, have been studied extensively, but their physiological roles in microbes and evolutionary relationships with other amylolytic enzymes remain unclear. Here, we report the biochemical properties of a thermostable archaeal MAase from Thermoplasma volcanium GSS1 (TpMA) for the first time. The primary structure and catalytic properties of TpMA were similar to those of MAases, such as possession of an extra domain at its N-terminal and preference for CD over starch. TpMA showed high thermostability and optimal activity at 75 °C and 80 °C for β-CD and soluble starch, respectively. The recombinant TpMA exists as a high oligomer in a solution and the oligomeric TpMA was dissociated into dimer and monomer mixture by a high concentration of NaCl. The substrate preference and thermostability of TpMA were significantly dependent on the oligomeric state of the enzyme. However, TpMA exhibited distinguishable characteristics from those of bacterial MAases. The transglycosylation pattern of TpMA was opposite to that of bacterial MAases. TpMA formed more α-1,4-glycosidic linked transfer product than α-1,6-linked products. Like as α-amylases, notably, TpMA has a longer subsite structure than those of other CD-degrading enzymes. Our findings in this study suggest that TpMA, the archaeal MAase, shares characteristics of both bacterial MAases and α-amylases, and locates in the middle of the evolutionary process between α-amylases and bacterial MAases.

Keywords: Thermoplasma volcanium; Maltogenic amylase; Thermostability; Oligomeric state; Subsite structure

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BBA - Proteins and Proteomics (v.1774, #5)