Characterization of alanine and malate dehydrogenases from a marine psychrophile strain PA-43

Alanine dehydrogenase (AlaDH: EC 1.4.1.1), malate dehydrogenase (MDH: EC 1.1.1.37), and glutamate dehydrogenase (EC 1.4.1.2), all NAD+ dependent, were detected in extracts from a psychrophilic bacterium, strain PA-43, isolated from a sea urchin off the Icelandic coast. Characterization tests suggested that the strain had a close relationship to Vibrio, but sequencing of part of the 16S rDNA gene placed the bacterium among Shewanella species in a constructed phylogenetic tree. The bacterium had an optimum growth temperature of 16.5 degrees C, and maximum dehydrogenase expression was obtained in a rich medium supplemented with NaCl. Both AlaDH and MDH were purified to homogeneity. AlaDH is a hexamer, with an approximate relative molecular mass of 260,000, whereas MDH is dimeric, with an apparent relative molecular mass of approximately 70,000. Both enzymes were thermolabile, and the optimum temperatures for activity were shifted toward lower temperatures than those found in the same enzymes from mesophiles, 37 degrees C for MDH and approximately 47 degrees C for AlaDH. The pH optima for AlaDH in the forward and reverse reactions were 10.5 and 9, respectively, whereas those for MDH were 10-10.2 and 8.8, respectively. Partial amino acid sequences, comprising approximately 30% of the total sequences from each enzyme, were determined for N-terminal, tryptic, and chymotryptic fragments of the enzymes. The AlaDH showed the highest similarity to AlaDHs from the psychrotroph Shewanella Ac10 and the mesophile Vibrio proteolyticus, whereas MDH was most similar to the MDHs from the mesophiles Escherichia coli and Haemophilus influenzae, with lower identity to the psychrophilic malate dehydrogenases from Vibrio 5710 and Photobacterium SS9.     

Temperature range for formic hydrogenlyase induction and activity in psychrophilic and mesophilic bacteria

The temperature range for induction of formic hydrogenlyase in cell suspensions of psychrophilic strain 82 was 0 C to 20 C compared to 15 C to 45 C for mesophilicEscherichia coli and 15 C to 40 C for mesophilicAerobacter aerogenes. The respective temperatures for maximum enzyme activity were 30 C, 45 C and 40 C, and for formation of the maximum amount of enzyme 15 C, 35 C and 30 C. The temperatures for all three enzyme functions, therefore, were considerably lower for the psychrophile than for the mesophiles.This research has been supported by a grant from the National Science Foundation.     

Nad+ Dependent Alcohol Dehydrogenase from Sulfolobus Solfataricus: Structural and Functional Features

Alcohol dehydrogenase, EC 1.1.1.1 (ADH), from the thermophilic archaebacterium Sulfolobus solfataricus (SsADH) is a strictly NAD⁺-dependent enzyme with an amino acid sequence related to those of horse liver, yeast and Thermoanaerobium brockii ADHs. This enzyme is remarkably thermophilic and thermostable; protein stability is strictly dependent on the presence of structural zinc in the molecule. For its broad substrate specificity, product stereoselectivity and acceptance of NAD⁺-macromolecular derivatives, SsADH appears suitable for laboratory-scale chemoselective synthesis. Moreover, it represents a useful protein model for studying the structure-function-stability relationships in thermophilic enzymes.     

Thermostable aldehyde dehydrogenase from psychrophile,Cytophaga sp. KUC-1: enzymological characteristics and functional properties

We found the occurrence of NAD(P)(+)-dependent aldehyde dehydrogenase (EC1.2.1.5) in the cells of a psychrophile from Antarctic seawater, Cytophaga sp. KUC-1, and purified to homogeneity. About 50% of the enzyme activity remained even after heating at 50 degrees C for 65min and the highest activity was observed in the range of 55-60 degrees C. The enzyme was thermostable and thermophilic, although it was derived from a psychrophile. The circular dichroism at 222nm of the enzyme showed a peak at 32 degrees C. This temperature was closely similar to the transition temperature in the Arrhenius plots. The stereospecificity for the hydride transfer at C4-site of nicotinamide moiety of NADH was pro-R. The gene encoding the enzyme consisted of an open reading frame of 1506-bp encoding a protein of 501 amino acid residues. The significant sequence identity (61%) was found between the Cytophaga and the Pseudomonas aeruginosa enzymes, although their thermostabilities are completely different.     

Purification and properties of histidinol dehydrogenases from psychrophilic, mesophilic and thermophilic bacilli.

As a first step in elucidating one molecular mechanism of adaptation to life at extreme temperatures, we purified and characterized the enzyme histidinol dehydrogenase (EC 1.1.1.23) from a number of bacilli whose growth temperatures range from 5 degrees t to 90 degrees C. The enzymes were purified by (NH4)2SO4 precipitation, ion-exchange chromatography on Sephadex, affinity chromatography on histamine- or histidine-Sepharose and preparative gradient gel electrophoresis. All had similar mol.wts. (29200), sedimentation coefficients (S20,w 2.56S), affinities for histidinol and NAD+ (Km = 48 micron and 0.2 mM respectively) and all had pH optima at 9.6. Marked differences were observed in stability with respect to temperature and the temperature at which the initial velocity for histidinol dehydrogenation was optimal. These optima range from 25 degrees C for the enzyme from the psychrophilic species through to 41 degrees C for the mesophiles to 85-92 degrees C for the extreme thermophiles. It is concluded that the ability of the enzymes to operate at their various optimum temperatures is an intrinsic property of their amino acid sequences.     

Improvement in thermostability and psychrophilicity of psychrophilic alanine racemase by site-directed mutagenesis

A psychrophilic alanine racemase from Bacillus psychrosaccharolyticus has a higher catalytic activity than a thermophilic alanine racemase from Bacillus stearothermophilus even at 60 °C in the presence of pyridoxal 5′-phosphate (PLP), although the thermostability of the former enzyme is lower than that of the latter one [FEMS Microbial. Lett. 192 (2000) 169]. In order to improve the thermostability of the psychrophilic enzyme, two hydrophilic amino acid residues (Glu150 and Arg151) at a surface loop surrounding the active site of the enzyme were substituted with the corresponding residues (Val and Ala) in the B. stearothermophilus alanine racemase. The mutant enzyme (ER150,151VA) showed a higher thermostability, and a markedly lower K_m value for PLP, than the wild type one. In addition, the catalytic activities at low temperatures and kinetic parameters of the two enzymes indicated that the mutant enzyme was more psychrophilic than the wild type one. Thus, the psychrophilic alanine racemase was improved in both psychrophilicity and thermostability by the site-directed mutagenesis. The mutant enzyme may be useful for the production of stereospecifically deuterated NADH and various -amino acids.     

Cloning and sequencing of the leucine dehydrogenase gene from Bacillus sphaericus IFO 3525 and importance of the C-terminal region for the enzyme activity

The structural gene (leudh) coding for leucine dehydrogenase from Bacillus sphaericus IFO 3525 was cloned into Escherichia coli cells and sequenced. The open reading frame coded for a protein of 39.8 kDa. The deduced amino acid sequence of the leucine dehydrogenase from B. sphaericus showed 76–79% identity with those of leucine dehydrogenases from other sources. About 16% of the amino acid residues of the deduced amino acid sequence were different from the sequence obtained by X-ray analysis of the B. sphaericus enzyme. The recombinant enzyme was purified to homogeneity with a 79% yield. The enzyme was a homooctamer (340 kDa) and showed the activity of 71.7 μmol·min⁻¹·mg⁻¹) of protein. The mutant enzymes, in which more than six amino acid residues were deleted from the C-terminal of the enzyme, showed no activity. The mutant enzyme with deletion of four amino acid residues from the C-terminal of the enzyme was a dimer and showed 4.5% of the activity of the native enzyme. The dimeric enzyme was more unstable than the native enzyme, and the K_m values for -leucine and NAD⁺ increased. These results suggest that the Asn-Ile-Leu-Asn residues of the C-terminal region of the enzyme play an important role in the subunit interaction of the enzyme.     

Structure and function of psychrophilic alanine racemase

We describe the structure and function of psychrophilic alanine racemases from Bacillus psychrosaccharolyticus and Pseudomonas fluorescens. These enzymes showed high catalytic activities even at 0°C and were extremely labile at temperatures over 35°C. The enzymes were also found to be less resistant to organic solvents than alanine racemases from thermophilic and mesophilic bacteria, both in vivo and in vitro. Both enzymes have a dimeric structure and contain 2 mol of pyridoxal 5′-phosphate (PLP) per mol as a coenzyme. The enzyme from B. psychrosaccharolyticus was found to have a markedly large K_m value (5.0 μM) for PLP in comparison with other reported alanine racemases, and was stable at temperatures up to 50°C in the presence of excess amounts of PLP. The dissociation of PLP from the P. fluorescens enzyme may trigger the unfolding of the secondary structure. The enzyme from B. psychrosaccharolyticus has a distinguishing hydrophilic region around residue no. 150 in its deduced amino acid sequence, whereas the corresponding regions of other Bacillus alanine racemases are hydrophobic. The position of this region in the three dimensional structure of this enzyme was predicted to be in a surface loop surrounding the active site. This hydrophilic region may interact with solvent, reduce the compactness of the active site, and destabilize the enzyme.     

Developments in industrially important thermostable enzymes: a review

Cellular components of thermophilic organisms (enzymes, proteins and nucleic acids) are also thermostable. Apart from high temperature they are also known to withstand denaturants of extremely acidic and alkaline conditions. Thermostable enzymes are highly specific and thus have considerable potential for many industrial applications. The use of such enzymes in maximising reactions accomplished in the food and paper industry, detergents, drugs, toxic wastes removal and drilling for oil is being studied extensively. The enzymes can be produced from the thermophiles through either optimised fermentation of the microorganisms or cloning of fast-growing mesophiles by recombinant DNA technology. In this review, the source microorganisms and properties of thermostable starch hydrolysing amylases, xylanases, cellulases, chitinases, proteases, lipases and DNA polymerases are discussed. The industrial needs for such specific thermostable enzyme and improvements required to maximize their application in the future are also suggested.     

Comparative void-volume analysis of psychrophilic and mesophilic enzymes: Structural bioinformatics of psychrophilic enzymes reveals sources of core flexibility

Background

Psychrophiles, cold-adapted organisms, have adapted to live at low temperatures by using a variety of mechanisms. Their enzymes are active at cold temperatures by being structurally more flexible than mesophilic enzymes. Even though, there are some indications of the possible structural mechanisms by which psychrophilic enzymes are catalytic active at cold temperatures, there is not a generalized structural property common to all psychrophilic enzymes.

Results

We examine twenty homologous enzyme pairs from psychrophiles and mesophiles to investigate flexibility as a key characteristic for cold adaptation. B-factors in protein X-ray structures are one way to measure flexibility. Comparing psychrophilic to mesophilic protein B-factors reveals that psychrophilic enzymes are more flexible in 5-turn and strand secondary structures. Enzyme cavities, identified using CASTp at various probe sizes, indicate that psychrophilic enzymes have larger average cavity sizes at probe radii of 1.4-1.5 Å, sufficient for water molecules. Furthermore, amino acid side chains lining these cavities show an increased frequency of acidic groups in psychrophilic enzymes.

Conclusions

These findings suggest that embedded water molecules may play a significant role in cavity flexibility, and therefore, overall protein flexibility. Thus, our results point to the important role enzyme flexibility plays in adaptation to cold environments.

     

Manipulations of AMP metabolic genes increase growth rate and cold tolerance in Escherichia coli: implications for psychrophilic evolution

Diverse organisms have adapted to thrive at low temperatures (i.e., <20 °C, termed psychrophiles), colonizing the majority of earth's biosphere. In contrast with mesophiles (20-40 °C thermal range), all observed psychrophiles increase intracellular adenosine 5'-triphosphate concentrations as temperatures decline; this phenomenon has been described as an important compensatory mechanism to deal with decreases in thermal energy and molecular motion. We considered purine metabolic pathways in class gammaproteobacteria (n = 115) to investigate metabolic and evolutionary bases of this process. A survey of the KEGG database indicated that psychrophilic purine metabolic pathways tend to be enriched with de novo adenosine 5'-monophosphate (AMP) synthetic enzymes, whereas mesophiles tend to be enriched with AMP degradative enzymes. Function of the observed psychrophilic pathway structure was tested by engineering the mesophilic gammaproteobacterium Escherichia coli to reflect psychrophilic purine metabolism, specifically by expressing adenylosuccinate synthetase (purA) from the psychrophilic gammaproteobacterium, Psychrobacter cryohalolentis, in an AMP nucleosidase (amn)-deficient background. Modified E. coli was capable of growing up to ～70% faster at low temperatures and became up to ～10-fold more cold tolerant relative to wild type. These findings highlight potentially important transitional steps in psychrophilic evolution.     

Improved high thermal stability of pullulanase from a newly isolated thermophilic Bacillus sp. AN-7

A thermophilic Bacillus sp. strain AN-7, isolated from a soil in India, produced an extracellular pullulanase upon growth on starch–peptone medium. The enzyme was purified to homogeneity by ammonium sulfate precipitation, anion exchange and gel filtration chromatography. The optimum temperature and pH for activity was 90 °C and 6.0. With half-life time longer than one day at 80 °C the enzyme proves to be thermostable in the pH range 4.5–7.0. The pullulanase from Bacillus strain lost activity rapidly when incubated at temperature higher than 105 °C or at pH lower than 4.5. Pullulanase was completely inhibited by the Hg²⁺ ions. Ca²⁺, dithiothreitol, and Mn²⁺ stimulated the pullulanase activity. Kinetic experiments at 80 °C and pH 6.0 gave V_max and K_m values of 154 U mg⁻¹ and 1.3 mg ml⁻¹. The products of pullulan were maltotriose and maltose. This proved that the purified pullulanase (pullulan-6-glucanohydrolase, EC 3.2.1.41) from Bacillus sp. AN-7 is classified under pullulanase type I. To our knowledge, this Bacillus pullulanase is the most highly thermostable type I pullulanase known to date.     

Enzymatical properties of psychrophilic phosphatase I

Phosphatase I purified from a psychrophile (Shewanella sp.) [Tsuruta et al. (1998) J. Biochem. 123, 219-225] dephosphorylated O-phospho-L-tyrosine and phospho-tyrosyl residues in phosphorylated poly(Glu4,Tyr1) random polymer (polyEY) and phosphorylated myelin basic protein (MBP) but not phosphoseryl and/or phosphothreonyl residues in phosphorylated histone H1, casein and phosphorylase a, indicating that the enzyme showed protein-tyrosine-phosphatase (PTPase, EC 3.1.3.48)-like activity in vitro. The enzyme was remarkably inhibited by diethylpyrocarbonate (DEPC), monoiodoacetic acid (MIAA), and monoiodoacetamide (MIAM). Binding of 1 mol of DEPC to 1 mol of the enzyme caused complete inhibition of the enzyme; and 0.88 mol of 1-carboxymethylated histidine per mole of the enzyme was found when 90% of enzyme activity was lost by modification with 14C-MIAA. These results indicated that this psychrophilic enzyme was a PTPase-like enzyme with histidine as its catalytic residue.     

Improving enzyme characteristics by gene shuffling; application to β-glucosidase

The genes of family 3 β-glucosidase enzymes consist of five distinct regions; the N-terminal residues, an N-terminal catalytic domain, a nonhomologous region, a C-terminal domain of unknown function and the C-terminal residues. The β-glucosidase genes derived from Cellvibrio gilvus (CG) and Agrobacterium tumefaciens (AT) have been subjected to gene deletion, truncation and shuffling. The folding information was found to be distributed unevenly across the different regions based on the gene manipulation results. Chimeric enzymes with improved enzyme characteristics were obtained only by gene shuffling at the C-terminal domain.     

温度对嗜冷酵母糖代谢途径某些关键酶的活性效应

对嗜冷酵母Y18和酿酒酵母细胞中EMP途径和TCA循环中一些关键酶的温度特性进行了比较研究。Y18细胞中,1,6二磷酸果糖醛缩酶、琥珀酸脱氢酶和己糖激酶对温度很敏感,符合Feller提出的冷活性的概念属于冷活性酶类。柠檬酸合成酶的温度特性类似于中温酶。Α酮戊二酸脱氢酶存在不同温度特性的同功酶。通过对嗜冷酵母和中温酶母细胞中琥珀酸脱氢酶的Km值进行比较,结果显示嗜冷酵母琥珀酸脱氢酶在20℃具有较低的Km值。另外还对嗜冷菌细胞中代谢酶类的一些特点进行了讨论。     

The fine structure of psychrophilic and mesophilic Rhodotorula rubra

Transmission electron microscopy of thin sections of two mesophilic and a psychrophilic Rhodotorula rubra revealed structures normally associated with yeast. The capsule of all three strains was thicker in the cells grown in glucose than those grown in glucose-free media. The cell wall of all three strains showed more lamellae in the cells grown in a medium containing glycerol. Budding was of the Rhodoturula type. Endoplasmic reticula running parallel to the nucleus were commonly observed in the psychrophiles but not in the mesophiles. In the psychrophile, in association with the plasmalemma, lomasomes and plasmalemmasomes were observed. Giant mitochondria were commonly seen in the cells grown in a fermentable carbohydrate-free medium. Vacuoles were mostly empty.     

Determination of the sites required for the allosteric inhibition of serine acetyltransferase by L-cysteine in plants.

Serine acetyltransferase (SATase; EC 2.3.1.30) catalyzes the formation of O-acetylserine from L-Ser and acetyl-CoA in plants and bacteria. In plants, two types of SATase have been described. One is allosterically inhibited by L-Cys, and the second is not sensitive to L-Cys inhibition. However, the allosteric site in SATase has not been identified. To understand better the mechanism of L-Cys inhibition of plant SATases, we constructed several chimeric SATase enzymes from watermelon SATase (WaSATase) (sensitive type) and Arabidopsis SAT-p (insensitive type). These enzymes were expressed in Escherichia coli, and inhibition of the mutated SATase activity by L-Cys was analyzed. Mutated WaSATase, in which Met280 was changed to Ile, was no longer inhibited by L-Cys. Analysis of the inhibition the chimeric enzymes indicated that the C-terminal region of WaSATase from Pro276 to Phe285, in which five amino acids are different from those of SAT-p, was responsible for the determination of the sensitivity to L-Cys. In particular, Gly277 in the C-terminal region of WaSATase was primarily responsible for the L-Cys inhibition. The N-terminal half of the protein, which does not contain the catalytic domain, was also important for the sensitivity to L-Cys. These results indicate that the sensitivity of SATase to L-Cys is due to the N-terminal and C-terminal regions rather than to the catalytic domain.     

Partial purification and characterization of the lipase of a facultatively psychrophilic bacterium (Acinetobacter O16).

The extracellular lipase(s) of the psychrophile Acinetobacter O16 was studied. When the enzyme was precipitated by (NH4)2SO4 and passed through a Sephadex G200 column, two peaks of lipase activity appeared. The larger peak, which behaved like a substance of high molecular weight, being eluted in the void volume, was purified 250-fold over the crude enzyme (culture supernatant) by passage through a DEAE-Sephadex column. When the enzyme was applied to a DEAE-cellulose column it could not be eluted unless it had first been treated with the detergent Titon X 100. It is suggested that lipids or phospholipids make up an important part of the molecule. The activity of the crude and partly purified enzymes was studied in relation to pH and temperature optima. Lipases from the psychrophilic Acinetobacter O16 and from the mesophilic Acinetobacter O4 reacted in the same way to temperature. The crude enzyme from Acinetobacter O16 was more temperature-stable than the purified enzyme.     

Comparative Effect of Temperature on the Oxidative Metabolism of Whole and Disrupted Cells of a Psychrophilic and a Mesophilic Species of Bacillus

We investigated the influence of temperature, in the range of 45 to 5 C, on the rate of oxidation of glucose and citrate by intact cells and cell-free extracts of psychrophilic Bacillus psychrophilus and mesophilic B. thuringiensis. Both glucose and endogenous oxidation by whole and disrupted cells of the psychrophile decreased more slowly with decrease in temperature than did glucose and endogenous oxidation by whole and disrupted cells of the mesophile. Similar results were obtained for the oxidation of citrate by cell-free extracts. Since substrate permeability is not involved in the oxidative metabolism of the cell-free extracts, we concluded that the internal enzymes of the psychrophile differ from those of the mesophile.     

C-Terminal region of human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase is involved in the interaction with prostaglandin substrates.

NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes the oxidation of the 15(S) hydroxyl group of prostaglandins to a 15-keto group resulting in a significant reduction of the biological activities of prostaglandins. Although the key residues involved in NAD+ binding and in catalytic activity have been partially identified, the sites of interaction of the enzyme with the prostaglandin substrates are yet to be determined. Homology analysis of the primary structures of 15-PGDH from human, mouse and rat indicates that the sequences are almost homologous except for two regions near the C-terminus. The involvement of the C-terminal region in catalytic activity was examined by studies on C-terminally truncated enzymes and on human/rat chimeric enzymes. When three to four amino acids were removed successively from the C-terminal end of human 15-PGDH, the truncated enzymes exhibited decreasing Vmax/Km ratios and increasing Km values for PGE2 as the chain was shortened. Similarly, when the C-terminal 14 amino acids of human 15-PGDH were replaced by the C-terminal 14 amino acids of rat 15-PGDH or vice versa, the Vmax/Km ratios and the Km values for prostaglandin E2 of the chimeric enzymes were in between those of the two wild-type enzymes. This indicates that the catalytic effectiveness of human 15-PGDH decreases as the C-terminal region is gradually removed or replaced by rat sequences. The C-terminal region appears to be more important for the interaction of the enzyme with the prostaglandin substrates than with the coenzyme.