Nucleotide sequence of the lipase gene lip2 from the antarctic psychrotroph Moraxella TA144 and site-specific mutagenesis of the conserved serine and histidine residues

The lip2 gene from the antarctic psychotroph Moraxella TA144 was sequenced. The primary structure of the Lip2 preprotein deduced from the nucleotide sequence is composed of 433 amino acids with a predicted Mr of 47,222. This enzyme contains a Ser-centered consensus sequence and a conserved His-Gly dipeptide found in most lipase amino-terminal domains. These sequences are involved in the lipase active site conformation since substitution of the conserved Ser or His residues by Ala and Gln, respectively, results in the loss of both lipase and esterase activities. Structural factors that would allow proper enzyme flexibility at low temperatures are discussed. It is suggested that only subtle changes in the primary structure of these psychrotrophic enzymes can account for their ability to catalyze lipolysis at temperatures close to 0 degrees C.     

A cold-active glucanase from the ruminal bacterium Fibrobacter succinogenes S85

We previously characterized two endoglucanases, CelG and EGD, from the mesophilic ruminal anaerobe Fibrobacter succinogenes S85. Further comparative experiments have shown that CelG is a cold-active enzyme whose catalytic properties are superior to those of several other intensively studied cold-active enzymes. It has a lower temperature optimum, of 25 degrees C, and retains about 70% of its maximum activity at 0 degrees C, while EGD has a temperature optimum of 35 degrees C and retains only about 18% of its maximal activity at 0 degrees C. When assayed at 4 degrees C, CelG exhibits a 33-fold-higher kcat value and a 73-fold-higher physiological efficiency (kcat/Km) than EGD. CelG has a low thermal stability, as indicated by the effect of temperature on its activity and secondary structure. The presence of small amino acids around the putative catalytic residues may add to the flexibility of the enzyme, thereby increasing its activity at cold temperatures. Its activity is modulated by sodium chloride, with an increase of over 1.8-fold at an ionic strength of 0.03. Possible explanations for the presence of a cold-active enzyme in a mesophile are that cold-active enzymes are more broadly distributed than previously expected, that lateral transfer of the gene from a psychrophile occurred, or that F. succinogenes originated from the marine environment.     

Cloning of cold-active alkaline phosphatase gene of a psychrophile,Shewanella sp., and expression of the recombinant enzyme

A psychrophilic alkaline phosphatase (EC 3.1.3.1) from Shewanella sp. is a cold-active enzyme that has high catalytic activity at low temperature [Ishida et al. (1998) Biosci. Biotechnol. Biochem., 62, 2246-2250]. Here, we identified the nucleotide sequence of a gene encoding the enzyme after cloning with the polymerase chain reaction (PCR) and inverted PCR techniques. The deduced amino acid sequence of the enzyme contained conserved amino acids found among mesophilic alkaline phosphatases and showed some structural characteristics including a high content of hydrophobic amino acid residues and the lack of single alpha-helix compared with the alkaline phosphatase of Escherichia coli, which were possibly efficient for catalytic reaction at low temperatures. The recombinant enzyme expressed in E. coli was purified to homogeneity with the molecular mass of 41 kDa. The recombinant enzyme had a specific activity of 1,500 units/mg and had high catalytic activity at low temperatures.     

Flexibility and enzymatic cold-adaptation: a comparative molecular dynamics investigation of the elastase family

Molecular dynamics simulations of representative mesophilic and psycrophilic elastases have been carried out at different temperatures to explore the molecular basis of cold adaptation inside a specific enzymatic family. The molecular dynamics trajectories have been compared and analyzed in terms of secondary structure, molecular flexibility, intramolecular and protein-solvent interactions, unravelling molecular features relevant to rationalize the efficient catalytic activity of psychrophilic elastases at low temperature. The comparative molecular dynamics investigation reveals that modulation of the number of protein-solvent interactions is not the evolutionary strategy followed by the psycrophilic elastase to enhance catalytic activity at low temperature. In addition, flexibility and solvent accessibility of the residues forming the catalytic triad and the specificity pocket are comparable in the cold- and warm-adapted enzymes. Instead, loop regions with different amino acid composition in the two enzymes, and clustered around the active site or the specificity pocket, are characterized by enhanced flexibility in the cold-adapted enzyme. Remarkably, the psycrophilic elastase is characterized by reduced flexibility, when compared to the mesophilic counterpart, in some scattered regions distant from the functional sites, in agreement with hypothesis suggesting that local rigidity in regions far from functional sites can be beneficial for the catalytic activity of psychrophilic enzymes.     

Identification of catalytically important amino acid residues of Streptomyces lividans acetylxylan esterase A from carbohydrate esterase family 4

Multiple sequence alignment of Streptomyces lividans acetylxylan esterase A and other carbohydrate esterase family 4 enzymes revealed the following conserved amino acid residues: Asp-12, Asp-13, His-62, His-66, Asp-130, and His-155. These amino acids were mutated in order to investigate a functional role of these residues in catalysis. Replacement of the conserved histidine residues by alanine caused significant reduction of enzymatic activity. Maintenance of ionizable carboxylic group in side chains of amino acids at positions 12, 13, and 130 seems to be necessary for catalytic efficiency. The absence of conserved serine excludes a possibility that the enzyme is a serine esterase, in contrast to acetylxylan esterases of carbohydrate esterase families 1, 5, and 7. On the contrary, total conservation of Asp-12, Asp-13, Asp-130, and His-155 along with dramatic decrease in enzyme activity of mutants of either of these residues lead us to a suggestion that acetylxylan esterase A from Streptomyces lividans and, by inference, other members of carbohydrate esterase family 4 are aspartic deacetylases. We propose that one component of the aspartate dyad/triad functions as a catalytic nucleophile and the other one(s) as a catalytic acid/base. The ester/amide bond cleavage would proceed via a double displacement mechanism through covalently linked acetyl-enzyme intermediate of mixed anhydride type.     

Crystallization and preliminary X-ray diffraction studies of alpha-amylase from the antarctic psychrophile Alteromonas haloplanctis A23.

A cold-active alpha-amylase was purified from culture supernatants of the antarctic psychrophile Alteromonas haloplanctis A23 grown at 4 degrees C. In order to contribute to the understanding of the molecular basis of cold adaptations, crystallographic studies of this cold-adapted enzyme have been initiated because a three-dimensional structure of a mesophilic counterpart, pig pancreatic alpha-amylase, already exists. alpha-Amylase from A. haloplanctis, which shares 53% sequence identity with pig pancreatic alpha-amylase, has been crystallized and data to 1.85 A have been collected. The space group is found to be C222(1) with a = 71.40 A, b = 138.88 A, and c = 115.66 A. Until now, a three-dimensional structure of a psychrophilic enzyme is lacking.     

Nucleotide and derived amino acid sequence of the subtilisin from the antarctic psychrotroph Bacillus TA39.

The nucleotide sequence of the subtilisin-encoding gene from the antarctic psychrotroph Bacillus TA39 was determined. The primary structure of the subtilisin precursor is composed of 420 amino acids giving rise to a mature enzyme of 309 amino acids. Asp-145, His-185 and Ser-361 are the proposed catalytic residues of the active site.     

Purification and characterization of the heat-labile alpha-amylase secreted by the psychrophilic bacterium TAC 240B.

A total of 59 bacteria samples from Antarctic sea water were collected and screened for their ability to produce alpha-amylase. The highest activity was recorded from an isolate identified as an Alteromonas species. The purified alpha-amylase shows a molecular mass of about 50,000 Da and a pI of 5.2. The enzyme is stable from pH 7.5 to 9 and has a maximal activity at pH 7.5. Compared with other alpha-amylases from mesophiles and thermophiles, the "cold enzyme" displays a higher activity at low temperature and a lower stability at high temperature. The psychrophilic alpha-amylase requires both Cl- and Ca2+ for its amylolytic activity. Br- is also quite efficient as an allosteric effector. The comparison of the amino acid composition with those of other alpha-amylases from various organisms shows that the cold alpha-amylase has the lowest content in Arg and Pro residues. This could be involved in the principle used by the psychrophilic enzyme to adapt its molecular structure to the low temperature of the environment.     

Molecular Dynamics Study of the Structure,Flexibility and Dynamics of Thermostable L1 Lipase at High Temperatures

Molecular Dynamics (MD) simulations have been used to understand how protein structure, dynamics, and flexibility are affected by adaptation to high temperature for several years. We report here the results of the high temperature MD simulations of Bacillus stearothermophilus L1 (L1 lipase). We found that the N-terminal moiety of the enzyme showed a high flexibility and dynamics during high temperature simulations which preceded and followed by clear structural changes in two specific regions; the small domain and the main catalytic domain or core domain of the enzyme. These two domains interact with each other through a Zn²⁺-binding coordination with Asp-61 and Asp-238 from the core domain and His-81 and His-87 from the small domain. Interestingly, the His-81 and His-87 were among the highly fluctuated and mobile residues at high temperatures. The results appear to suggest that tight interactions of Zn²⁺-binding coordination with specified residues became weak at high temperature which suggests the contribution of this region to the thermostability of the enzyme. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A cold-adapted esterase from psychrotrophic <Emphasis Type="Italic">Pseudoalteromas</Emphasis> sp. strain 643A

A psychrotrophic bacterium producing a cold-adapted esterase upon growth at low temperatures was isolated from the alimentary tract of Antarctic krill Euphasia superba Dana, and classified as Pseudoalteromonas sp. strain 643A. A genomic DNA library of strain 643A was introduced into Escherichia coli TOP10F', and screening on tributyrin-containing agar plates led to the isolation of esterase gene. The esterase gene (estA, 621 bp) encoded a protein (EstA) of 207 amino acid residues with molecular mass of 23,036 Da. Analysis of the amino acid sequence of EstA suggests that it is a member of the GDSL-lipolytic enzymes family. The purification and characterization of native EstA esterase were performed. The enzyme displayed 20-50% of maximum activity at 0-20 degrees C. The optimal temperature for EstA was 35 degrees C. EstA was stable between pH 9 and 11.5. The enzyme showed activity for esters of short- to medium-chain (C(4) and C(10)) fatty acids, and exhibited no activity for long-chain fatty acid esters like that of palmitate and stearate. EstA was strongly inhibited by phenylmethylsulfonyl fluoride, 2-mercaptoethanol, dithiothreitol and glutathione. Addition of selected divalent ions e.g. Mg(2+), Co(2+) and Cu(2+) led to the reduction of enzymatic activity and the enzyme was slightly activated ( approximately 30%) by Ca(2+) ions.     

Properties and active center of the thermostable branching enzyme from Bacillus stearothermophilus.

Although the branching enzyme (EC 2.4.1.18) is a member of the alpha-amylase family, the characteristics are not understood. The thermostable branching enzyme gene from Bacillus stearothermophilus TRBE14 was cloned and expressed in Escherichia coli. The branching enzyme was purified to homogeneity, and various enzymatic properties were analyzed by our improved assay method. About 80% of activity was retained when the enzyme was heated at 60 degrees C for 30 min, and the optimum temperature for activity was around 50 degrees C. The enzyme was stable in the range of pH 7.5 to 9.5, and the optimum pH was 7.5. The nucleotide sequence of the gene was determined, and the active center of the enzyme was analyzed by means of site-directed mutagenesis. The catalytic residues were tentatively identified as two Asp residues and a Glu residue by comparison of the amino acid sequences of various branching enzymes from different sources and enzymes of the alpha-amylase family. When the Asp residues and Glu were replaced by Asn and Gln, respectively, the branching enzyme activities disappeared. The results suggested that these three residues are the catalytic residues and that the catalytic mechanism of the branching enzyme is basically identical to that of alpha-amylase. On the basis of these results, four conserved regions including catalytic residues and most of the substrate-binding residues of various branching enzymes are proposed.     

Role of aspartic acid 121 in human pancreatic ribonuclease catalysis

Bovine pancreatic ribonuclease (RNase A) is one of the most well studied enzymes of the ribonuclease family, unlike its human counterpart, the human pancreatic ribonuclease (HPR), whose physiological role in the body is not clearly understood. Human pancreatic ribonuclease consists of 128 amino acids and the main residues located in the active site of RNase A are also conserved in HPR. In the current study, to investigate the role of Asp-121 in the catalytic activity of human pancreatic ribonuclease, several variants were generated in which Asp-121 was either mutated to an alanine or C-terminal residues beyond Asp-121, and Phe-120 were deleted. The HPR mutants were cloned, expressed in E. coli and purified to homogeneity, and functionally characterized. The mutation D121A in HPR significantly decreased the rate of the enzymatic reaction, however this decrease was not universally observed for all substrates studied. Removal of the seven C-terminal amino acid residues thereby exposing Asp-121 yielded an HPR mutant with enhanced activity, however a further deletion removing Asp-121 resulted in the complete inactivation of HPR. Our results indicate that Asp-121 is crucial for the catalytic activity of HPR and may be involved in the depolymerization activity of the enzyme.     

Nucleotide sequence of the lipase gene lip3 from the antarctic psychotroph Moraxella TA144

A lipase gene (lip3) from the psychotrophic strain Moraxella TA144 has been cloned and sequenced. The deduced primary structure of the lipase preprotein is composed of 315 amino acids with a predicted Mr of 34,772. This enzyme contains two consensus peptides showing cluster of glycine residues that may be involved in domain flexibility. The cloned gene product conserves the low temperature activity and the thermolability properties of the wild enzyme.     

Site-directed mutagenesis study of yeast peptide:N-glycanase. Insight into the reaction mechanism of deglycosylation.

Yeast peptide:N-glycanase (Png1p; PNGase), a deglycosylation enzyme involved in the proteasome dependent degradation of proteins, has been reported to be a member of the transglutaminase superfamily based on sequence alignment. In this study we have investigated the structure-function relationship of Png1p by site-directed mutagenesis. Cys-191, His-218, and Asp-235 of Png1p are conserved in the sequence of factor XIIIa, where these amino acids constitute a catalytic triad. Point mutations of these residues in Png1p resulted in complete loss in activity, consistent with a role for each in catalyzing deglycosylation of glycoproteins. Other conserved amino acid residues, Trp-220, Trp-231, Arg-210, and Glu-222, were also vitally important for folding and structure stability of the enzyme as revealed by circular dichroism analysis. The potential effects of the mutations were predicted by mapping the conserved amino acids of Png1p within the known three-dimensional structure of factor XIIIa. Our data suggest that the lack in enzyme activity when any of the catalytic triad residues is mutated is either due to the absence of charge relay in the case of the triad or due to the disruption of the native fold of the enzyme. These findings strongly suggest a common evolutionary lineage for the PNGases and transglutaminases.     

Thermodynamic stability of a cold-active alpha-amylase from the Antarctic bacterium Alteromonas haloplanctis

The thermal stability of the cold-active alpha-amylase (AHA) secreted by the Antarctic bacterium Alteromonas haloplanctis has been investigated by intrinsic fluorescence, circular dichroism, and differential scanning calorimetry. It was found that this heat-labile enzyme is the largest known multidomain protein exhibiting a reversible two-state unfolding, as demonstrated by the recovery of DeltaHcal values after consecutive calorimetric transitions, a DeltaHcal/DeltaHeff ratio close to unity, and the independence of unfolding thermodynamic parameters of scan rates. By contrast, the mesophilic alpha-amylases investigated here (from porcine pancreas, human salivary glands, yellow meal beetle, Bacillus amyloliquefaciens, and Bacillus licheniformis) unfold irreversibly according to a non-two-state mechanism. Unlike mesophilic alpha-amylases, the melting point of AHA is independent of calcium and chloride binding while the allosteric and structural functions of these ions are conserved. The thermostability of AHA at optimal conditions is characterized by a Tm of 43.7 degrees C, a DeltaHcal of 238 kcal mol-1, and a DeltaCp of 8.47 kcal mol-1 K-1. These values were used to calculate the Gibbs free energy of unfolding over a wide range of temperatures. This stability curve shows that (a) the specific DeltaGmax of AHA [22 cal (mol of residue)-1] is 4 times lower than that of mesophilic alpha-amylases, (b) group hydration plays a crucial role in the enzyme flexibility at low temperatures, (c) the temperature of cold unfolding closely corresponds to the lower limit of bacterial growth, and (d) the recombinant heat-labile enzyme can be expressed in mesophilic hosts at moderate temperatures. It is also argued that the cold-active alpha-amylase has evolved toward the lowest possible conformational stability of its native state.     

Cloning, expression and characterization of trehalose-6-phosphate phosphatase from a psychrotrophic bacterium, Arthrobacter strain A3

A trehalose-6-phosphate phosphatase (TPP) gene, otsB, from a psychrotrophic bacterium, Arthrobacter strain A3, was identified. The product of this otsB gene is 266 amino acids in length with a calculated molecular weight of 27,873 Da. The protein was expressed in Escherichia coli and purified to apparent homogeneity. The purified recombinant TPP catalyzed the dephosphorylation of trehalose-6-phosphate to form trehalose and showed a broad optimum pH range from 5.0 to 7.5. This enzyme also showed an absolute requirement for Mg(2+) or Co(2+) for catalytic activity. The recombinant TPP had a maximum activity at 30 °C and maintained activity over a temperature range of 4-30 °C. TPP was generally heat-labile, losing 70 % of its activity when subjected to heat treatment at 50 °C for 6 min. Kinetic analysis of the Arthrobacter strain A3 TPP showed ~tenfold lower K (m) values when compared with values derived from other bacterial TPP enzymes. The highest k (cat)/K (m) value was 37.5 mM(-1) s(-1) (repeated three times), which is much higher than values published for mesophilic E. coli TPP, indicating that the Arthrobacter strain A3 TPP possessed excellent catalytic activity at low temperatures. Accordingly, these characteristics suggest that the TPP from the Arthrobacter strain A3 is a new cold-adapted enzyme. In addition, this is the first report characterizing the enzymatic properties of a TPP from a psychrotrophic organism.     

Characteristics of two forms of alpha-amylases and structural implication.

Complete (Ba-L) and truncated (Ba-S) forms of alpha-amylases from Bacillus subtilis X-23 were purified, and the amino- and carboxyl-terminal amino acid sequences of Ba-L and Ba-S were determined. The amino acid sequence deduced from the nucleotide sequence of the alpha-amylase gene indicated that Ba-S was produced from Ba-L by truncation of the 186 amino acid residues at the carboxyl-terminal region. The results of genomic Southern analysis and Western analysis suggested that the two enzymes originated from the same alpha-amylase gene and that truncation of Ba-L to Ba-S occurred during the cultivation of B. subtilis X-23 cells. Although the primary structure of Ba-S was approximately 28% shorter than that of Ba-L, the two enzyme forms had the same enzymatic characteristics (molar catalytic activity, amylolytic pattern, transglycosylation ability, effect of pH on stability and activity, optimum temperature, and raw starch-binding ability), except that the thermal stability of Ba-S was higher than that of Ba-L. An analysis of the secondary structure as well as the predicted three-dimensional structure of Ba-S showed that Ba-S retained all of the necessary domains (domains A, B, and C) which were most likely to be required for functionality as alpha-amylase.     

Analysis of the psychrotolerant property of hormone-sensitive lipase through site-directed mutagenesis

Mammalian hormone-sensitive lipase (HSL) has given its name to a family of primarily prokaryotic proteins which are structurally related to type B carboxylesterases. In many of these alpha/beta hydrolases, a conserved HG-dipeptide flanks the catalytic pocket. In HSL this dipeptide is followed by two additional glycine residues. Through site-directed mutagenesis, we have investigated the importance of this motif for enzyme activity. Since the presence of multiple glycine residues in a critical region could contribute to cold adaptation by providing local flexibility, we studied the effect of mutating these residues on the psychrotolerant property of HSL. Any double mutation rendered the enzyme completely inactive, without any major effect on the enzyme stability. The partially active single mutants retained the same proportion of activity at reduced temperatures as the wild-type enzyme. These results do not support a role for the HGGG motif in catalysis at low temperatures, but provide further validation of the current three-dimensional model of HSL. Rat HSL was found to be relatively more active than human HSL at low temperatures. This difference was, however, not due to the 12 amino acids which are present in the regulatory module of the rat enzyme but absent in human HSL.     

Mutagenesis of human acetylcholinesterase. Identification of residues involved in catalytic activity and in polypeptide folding.

Evidence for the involvement of Ser-203, His-447, and Glu-334 in the catalytic triad of human acetylcholinesterase was provided by substitution of these amino acids by alanine residues. Of 20 amino acid positions mutated so far in human acetylcholinesterase (AChE), these three were unique in abolishing detectable enzymatic activity (less than 0.0003 of wild type), yet allowing proper production, folding, and secretion. This is the first biochemical evidence for the involvement of a glutamate in a hydrolase triad (Schrag, J.D., Li, Y., Wu, M., and Cygler, M. (1991) Nature 351, 761-764), supporting the x-ray crystal structure data of the Torpedo californica acetylcholinesterase (Sussman, J.L., Harel, M., Frolow, F., Oefner, C., Goldman, A., Toker, L. and Silman, I. (1991) Science 253, 872-879). Attempts to convert the AChE triad into a Cys-His-Glu or Ser-His-Asp configuration by site-directed mutagenesis did not yield effective AChE activity. Another type of substitution, that of Asp-74 by Gly or Asn, generated an active enzyme with increased resistance to succinylcholine and dibucaine; thus mimicking in an AChE molecule the phenotype of the atypical butyrylcholinesterase natural variant (D70G mutation). Mutations of other carboxylic residues Glu-84, Asp-95, Asp-333, and Asp-349, all conserved among cholinesterases, did not result in detectable alteration in the recombinant AChE, although polypeptide productivity of the D95N mutant was considerably lower. In contrast, complete absence of secreted human AChE polypeptide was observed when Asp-175 or Asp-404 were substituted by Asn. These two aspartates are conserved in the entire cholinesterase/thyroglobulin family and appear to play a role in generating and/or maintaining the folded state of the polypeptide. The x-ray structure of the Torpedo acetylcholinesterase supports this assumption by revealing the participation of these residues in salt bridges between neighboring secondary structure elements.     

Extracellular protease from the antarctic yeast Candida humicola.

The psychrotrophic, dimorphic yeast Candida humicola, isolated from Antarctic soil, secretes an acidic protease into the medium. The secretion of this protease by C. humicola was found to be dependent on the composition of the medium. In YPD or yeast nitrogen base medium containing either amino acids or ammonium sulfate as the nitrogen source, the activity of the protease in the medium was low (basal level). However, when yeast nitrogen base medium was depleted of amino acids or ammonium sulfate and supplemented with proteins, the activity of the enzyme increased. The secretion of the enzyme was greater during exponential growth at low temperatures than during growth at higher temperatures. The purified protease had a molecular mass of 36,000 Da and was inhibited by pepstatin, iodoacetamide, and sodium dodecyl sulfate. Despite the prevalent cold temperatures in Antarctica, this extracellular protease of the psychrotrophic yeast C. humicola was active at temperatures ranging from 0 to 45 degrees C, with an optimum activity at 37 degrees C.