Isolation of a Bacillus stearothermophilus mutant exhibiting increased thermostability in its restriction endonuclease.

A procedure was developed for the selection of spontaneous mutants of Bacillus stearothermophilus NUB31 that are more efficient than the wild type in the restriction of phage at elevated temperatures. Inactivation studies revealed that two mutants contained a more thermostable restriction enzyme and one mutant contained three times more enzyme than the wild type. The restriction endonucleases from the wild type and one of the mutants were purified to apparent homogeneity. The mutant enzyme was more thermostable than the wild-type enzyme. The subunit molecular weight, amino acid composition, N-terminal and C-terminal amino acid residues, tryptic peptide map, and catalytic properties of the two enzymes were determined. The two enzymes have similar catalytic properties, but the molecular size of the mutant enzyme is approximately 6 to 7 kilodaltons larger than that of the wild-type enzyme. The mutant enzyme contains 54 additional amino acid residues, of which 26 to 28 are aspartate/asparagine, 8 to 15 are glutamate/glutamine, and 8 to 9 are tyrosine residues. The two enzymes contained similar amounts of the other amino acids, identical N-terminal residues, and different C-terminal residues. Tryptic peptide analyses revealed a high degree of homology between the two enzymes. The increased thermostability observed in the mutant enzyme appears to have been achieved by a mutation that resulted in the addition of amino acid residues to the wild-type enzyme. A number of mechanisms are discussed that could account for the observed difference between the mutant and wild-type enzymes.     

Deduced amino acid sequence of Escherichia coli adenosine deaminase reveals evolutionarily conserved amino acid residues: implications for catalytic function

The goal of the research reported here is to identify evolutionarily conserved amino acid residues associated with enzymatic deamination of adenosine. To do this, we isolated molecular clones of the Escherichia coli adenosine deaminase gene by functional complementation of adenosine deaminase deficient bacteria and deduced the amino acid sequence of the enzyme from the nucleotide sequence of the gene. Nucleotide sequence analysis revealed the presence of a 996-nucleotide open reading frame encoding a protein of 332 amino acids having a molecular weight of 36,345. The deduced amino acid sequence of the E. coli enzyme has approximately 33% identity with those of the mammalian adenosine deaminases. With conservative amino acid substitutions the overall sequence homology approaches 50%, suggesting that the structures and functions of the mammalian and bacterial enzymes are similar. Additional amino acid sequence analysis revealed specific residues that are conserved among all three adenosine deaminases and four AMP deaminases for which sequence information is currently available. In view of previously published enzymological data and the conserved amino acid residues identified in this study, we propose a model to account for the enzyme-catalyzed hydrolytic deamination of adenosine. Potential catalytic roles are assigned to the conserved His 214, Cys 262, Asp 295, and Asp 296 residues of mammalian adenosine deaminases and the corresponding conserved amino acid residues in bacterial adenosine deaminase and the eukaryotic AMP deaminases.     

Molecular cloning, expression, purification and characterization of calcineurin from bovine cardiac muscle

Calcineurin (CaN), also known as calmodulin-dependent phosphatase, was cloned from bovine cardiac muscle and the deduced amino acid sequences of CaN A revealed that it had an open reading frame of 511 amino acid residues. As compared to bovine brain CaN A, the cardiac enzyme contains a 10 amino acid (ATVEAIEADE) deletion before the autoinhibitory region. A deletion analysis of the catalytic domain revealed a 20% decrease in phosphatase activity when the N-terminal 200 amino acids were removed from CaN A as compared to the wild type enzyme. The C-terminal deletions of CaN A revealed that in addition to the autoinhibitory domain (residues 457-480), additional adjacent residues (407-456) also inhibited CaN activity. These results point to either a second autoinhibitory region within CaN A or an extension of the previously noted autoinhibitory region within the cardiac CaN A enzyme.     

Expression and characterization of catalytic and regulatory domains of rat tyrosine hydroxylase.

Phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase constitute a family of tetrahydropterin-dependent aromatic amino acid hydroxylases. Comparison of the amino acid sequences of these three proteins shows that the C-terminal two-thirds are homologous, while the N-terminal thirds are not. This is consistent with a model in which the C-terminal two-thirds constitute a conserved catalytic domain to which has been appended discrete regulatory domains. To test such a model, two mutant proteins have been constructed, expressed in Escherichia coli, purified, and characterized. One protein contains the first 158 amino acids of rat tyrosine hydroxylase. The second lacks the first 155 amino acid residues of this enzyme. The spectral properties of the two domains suggest that their three-dimensional structures are changed only slightly from intact tyrosine hydroxylase. The N-terminal domain mutant binds to heparin and is phosphorylated by cAMP-dependent protein kinase at the same rate as the holoenzyme but lacks any catalytic activity. The C-terminal domain mutant is fully active, with Vmax and Km values identical to the holoenzyme; these results establish that all of the catalytic residues of tyrosine hydroxylase are located in the C-terminal 330 amino acids. The results with the two mutant proteins are consistent with these two segments of tyrosine hydroxylase being two separate domains, one regulatory and one catalytic.     

Probing the essential catalytic residues and substrate affinity in the thermoactive Bacillus stearothermophilus US100 L-arabinose isomerase by site-directed mutagenesis

The L-arabinose isomerase (L-AI) from Bacillus stearothermophilus US100 is characterized by its high thermoactivity and catalytic efficiency. Furthermore, as opposed to the majority of l-arabinose isomerases, this enzyme requires metallic ions for its thermostability rather than for its activity. These features make US100 L-AI attractive as a template for industrial use. Based on previously solved crystal structures and sequence alignments, we identified amino acids that are putatively important for the US100 L-AI isomerization reaction. Among these, E306, E331, H348, and H447, which correspond to the suggested essential catalytic amino acids of the L-fucose isomerase and the L-arabinose isomerase from Escherichia coli, are presumed to be the active-site residues of US100 L-AI. Site-directed mutagenesis confirmed that the mutation of these residues resulted in totally inactive proteins, thus demonstrating their critical role in the enzyme activity. A homology model of US100 L-AI was constructed, and its analysis highlighted another set of residues which may be crucial for the recognition and processing of substrates; hence, these residues were subjected to mutagenesis studies. The replacement of the D308, F329, E351, and H446 amino acids with alanine seriously affected the enzyme activities, and suggestions about the roles of these residues in the catalytic mechanism are given. The mutation F279Q strongly increased the enzyme's affinity for L-fucose and decreased the affinity for L-arabinose compared to that of the wild-type enzyme, showing the implication of this amino acid in substrate recognition.     

A new carboxylesterase from Brevibacterium linens IFO 12171 responsible for the conversion of 1,4-butanediol diacrylate to 4-hydroxybutyl acrylate: purification, characterization, gene cloning, and gene expression in Escherichia coli.

A carboxylesterase that is responsible for conversion of 1,4-butanediol diacrylate (BDA) to 4-hydroxybutyl acrylate (4HBA) was found in Brevibacterium lines IFO 12171, and purified to homogeneity. The purified enzyme was active toward a variety of diesters of ethylene glycol, 1,4-butanediol, and 1,6-hexanediol. The K(m) and kcat of the enzyme for BDA were 3.04 mM and 203,000 s-1, respectively. The reaction with the purified enzyme gave 98 mM 4HBA from 100 mM BDA for 60 min. The enzyme gene was cloned from the chromosomal DNA of the bacterium. The open reading frame encoding the enzyme was 1176 bp long, corresponding to a protein of 393 amino acid residues (molecular mass = 42,569 Da). The deduced amino acid sequence contained the tetra peptide motif sequence, STTK, and the serine residue was confirmed to be the catalytic center of BDA esterase by site-directed mutagenesis for several amino acid residues. The gene was expressed in Escherichia coli under the control of the lac promoter, and the gene product (a fusion protein with 6 amino acid residues from beta-galactosidase) showed the same catalytic properties as the enzyme from the parent strain.     

The chemistry of protein catalysis

We report, for the first time, on the statistics of chemical mechanisms and amino acid residue functions that occur in enzyme reaction sequences using the MACiE database of 202 distinct enzyme reaction mechanisms as a knowledge base. MACiE currently holds representatives from each Enzyme Commission sub-subclass where there is an available crystal structure and sufficient evidence in the primary literature for a mechanism. Each catalytic step of every reaction sequence in MACiE is fully annotated, so that it includes the function of the catalytic residues involved in the reaction and the chemical mechanisms by which substrates are transformed into products. We show that the most catalytic amino acid residues are histidine, cysteine and aspartate, which are also the residues whose side-chains are more likely to serve as reactants, and that have the greatest versatility of function. We show that electrophilic reactions in enzymes are very rare, and the majority of enzyme reactions rely upon nucleophilic and general acid/base chemistry. However, although rare, radical (homolytic) reactions are much more common than electrophilic reactions. Thus, the majority of amino acid residues perform stabilisation roles (as spectators) or proton shuttling roles (as reactants). The analysis presented provides a better understanding of the mechanisms of enzyme catalysis and may act as an initial step in the validation and prediction of mechanism in an enzyme active site.     

The kinetic analysis of recognition of the damaged nucleotides by mutant forms of the 8-oxoguanine DNA glycosylase hOGG1

We have investigated the role of Tyr-203, His-270, and Lys-249 amino acid residues from the 8-oxoguanine glycosylase (hOGG1) active site in the process of recognition of 7,8-dihydro-8-oxoguanine (oxoG) damaged nucleotide and in the catalytic stages of enzymatic reaction. The pre-steady state kinetic analysis of conformational transitions of mutant forms of the enzyme and model DNA substrates during the enzymatic process revealed that the studied amino acid residues are involved in the specific binding of DNA substrates. The Tyr-203 is responsible for recognition of the damaged nucleotide; interaction between His-270 and DNA is necessary for the formation of the catalytically active complex with the oxoG-containing DNA. The Lys-249 acts not only as one of the catalytically important amino acids of the active site of the enzyme, but also plays a significant role in the formation of specific enzyme–substrate complex. The present study significantly complements the molecular-kinetic model of the enzymatic reaction and helps to clarify the origin of the high specificity of hOGG1 to oxidized bases in DNA.     

Site-directed mutagenesis of glutathione S-transferase YaYa: nonessential role of histidine in catalysis

A cDNA encoding a rat liver glutathione S-transferase Ya subunit has been expressed in Escherichia coli and the expressed enzyme purified to homogeneity. In order to examine the catalytic role of histidine in the glutathione S-transferase Ya homodimer, site-directed mutagenesis was used to replace all three histidine residues (at positions 8, 143, and 159) by other amino acid residues. The replacement of histidine 8 or histidine 143 with valine did not affect the 1-chloro-2,4-dinitrobenzene-conjugating activity nor the isomerase activity. However, the replacement of histidine with valine at position 159 produced the mutant GST which exhibited only partial activity. A greater decrease in catalytic activity was observed by histidine----tyrosine or histidine----lysine replacement at position 159. On the other hand, the histidine 159----asparagine mutant retained full catalytic activity. Our results indicate that histidine residues in the Ya homodimer are not essential for catalytic activity. However, histidine 159 might be critical in maintaining the proper conformation of this enzyme since replacement of this amino acid by either lysine or tyrosine did result in significant loss of enzymatic activity.     

Structural modeling and site-directed mutagenesis of the actinorhodin beta-ketoacyl-acyl carrier protein synthase

A three-dimensional model of the Streptomyces coelicolor actinorhodin beta-ketoacyl synthase (Act KS) was constructed based on the X-ray crystal structure of the related Escherichia coli fatty acid synthase condensing enzyme beta-ketoacyl synthase II, revealing a similar catalytic active site organization in these two enzymes. The model was assessed by site-directed mutagenesis of five conserved amino acid residues in Act KS that are in close proximity to the Cys169 active site. Three substitutions completely abrogated polyketide biosynthesis, while two replacements resulted in significant reduction in polyketide production. (3)H-cerulenin labeling of the various Act KS mutant proteins demonstrated that none of the amino acid replacements affected the formation of the active site nucleophile.     

酸性木聚糖酶XynⅡ活性中心关键氨基酸残基的鉴定

目的:鉴定来源于宇佐美曲霉(Aspergillus usamii)E001的酸性木聚糖酶XynⅡ活性中心关键氨基酸残基。方法:对XynⅡ进行SWISS-MODEL同源建模和BLAST序列比较,分析XynⅡ中所有可能作为催化残基的保守氨基酸,采用定点突变手段对其进行鉴定研究。结果:只有Glu-79和Glu-170位于酶与底物作用的活性中心,它们分别位于β折叠股B6和B4上,推测Glu-79和Glu-170为XynⅡ活性中心关键氨基酸残基。将Glu-79和Glu-170突变为酸性的Gln,突变酶E79Q,E170Q在大肠杆菌和毕赤酵母中表达后,活性均丧失。结论:79位、170位Glu是木聚糖酶XynⅡ活性中心的关键氨基酸残基,为该酶进一步的结构与功能研究提供了理论基础。     

Cloning and sequencing of the gene coding for alcohol dehydrogenase of Bacillus stearothermophilus and rational shift of the optimum pH.

Using Bacillus subtilis as a host and pTB524 as a vector plasmid, we cloned the thermostable alcohol dehydrogenase (ADH-T) gene (adhT) from Bacillus stearothermophilus NCA1503 and determined its nucleotide sequence. The deduced amino acid sequence (337 amino acids) was compared with the sequences of ADHs from four different origins. The amino acid residues responsible for the catalytic activity of horse liver ADH had been clarified on the basis of three-dimensional structure. Since those catalytic amino acid residues were fairly conserved in ADH-T and other ADHs, ADH-T was inferred to have basically the same proton release system as horse liver ADH. The putative proton release system of ADH-T was elucidated by introducing point mutations at the catalytic amino acid residues, Cys-38 (cysteine at position 38), Thr-40, and His-43, with site-directed mutagenesis. The mutant enzyme Thr-40-Ser (Thr-40 was replaced by serine) showed a little lower level of activity than wild-type ADH-T did. The result indicates that the OH group of serine instead of threonine can also be used for the catalytic activity. To change the pKa value of the putative system, His-43 was replaced by the more basic amino acid arginine. As a result, the optimum pH of the mutant enzyme His-43-Arg was shifted from 7.8 (wild-type enzyme) to 9.0. His-43-Arg exhibited a higher level of activity than wild-type enzyme at the optimum pH.     

Understanding the Functional Roles of Amino Acid Residues in Enzyme Catalysis

The MACiE database contains 223 distinct step-wise enzyme reaction mechanisms and holds representatives from each EC sub-subclass where there is a crystal structure and sufficient evidence in the literature to support a mechanism. Each catalytic step of every reaction sequence in MACiE is fully annotated so that it includes the function of the catalytic residues involved in the reaction and the mechanism by which substrates are transformed into products. Using MACiE as a knowledge base, we have seen that the top 10 most catalytic residues are histidine, aspartate, glutamate, lysine, cysteine, arginine, serine, threonine, tyrosine and tryptophan. Of these only seven (cysteine, histidine, aspartate, lysine, serine, threonine and tyrosine) dominate catalysis and provide essentially five functional roles that are essential. Stabilisation is the most common and essential role for all classes of enzyme, followed by general acid/base (proton acceptor and proton donor) functionality, with nucleophilic addition following closely behind (nucleophile and nucleofuge). We investigated the occurrence of these residues in MACiE and the Catalytic Site Atlas and found that, as expected, certain residue types are associated with each functional role, with some residue types able to perform diverse roles. In addition, it was seen that different EC classes of enzyme have a tendency to employ different residues for catalysis. Further, we show that whilst the differences between EC classes in catalytic residue composition are not immediately obvious from the general classes of Ingold mechanisms, there is some weak correlation between the mechanisms involved in a given EC class and the functions that the catalytic amino acid residues are performing. The analysis presented here provides a valuable insight into the functional roles of catalytic amino acid residues, which may have applications in many aspects of enzymology, from the design of novel enzymes to the prediction and validation of enzyme reaction mechanisms.     

Comparison of amino acid sequences between phosphoenolpyruvate carboxylases from Escherichia coli (allosteric) and Anacystis nidulans (non-allosteric): identification of conserved and variable regions

Amino acid sequences of phosphoenolpyruvate carboxylases of Escherichia coli (allosteric) and a cyanobacterium Anacystis nidulans (non-allosteric) were aligned. The pattern of homology suggests that the enzyme molecule is comprised of two distinct regions, namely, a conserved region (C-terminal half) and a variable region (N-terminal half). Among the amino acid residues which have previously been presumed essential for the catalytic activity, three histidine residues were found to be conserved, but cysteine residues were not. Furthermore, the conserved sequence unique to the enzyme was identified by comparison of the enzyme sequence with amino acid sequences in our data bank.     

Divergent allosteric patterns verify the regulatory paradigm for aspartate transcarbamylase

The native Escherichia coli aspartate transcarbamoylase (ATCase, E.C. 2.1.3.2) provides a classic allosteric model for the feedback inhibition of a biosynthetic pathway by its end products. Both E. coli and Erwinia herbicola possess ATCase holoenzymes which are dodecameric (2(c3):3(r2)) with 311 amino acid residues per catalytic monomer and 153 and 154 amino acid residues per regulatory (r) monomer, respectively. While the quaternary structures of the two enzymes are identical, the primary amino acid sequences have diverged by 14 % in the catalytic polypeptide and 20 % in the regulatory polypeptide. The amino acids proposed to be directly involved in the active site and nucleotide binding site are strictly conserved between the two enzymes; nonetheless, the two enzymes differ in their catalytic and regulatory characteristics. The E. coli enzyme has sigmoidal substrate binding with activation by ATP, and inhibition by CTP, while the E. herbicola enzyme has apparent first order kinetics at low substrate concentrations in the absence of allosteric ligands, no ATP activation and only slight CTP inhibition. In an apparently important and highly conserved characteristic, CTP and UTP impose strong synergistic inhibition on both enzymes. The co-operative binding of aspartate in the E. coli enzyme is correlated with a T-to-R conformational transition which appears to be greatly reduced in the E. herbicola enzyme, although the addition of inhibitory heterotropic ligands (CTP or CTP+UTP) re-establishes co-operative saturation kinetics. Hybrid holoenzymes assembled in vivo with catalytic subunits from E. herbicola and regulatory subunits from E. coli mimick the allosteric response of the native E. coli holoenzyme and exhibit ATP activation. The reverse hybrid, regulatory subunits from E. herbicola and catalytic subunits from E. coli, exhibited no response to ATP. The conserved structure and diverged functional characteristics of the E. herbicola enzyme provides an opportunity for a new evaluation of the common paradigm involving allosteric control of ATCase.     

DNA aptamers as potential anti-HIV agents

Guanine (G)-rich DNA sequences can adopt stable G-quadruplex structures by G-tetrad hydrogen-bonding and hydrophobic stacking. Recently, it has been shown that a DNA sequence forms an aptamer (termed 93del) and adopts a novel dimeric quadruplex folding topology in K+ solution. This aptamer exhibits anti-HIV1 integrase activity in the nanomolar range in vitro. A docking-based model of the 93del-integrase complex positions the DNA aptamer within a channel of the tetrameric integrase. This mutual fitting blocks several catalytic amino acid residues that are essential for integrase function, and accounts for the anti-HIV1 activity of the 93del aptamer.     

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.     

A self-stabilized model of the chymotrypsin catalytic pocket. The energy profile of the overall catalytic cycle

A model of the catalytic triad of chymotrypsin is built assuring the arrangement and properties as they are within the complete enzyme. The model contains 18 amino acid residues of chymotrypsin and its substrate. A total of 135 atoms (including 70 heavy atoms) were subjected to full ab initio geometry optimizations through 127 individual steps along the reaction coordinate of the complete catalytic mechanism. It was shown that the described model of the catalytic apparatus forms a self-stabilized molecule ensemble without the rest of the enzyme and substrate. According to the calculations, the formations of the first and second tetrahedral intermediates in the model have 20.3 and 15.7 kcal/mol activation energy barriers, respectively. Removing elements of the catalytic apparatus such as the (1) catalytic aspartate or (2) the anion hole, as well as (3) inserting a water molecule "early" in the catalytic process, or (4) introducing conformational rigidity of the substrate, results in an increase of the above energy barrier of the first catalytic step in the model by 6.4, 13.7, 3.7, and 4.1 kcal/mol, respectively. Based on the calculated process one can conclude that the catalytic reaction in this model is much more similar to the reaction in the enzyme than to the reference reaction. To our knowledge, this is the first model system that mimics the complete catalytic mechanism.     

Active site residue involvement in monoamine or diamine oxidation catalysed by pea seedling amine oxidase

The structures of copper amine oxidases from various sources show good similarity, suggesting similar catalytic mechanisms for all members of this enzyme family. However, the optimal substrates for each member differ, depending on the source of the enzyme and its location. The structural factors underlying substrate selectivity still remain to be discovered. With this in view, we examined the kinetic behaviour of pea seedling amine oxidase with cadaverine and hexylamine, the first bearing two, and the second only one, positively charged amino group. The dependence of K(m) and catalytic constant (k(c)) values on pH, ionic strength and temperature indicates that binding of the monoamine is driven by hydrophobic interactions. Instead, binding of the diamine is strongly facilitated by electrostatic factors, controlled by polar side-chains and two titratable residues present in the active site. The position of the docked substrate is also essential for the participation of titratable amino acid residues in the following catalytic steps. A new mechanistic model explaining the substrate-dependent kinetics of the reaction is discussed.     

Analysis of the mechanism of the Serratia nuclease using site-directed mutagenesis.

Based on crystal structure analysis of the Serratia nuclease and a sequence alignment of six related nucleases, conserved amino acid residues that are located in proximity to the previously identified catalytic site residue His89 were selected for a mutagenesis study. Five out of 12 amino acid residues analyzed turned out to be of particular importance for the catalytic activity of the enzyme: Arg57, Arg87, His89, Asn119 and Glu127. Their replacement by alanine, for example, resulted in mutant proteins of very low activity, < 1% of the activity of the wild-type enzyme. Steady-state kinetic analysis of the mutant proteins demonstrates that some of these mutants are predominantly affected in their kcat, others in their Km. These results and the determination of the pH and metal ion dependence of selected mutant proteins were used for a tentative assignment for the function of these amino acid residues in the mechanism of phosphodiester bond cleavage by the Serratia nuclease.