Creation of a ribonuclease abzyme through site-directed mutagenesis

The development of abzymes (antibody/enzymes) is one method of creating reagents with novel catalytic activity. To date, most abzymes have been obtained by immunization with transition state analogs. We have chosen to start with an existing antibody and convert it into an enzyme by the addition of catalytic residues to the binding site. We have introduced a histidine residue into antibody Jel 103 and converted it into an abzyme that cleaves poly(rI) with a kinetic efficiency of about 100 M(-1) sec(-1).     

Catalytic efficiency of expressed aromatase following site-directed mutagenesis

A solid and cystic tumor (SCT) of the pancreas occurring in a 35-yr-old male is reported. Cut sections of the specimen revealed a solid, ill-defined mass measuring 2.5 x 2.3 x 2.0 cm, without cystic or necrotic changes. Histologically, the solid tumor consisted of small, round acidophilic cells invading the surrounding pancreatic parenchyma. The tumor cells were positive for alpha-1-antitrypsin and neuron-specific-enolase. Ultrastructural studies revealed clear nuclei with no zymogen, but immature secretory granules in the cytoplasm of the tumor cells, which had a junctional complex-like structure. These findings were consistent with the so-called solid and cystic tumor of the pancreas. There was neither a capsule surrounding the tumor nor a papillary structure, known to be characteristic findings of the SCT tumor. The small tumor reported in the present article might represent an early-stage SCT of the pancreas.     

Computational site-directed mutagenesis of haloalkane dehalogenase in position 172

Laparoscopic cholecystectomy is considered as the new gold standard operation for removal of the gallbladder, and has several advantages over the traditional open cholecystectomy. However, in the last few years there is an increasing number of case reports of port site metastases following laparoscopic cholecystectomy for unsuspected carcinoma of the gallbladder. Two case reports of trocar site metastases are presented, and they further highlight the concern of the role of minimal invasive surgery in the presence of unsuspected carcinoma of the gallbladder. In this review we speculate on the mechanisms which may be responsible for metastatic deposits during laparoscopic cholecystectomy and suggest certain recommendations.     

Human lysosomal acid lipase/cholesteryl ester hydrolase and human gastric lipase: site-directed mutagenesis of Cys227 and Cys236 results in substrate-dependent reduction of enzymatic activity

Chemical modification studies and site-directed mutagenesis experiments have provided evidence that human lysosomal acid lipase/cholesteryl ester hydrolase (HLAL), human gastric lipase (HGL), and rat lingual lipase (RLL) are serine esterases. Loss of HLAL and HGL activity was also observed in the presence of sulfhydryl-reactive substances, suggesting that cysteines are likewise essential for substrate hydrolysis. To study the functional role of the HLAL and HGL cysteine residues, we replaced these amino acids with alanine by site-directed mutagenesis. Substitutions at positions 227 and 236, alone or together, drastically reduced hydrolytic activity in a substrate-dependent manner while the other mutants were not affected to any great extent. HLAL(Cys227-->Ala), HLAL(Cys236-->Ala), and HLAL(Cys227-->Ala, Cys236-->Ala) were essentially inactive against cholesteryl oleate, but retained about 23-39%, 28-37%, and 13-17% of catalytic activity for both triolein and tributyrin, respectively. The data obtained with the corresponding HGL mutants confirmed the importance of residues 227 and 236 in maintaining enzymatic activity towards long- and short-chain triglycerides. In order to assess the contribution of the eight amino acids delimited by Cys227 and Cys236 to lipolysis, we generated HLAL replacement mutants containing the corresponding residues 228-235 of HGL or RLL. Both HLAL chimeras were catalytically active towards all three substrates analyzed, indicating that these amino acids do not determine HLAL substrate specificity. Deletion of the eight-amino acid alpha-helix as well as disruption of its hydrophobic surface, in contrast, abolished enzymatic activity. Our studies suggest that Cys227, Cys236, and the amphipathic helix formed by residues 228-235 are essential for HLAL- and HGL-mediated neutral lipid catabolism.     

Production of extracellular acidic lipase by Rhizopus arrhizus as a function of culture conditions

Thermophilic strain of Rhizopus arrhizus accumulates an acidic lipase in culture fluid when grown in a medium containing ground nut oil, milk powder and inorganic salts. Addition of 2.0% ground nut oil yielded the highest productivity of enzyme. Soyabean meal and arabinose were found to be the best nitrogen and carbon sources for enzyme production respectively. Addition of metal ions such as MnCl2, SnCl2 and CaCl2 increased the enzyme productivity by 4 fold. The enzyme productivity in the fermenter was much higher (310 U/ml) than in shake-flask (180 U/ml). Crude lipase preparation showed pH and temperature activity optima at 3.5 and 45 degrees C respectively. The enzyme is thermostable and highly active in hydrolysing triglycerides and failed to hydrolyse-methyl esters of caprylate and palmitate.     

Escherichia coli inorganic pyrophosphatase: site-directed mutagenesis of the metal binding sites

Aspartic acids 65, 67, 70, 97 and 102 in the inorganic pyrophosphatase of Escherichia coli, identified as evolutionarily conserved residues of the active site, have been replaced by asparagine. Each mutation was found to decrease the k(app) value by approx. 2-3 orders of magnitude. At the same time, the Km values changed only slightly. Only minor changes take place in the pK values of the residues essential for both substrate binding and catalysis. All mutant variants have practically the same affinity to Mg2+ as the wild-type pyrophosphatase.     

Absence of opioid stress-induced analgesia in mice lacking beta-endorphin by site-directed mutagenesis

A physiological role for beta-endorphin in endogenous pain inhibition was investigated by targeted mutagenesis of the proopiomelanocortin gene in mouse embryonic stem cells. The tyrosine codon at position 179 of the proopiomelanocortin gene was converted to a premature translational stop codon. The resulting transgenic mice display no overt developmental or behavioral alterations and have a normally functioning hypothalamic-pituitary-adrenal axis. Homozygous transgenic mice with a selective deficiency of beta-endorphin exhibit normal analgesia in response to morphine, indicating the presence of functional mu-opiate receptors. However, these mice lack the opioid (naloxone reversible) analgesia induced by mild swim stress. Mutant mice also display significantly greater nonopioid analgesia in response to cold water swim stress compared with controls and display paradoxical naloxone-induced analgesia. These changes may reflect compensatory upregulation of alternative pain inhibitory mechanisms.     

Transposon-mediated random insertions and site-directed mutagenesis prevent the trafficking of a mouse mammary tumor virus superantigen

Mouse mammary tumor viruses (MMTVs) encode superantigens (Sags) which are critical to the life cycle of infectious virus and can mediate extensive deletion of T lymphocytes when expressed by endogenous proviruses. Little is known about the structure, intracellular trafficking, or nature of Sag association with major histocompatibility (MHC) class II products. In order to gain a better understanding of Sag structure-function relationships, we extensively mutagenized this type II glycoprotein using two different approaches: transposon-mediated random in-frame insertion mutagenesis and site-directed mutagenesis targeting clusters of charged residues. We find that 31 codon insertions are infrequently tolerated in Mtv-7 Sag, with just 1 of 14 insertion mutants functionally presented on the surface of B cells. Surprisingly, similar effects were observed with Sag mutants with substitutions at pairs of charged residues; only 2 of 6 mutants trafficked to the plasma membrane and stimulated T cells, 1 with a temperature-sensitive phenotype. The data suggest that the nonfunctional Mtv-7 Sag mutants are stringently retained in the endoplasmic reticulum due to conformational defects rather than disrupted interactions with MHC class II, thus identifying charged amino acids critical to the structural stability of viral superantigens.     

Prolyl aminopeptidase is not a sulfhydryl enzyme: identification of the active serine residue by site-directed mutagenesis

Prolyl aminopeptidase (PAP) has been classified as a sulfhydryl enzyme on the basis of its high sensitivity to p-chloromercuribenzoate and heavy metals. Recently, however, the possibility of PAP being instead a serine enzyme has been raised as a result of two observations--the conservation of some residues among the PAPs hitherto sequenced, and a similarity to some serine hydrolases. This is the first report describing an attempt to identify the active residue by site-directed mutagenesis. The pap genes from Bacillus coagulans and Aeromonas sobria, were used for the study. The changes made were Cys62Ser and Ser101Ala for the first enzyme, and Thr92Ala and Ser146Ala for the second. For both enzymes, only the changes made on the serine residues resulted in their complete inactivation, indicating that PAP is a serine peptidase.     

N-terminal protease of pestiviruses: identification of putative catalytic residues by site-directed mutagenesis

Pestiviruses are the only members of the Flaviviridae that encode a nonstructural protease at the N terminus of their polyproteins. This N-terminal protease (Npro) cleaves itself off of the nascent polyprotein autocatalytically and thereby generates the N terminus of the adjacent viral capsid protein C. In previous reports, sequence similarities between Npro and the catalytic residues of papain-like cysteine proteases were put forward. To test this hypothesis, substitutions of cysteine and histidine residues within Npro were carried out by site-directed mutagenesis. Translation of the mutagenized Npro-C proteins in cell-free lysates confirmed that only the predicted Cys69 was an essential amino acid for proteolysis, not His130. Further essential residues were identified with His49 and Glu22. While it remains speculative whether Glu22-His49-Cys69 actually build a catalytic triad, these results invalidate the assumption that Npro is a papain-like cysteine protease.     

Identification of the veratryl alcohol binding site in lignin peroxidase by site-directed mutagenesis

Site-directed mutagenesis was used to identify the veratryl alcohol binding site of lignin peroxidase. The cDNA encoding isozyme H8 was mutated at Glu146 to both an Ala and a Ser residue. The H8 polypeptide was produced by E. coli as inclusion bodies and refolded to yield active enzyme. The wild type recombinant enzyme and the mutants were purified to homogeneity and characterized by steady state kinetics. The kcat is decreased for both mutants of Glu146. The reactivity of mutants (kcat/Km) toward H2O2 were not affected. In contrast, the kcat/Km of the mutants for veratryl alcohol were decreased by at least half. The oxidation of guaiacol by these mutants were more significantly affected. These results collectively suggest that E146 plays a central role in the binding of veratryl alcohol by lignin peroxidase.     

Functional characterization of the N-glycosylation sites of human acid sphingomyelinase by site-directed mutagenesis

Most soluble lysosomal enzymes require a mannose-6-phosphate recognition marker present on asparagine-linked oligosaccharides for proper targeting to lysosomes. We have determined the influence of the six potential N-linked oligosaccharide chains of human acid sphingomyelinase (ASM) on catalytic activity, targeting, and processing of the enzyme. Each N-glycosylation site was modified by site-directed mutagenesis and subsequently expressed in COS-1 cells. Evidence is presented that five of these sites are used. Elimination of the four N-terminal glycosylation sites does not disturb lysosomal targeting, processing, or enzymatic activity. However, removal of the two C-terminal N-glycosylation sites inhibits the formation of mature enzyme. Absence of glycosylation site five resulted in rapid cleavage of the primary translation product to an enzymatically inactive protein which accumulated inside the endoplasmic reticulum/Golgi, whereas deletion of glycosylation site six led to the formation of an inactive ASM precursor, also retained inside the endoplasmic reticulum/Golgi. Our results also provide evidence that the site of early proteolytic cleavage of newly synthesized ASM must be located between the second and third glycosylation sites.     

Structure and properties of pyruvate decarboxylase and site-directed mutagenesis of the Zymomonas mobilis enzyme

Pyruvate decarboxylase (EC 4.1.1.1) is a thiamin diphosphate-dependent enzyme that catalyzes the penultimate step in alcohol fermentation. The enzyme is widely distributed in plants and fungi but is rare in prokaryotes and absent in animals. Here we review its structure and properties with particular emphasis on how site-directed mutagenesis of the enzyme from Zymomonas mobilis has assisted us to understand the function of critical residues.     

ATP-binding site of human brain hexokinase as studied by molecular modeling and site-directed mutagenesis

The interaction of ATP with the active site of hexokinase is unknown since the crystal structure of the hexokinase-ATP complex is unavailable. It was found that the ATP binding site of brain hexokinase is homologous to that of actin, heat shock protein hsc70, and glycerol kinase. On the basis of these similarities, the ATP molecule was positioned in the catalytic domain of human brain hexokinase, which was modeled from the X-ray structure of yeast hexokinase. Site-directed mutagenesis was performed to test the function of residues presumably involved in interaction with the tripolyphosphoryl moiety of ATP. Asp532, which is though to be involved in binding the Mg2+ ion of the MgATP2- complex, was mutated to Lys and Glu. The kcat values decreased 1000- and 200-fold, respectively, for the two mutants. Another residue, Thr680 was proposed to interact with the gamma-phosphoryl group of ATP through hydrogen bonds and was mutated to Val and Ser. The kcat value of the Thr680Val mutant decreased 2000-fold, whereas the kcat value of the Thr680Ser decreased only 2.5-fold, implying the importance of the hydroxyl group. The Km and dissociation constant values for either ATP or glucose of all the above mutants showed little or no change relative to the wild-type enzyme. The Ki values for the glucose 6-phosphate analogue 1,5-anhydroglucitol 6-phosphate, were the same as that of the wild-type enzyme, and the inhibition was reversed by inorganic phosphate (Pi) for all four mutants. The circular dichroism spectra of the mutants were the same as that of the wild-type enzyme. The results from the site-directed mutagenesis demonstrate that the presumed interactions of investigated residues with ATP are important for the stabilization of the transition state.     

Isolation of Cry1Ab protein mutants of Bacillus thuringiensis by a highly efficient PCR site-directed mutagenesis system

A site-directed mutagenesis method was designed and used to create Cry1Ab mutant proteins in two of the five highly conserved blocks present in the Cry protein family. Region 1 comprises the central alpha-helix 5 of domain I and has been implicated in the pore formation activity of the toxin. Substitution of arginine by serine at position 173 (R173S) affects neither structural integrity nor toxicity. Region 2 comprises the major part of the domain I/domain II interface, characterized by the presence of numerous hydrogen bonds and electrostatic interactions. Mutations in the salt bridge formed by aspartic acid 242 and arginine 265 (D242N, D242C, R265C, and D242C/R265C) resulted in structurally unstable mutant proteins as is shown by their increased protease sensitivity and lack of biological activity.     

Investigation of a catalytic zinc binding site in Escherichia coli L-threonine dehydrogenase by site-directed mutagenesis of cysteine-38

We studied block of the internal pore of the ROMK1 inward-rectifier K+ channel by Mg2+ and five quaternary ammoniums (tetramethylammonium, tetraethylammonium, tetrapropylammonium, tetrabutylammonium, and tetrapentylammonium). The apparent affinity of these blockers varied as a function of membrane voltage. As a consequence, the channel conducted K+ current more efficiently in the inward than the outward direction; i.e., inward rectification. Although the size of some monovalent quaternary ammoniums is rather large, the zdelta values (which measure voltage dependence of their binding to the pore) were near unity in symmetric 100 mM K+. Furthermore, we observed that not only the apparent affinities of the blockers themselves, but also their dependence on membrane voltage (or zdelta), varied as a function of the concentration of extracellular K+. These results suggest that there is energetic coupling between the binding of blocking and permeating (K+) ions, and that the voltage dependence of channel blockade results, at least in part, from the movement of K+ ions in the electrical field. A further quantitative analysis of the results explains why the complex phenomenon of inward rectification depends on both membrane voltage and the equilibrium potential for K+.     

Nucleoside diphosphate kinase. Investigation of the intersubunit contacts by site-directed mutagenesis and crystallography

NDP kinase from Dictyostelium was mutated by site-directed mutagenesis at positions indicated by structural data to be involved in the trimer interface. The mutants were substitutions at residue Pro-100 (P100S and P100G) and deletions of 1-5 residues at the C terminus. Single mutants yielded proteins that kept both activity and hexameric structure. However, they were severely affected in their stability toward temperature and urea denaturation. When the P100S mutation was combined with any of the C-terminal deletions, the enzyme lost most of its activity and dissociated into dimers. Crystallographic analysis of the P100S protein was performed at 2.6 A resolution. The x-ray structure showed no direct alteration of intersubunits contacts at residue 100, but an induced disruption of the interaction between Asp-115 and the C terminus of another subunit. The substitution of proline 100 to serine corresponds to the Killer-of-prune mutation in Drosophila. Consequences of the mutation are discussed in view of the structural and biochemical properties observed in the mutant Dictyostelium protein.     

The structure of serine hydroxymethyltransferase as modeled by homology and validated by site-directed mutagenesis

We describe a model for the three-dimensional structure of E. coli serine hydroxymethyltransferase based on its sequence homology with other PLP enzymes of the alpha-family and whose tertiary structures are known. The model suggests that certain amino acid residues at the putative active site of the enzyme can adopt specific roles in the catalytic mechanism. These proposals were supported by analysis of the properties of a number of site-directed mutants. New active site features are also proposed for further experimental testing.     

High-level production, chemical modification and site-directed mutagenesis of a cephalosporin C acylase from Pseudomonas strain N176

A cephalosporin acylase from Pseudomonas strain N176 hydrolyses both 7-beta-(4-carboxybutanamido)-cephalosporanic acid (glutarylcephalosporanic acid) and cephalosporin C to 7-amino-cephalosporanic acid. However, its productivity in the original host was low and its activity against cephalosporin C was not sufficient for direct large-scale production of 7-amino-cephalosporanic acid. In order to overcome these problems, we established a high-level expression system for the acylase in Escherichia coli. Tyr270 in the acylase is reported to play an important role in the interaction with glutarylcephalosporanic acid, as determined from the reaction with an affinity-label reagent, 7 beta-(6-bromohexanoylamido) cephalosporanic acid [Ishii, Y., Saito, Y., Sasaki, H., Uchiyama, F., Hayashi, M., Nakamura, S. & Niwa, M. (1994) J. Ferment. Bioeng. 77, 598-603] and modification with tetranitromethane [Nobbs, T. J., Ishii, Y., Fujimura, T., Saito, Y. & Niwa, M. (1994) J. Ferment. Bioeng. 77, 604-609]. From carbamoylation with potassium cyanate and site-directed point mutagenesis of the cephalosporin C acylase, we have deduced that Tyr270 exists at a position where it can interact with a residue (possibly Ser239) corresponding to inactivation by carbamoylation. We mutated Met269 and Ala271 of the acylase and found that mutation of Met269 to Tyr or Phe caused a 1.6-fold and 1.7-fold increase, respectively, of specific activity against cephalosporin C as compared to that of the wild-type enzyme. Kinetic studies of these mutants revealed that their kcat values increased, although their Km values against cephalosporin C were not changed. These data indicate that the mutation of Met269 near Tyr270 induces a minor conformational change to increase the stability of the activated complex with the enzyme and cephalosporin C. In particular, a mutant in which Met269 was replaced by Tyr was 2.5-fold more efficient in converting cephalosporin C to 7-amino-cephalosporanic acid than the wild-type enzyme under conditions similar to those in a bio-reactor system.     

Analysis of glutamines in catalysis in Cephalosporium acremonium isopenicillin N synthase by site-directed mutagenesis

Isopenicillin N synthase (IPNS), an important enzyme in the beta-lactam antibiotic biosynthetic pathway, is responsible for the catalytic conversion of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N. Three catalytic ligands essential for IPNS activity have already been determined. Based on an Aspergillus nidulans IPNS crystal structure, the probable involvement of a fourth amino acid as a catalytic ligand was previously revealed. To continue the search for the fourth catalytic ligand, we report investigations on whether or not glutamines play a role in the catalytic action of Cephalosporium acremonium IPNS (cIPNS). Three glutamine residues were targeted for modification based on the previous revelation of one (Q337) via crystal structure coordinates, the conservation of one (Q234) in isozyme alignment and the proximity of one (Q227) to the catalytic centre. Analysis of the biotransformed mutant enzymes showed retention of activity, thereby rejecting the involvement of a possible glutamine as a catalytic ligand in cIPNS catalysis.