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PubMed1998

The enhancing of a cysteine proteinase activity at acidic pH by protein engineering, the role of glutamic 50 in the enzyme mechanism of caricain.

Carica papaya produces four cysteine proteinases. Calculations show that the Cys25, His159 essential ion pair is fully ionised at pH 2.99, where activity cannot be detected, but apparently an additional ionisation with a pKa of 4 is essential for activity (an electrostatic switch). Caricain (EC 3.4.22.30) wt and D158E genetic backgrounds were used to study the contribution of E50A to activity. E50 or E135 are candidates for the switch, E50A would be expected to reduce activity. However, activity increased at pH 5.0 in both backgrounds and at the pH optimum in D158E E50A but decreased slightly in the wt background. This challenges the hypothesis of an electrostatic switch.

Binding Sites↗
PubMed2002

Thermostabilization of cellulosomal endoglucanase EngB from Clostridium cellulovorans by in vitro DNA recombination with non-cellulosomal endoglucanase EngD.

Enhancement of enzyme thermostability by protein engineering gives us information about the thermostabilization mechanism as well as advantages for industrial use of enzymes. In this study, we enhanced the thermostability of endoglucanase EngB, one component of the cellulase complex (cellulosome) from Clostridium cellulovorans, by the directed evolution technique. The library was constructed by in vitro recombination of the genes for EngB and non-cellulosomal cellulase EngD, based on the fact that the catalytic domains of both cellulases were highly homologous. To obtain thermostable clones without loss of activity, the library was screened by a combination of activity and thermostability screening. We obtained three mutants out of 8000 selected clones that showed significantly higher thermostability than those of EngB and EngD without compromising their endoglucanase activities. One of the mutants possessed a sevenfold higher thermostability than EngB. The possible mechanisms of thermostabilization are discussed.

Amino Acid Sequence↗
PubMed1987

A genetically engineered P450 monooxygenase: construction of the functional fused enzyme between rat cytochrome P450c and NADPH-cytochrome P450 reductase.

A hybrid cDNA encoding a fused enzyme consisting of rat cytochrome P450c and rat NADPH-cytochrome P450 reductase was constructed by combining the cytochrome P450c cDNA with the cDNA fragment encoding the protease-solubilized moiety of the NADPH-cytochrome P450 reductase. The hybrid cDNA was inserted between the yeast alcohol dehydrogenase I promoter and terminator of the expression vector pAAH5 to yield expression plasmid pAMP19. Saccharomyces cerevisiae AH22 cells transformed with the expression plasmid pAMP19 produced a 130-kD protein reactive with both anti-cytochrome P450c Ig and antireductase Ig. The yeast cells containing the fused enzyme exhibited about four times higher monooxygenase activity toward 7-ethoxycoumarin than those containing rat cytochrome P450c alone. The fused enzyme was purified from the yeast microsomal fraction by sequential chromatography with DEAE-cellulose and 2',5'-ADP Sepharose 4B columns. The preparation had an apparent molecular weight of 130 kD and the same sequence of the 10 amino-terminal amino acids as that of rat cytochrome P450c. Spectral properties of the fused enzyme indicated the presence of a protoheme, flavin adenine dinucleotide, and flavin mononucleotide in the molecule. The reaction mechanism of the fused enzyme followed first-order kinetics. These results clearly indicate that the fused enzyme is a new self-catalytic P450 monooxygenase. Trypsin treatment of yeast microsomes containing the fused enzyme suggested that the P450 moiety is embedded in the microsomal membrane with the reductase moiety lying on the cytoplasmic side.

Animals↗
PubMed2005

Structure-based in vitro engineering of the anthranilate synthase, a metabolic key enzyme in the plant tryptophan pathway.

Rice (Oryza sativa) anthranilate synthase alpha-subunit, OASA2, was modified by in vitro mutagenesis based on structural information from bacterial homologs. Twenty-four amino acid residues, predicted as putative tryptophan binding sites or their proximal regions in the OASA2 sequence, were selected and 36 mutant OASA2 genes were constructed by PCR-based site-directed mutagenesis. Corresponding mutant proteins were synthesized in a combination of two in vitro systems, transcription with a bacteriophage SP6 RNA polymerase and translation with a wheat-embryo cell-free system. Enzymatic functions of the mutant proteins were simultaneously examined, and we found six mutants with elevated catalytic activity and five mutants with enhanced tolerance to feedback inhibition by tryptophan. Moreover, we observed that some sets of specific combinations of the novel mutations additively conferred both characteristics to the mutant enzymes. The functions of the mutant enzymes were confirmed in vivo. The free tryptophan content of mutant rice calli expressing OASA2 enzyme with a double mutation was 30-fold of that of untransformed calli. Thus, our in vitro approach utilizing structural information of bacterial homologs is a potent technique to generate designer enzymes with predefined functions.

Amino Acid Sequence↗
PubMed2004

An engineered cocaine hydrolase blunts and reverses cardiovascular responses to cocaine in rats.

There is increasing evidence that human plasma butyrylcholinesterase can lower the toxicity of cocaine overdose. Recently, with structure-based protein engineering, we converted this enzyme into a more efficient cocaine hydrolase (CocE). When tested in rats, CocE shortened cocaine's plasma half-life and decreased drug accumulation in heart and brain. Here, we have investigated the potential of CocE to antagonize cardiovascular responses to cocaine. Anesthetized rats were instrumented for continuous recording of blood pressure from the femoral artery. Cocaine (7 mg/kg i.v.) caused blood pressure to rise within 30 s by 25 to 37 mmHg, but pressure returned to baseline within 60 s. These transient pressor responses were prolonged up to 5 min when vagal reflexes were blocked with atropine (1 mg/kg). Under such conditions, pretreatment with CocE (3 mg/kg i.v.) reduced cocaine's pressor effect, whereas delayed treatment with CocE rapidly restored normal mean blood pressure. CocE had no hemodynamic effects in control animals not treated with cocaine. The finding that CocE can oppose pre-established physiologic actions of cocaine suggests that similar or improved hydrolases might help rescue patients from the life-threatening toxicity of drug overdose.

Animals↗
PubMed2004

Synergistic saccharification, and direct fermentation to ethanol, of amorphous cellulose by use of an engineered yeast strain codisplaying three types of cellulolytic enzyme.

A whole-cell biocatalyst with the ability to induce synergistic and sequential cellulose-degradation reaction was constructed through codisplay of three types of cellulolytic enzyme on the cell surface of the yeast Saccharomyces cerevisiae. When a cell surface display system based on alpha-agglutinin was used, Trichoderma reesei endoglucanase II and cellobiohydrolase II and Aspergillus aculeatus beta-glucosidase 1 were simultaneously codisplayed as individual fusion proteins with the C-terminal-half region of alpha-agglutinin. Codisplay of the three enzymes on the cell surface was confirmed by observation of immunofluorescence-labeled cells with a fluorescence microscope. A yeast strain codisplaying endoglucanase II and cellobiohydrolase II showed significantly higher hydrolytic activity with amorphous cellulose (phosphoric acid-swollen cellulose) than one displaying only endoglucanase II, and its main product was cellobiose; codisplay of beta-glucosidase 1, endoglucanase II, and cellobiohydrolase II enabled the yeast strain to directly produce ethanol from the amorphous cellulose (which a yeast strain codisplaying beta-glucosidase 1 and endoglucanase II could not), with a yield of approximately 3 g per liter from 10 g per liter within 40 h. The yield (in grams of ethanol produced per gram of carbohydrate consumed) was 0.45 g/g, which corresponds to 88.5% of the theoretical yield. This indicates that simultaneous and synergistic saccharification and fermentation of amorphous cellulose to ethanol can be efficiently accomplished using a yeast strain codisplaying the three cellulolytic enzymes.

Cellulase↗
PubMed1995

Engineering human monoclonal antibody fragments: a recombinant enzyme-linked Fab.

A new plasmid vector, pCRP, allowing the expression of human recombinant monoclonal antibody Fab fragments fused with a bacterial acid phosphatase has been constructed. pCRP can accept heavy- and light-chain cDNAs cloned from combinatorial antibody libraries displayed on filamentous phages with the pCombIII system and is able to direct expression to soluble Fabs in which the carboxy-terminus of the heavy chain is fused to the amino-terminus of the mature PhoN nonspecific acid phosphatase of Providencia stuartii. Using the pCRP vector, we expressed two different human recombinant Fabs cloned from combinatorial libraries (one anti-tetenus toxoid and the other anti-HIV-1 gp120) fused with the acid phosphatase. In both cases chimeric antibodies were obtained which retained the antigen-binding ability and the enzymatic activity. Similar Fab-enzyme fusions can be successfully used, even unpurified, in enzyme immunoassays.

Acid Phosphatase↗
PubMed1997

Lipase engineering: a window into structure-function relationships.

Utilization of genetic engineering techniques to create novel functional lipases has increased knowledge of structure-function relationships in this important class of enzymes. The examples of engineered lipases presented in this chapter addressed the investigation of domain-specific properties, heparin binding, and subunit orientation. Conclusions reached are credible because the designed lipases retained catalytic activity, implying native, or near-native, conformation. This approach has demonstrated vigor by determining the domain location of several important enzyme functions and by providing the first evidence that LPL subunits are arranged in a head-to-tail orientation. In conjunction with physical techniques, such as crystallography and nuclear magnetic resonance spectroscopy, the engineered lipase approach could reveal new insights into the mechanism by which lipolysis is accomplished. The studies described here represent only the first attempts to explore that subject; more sophisticated lipase engineering will be used in future as a window into structure-function relationships.

Amino Acid Sequence↗
PubMedDate not supplied

Genetically engineered P450 monooxygenases: construction of bovine P450c17/yeast reductase fused enzymes.

Seven P450/reductase fused enzymes were produced in Saccharomyces cerevisiae by expressing fused cDNAs consisting of bovine cytochrome P450c17 (P450c17) and yeast NADPH-cytochrome P450 reductase (reductase). These fused enzymes differed in the length and amino acid sequence of the hinge region between the P450 and reductase moieties. Expression of the fused constructs under the control of the yeast alcohol dehydrogenase I promoter and terminator of expression vector pAAH5 in S. cerevisiae AH22 cells resulted in the production of about 2-8 X 10(4) molecules per cell of the seven corresponding fused enzymes. Six of the fused enzymes incorporated a protoheme, as confirmed by reduced CO-difference spectra. Recombinant yeast strains producing each of the fused hemoproteins showed P450c17-dependent 17 alpha-hydroxylase activity toward progesterone. The most active fused enzyme, delta N23FE, which lacked the amino-terminal 23 amino acids of the reductase, showed about 10 times higher 17 alpha-hydroxylase activity than bovine P450c17, although the fused enzyme (delta N23FE)' with an amino acid sequence in the hinge region different from delta N23FE was less active than delta N23FE. The fused enzyme delta N0FE, consisting of P450c17 and whole reductase, showed about 1.8 times higher activity than bovine P450c17. No activity was found with delta N84FE lacking the amino-terminal 84 amino acids of the reductase moiety. P450c17-dependent C17,(20)-lyase activity toward 17 alpha-hydroxyprogesterone was detected to lesser extents in the recombinant yeast. Fused bovine P450c17/yeast reductase enzymes show enhanced 17 alpha-hydroxylase activity, and the length and amino acid sequence in the hinge region between the P450c17 and yeast reductase moieties can be important for efficient intramolecular electron transfer in the fused enzymes.

Animals↗
PubMed2004

EFICAz: a comprehensive approach for accurate genome-scale enzyme function inference.

EFICAz (Enzyme Function Inference by Combined Approach) is an automatic engine for large-scale enzyme function inference that combines predictions from four different methods developed and optimized to achieve high prediction accuracy: (i) recognition of functionally discriminating residues (FDRs) in enzyme families obtained by a Conservation-controlled HMM Iterative procedure for Enzyme Family classification (CHIEFc), (ii) pairwise sequence comparison using a family specific Sequence Identity Threshold, (iii) recognition of FDRs in Multiple Pfam enzyme families, and (iv) recognition of multiple Prosite patterns of high specificity. For FDR (i.e. conserved positions in an enzyme family that discriminate between true and false members of the family) identification, we have developed an Evolutionary Footprinting method that uses evolutionary information from homofunctional and heterofunctional multiple sequence alignments associated with an enzyme family. The FDRs show a significant correlation with annotated active site residues. In a jackknife test, EFICAz shows high accuracy (92%) and sensitivity (82%) for predicting four EC digits in testing sequences that are <40% identical to any member of the corresponding training set. Applied to Escherichia coli genome, EFICAz assigns more detailed enzymatic function than KEGG, and generates numerous novel predictions.

Amino Acid Sequence↗
PubMed2005

Directed evolution of copy number of a broad host range plasmid for metabolic engineering.

Random mutagenesis and directed evolution has been successfully used to improve desired properties of enzymes for biocatalysis and metabolic engineering. Here we employ the method to increase copy number of a pBBR-based broad host range plasmid, which can be used to express desired enzymes in a variety of microbial hosts. Localized random mutagenesis was performed in the replication control region of a pBBR-derived plasmid containing a beta-carotene reporter. Mutant plasmids were isolated that showed increased beta-carotene production. Real-time PCR analysis confirmed that the copy number of the mutant plasmids increased 3-7 fold. Sequence of the 10 mutant plasmids indicated that each plasmid contained single or multiple mutations in the rep gene or the flanking regions. Single amino acid change of serine to leucine at codon 100 of the replication protein and single nucleotide change of C to T at 46 bp upstream of the rep gene caused the increase of plasmid copy number. The utility of the mutant plasmids for metabolic engineering were further demonstrated by increased beta-carotene production, when an isoprenoid pathway gene (dxs) was co-expressed on a compatible plasmid. The mutant plasmids were tested in Agrobacterium tumefaciens. Increase of plasmid copy number and beta-carotene production was also observed in the non-Escherichia coli host.

Directed Molecular Evolution↗
PubMed2006

Protein engineering of class-A non-specific acid phosphatase (PhoN) of Salmonella typhimurium: modulation of the pH-activity profile.

Engineering of the PhoN enzyme of Salmonella typhimurium due to its superior characteristics for bioremediation of heavy metals has been advocated by Macaskie and colleagues [Basnakova, G., Stephens, E.R., Thaller, M.C., Rossolini, G.M., Macaskie, L.E., 1998. The use of Escherichia coli bearing a phoN gene for the removal of uranium and nickel from aqueous flows. Appl. Microbiol. Biotechnol. 50, 266-272]. The native enzyme hydrolyzes disparate organophosphates and exhibits optimal phosphatase activity at pH 5.5, for instance, with substrate p-nitrophenyl phosphate. Structurally guided Ile-78 was mutated using site-directed mutagenesis to Ala, Asp and His residues, with an aim to shift the optimum pH of the PhoN enzyme. Encouragingly, the I78A mutant displays significantly higher (as high as 160%) enzymatic efficiency over a broad pH range of 3.0-9.0, compared to the wild-type PhoN. The higher catalytic efficiency is due to the increase in k(cat), and can be mainly attributed to a deshielding of catalytic His-158 from the bulk-solvent. The I78D mutant possesses nearly twice the specific activity at the optimum pH of 7.0. The alkaline shift of the pH-activity profile agrees well with reasoning based on electrostatics. An increase in K(m), however, lowers the catalytic efficiency of the I78D mutant at the optimum pH. The I78H mutant, counter-intuitively, also exhibits an alkaline shift in the pH-optimum. Nonetheless, the active site scaffold in I78H mutant may not be disturbed, as similar steady-state kinetic parameters are observed for both I78H mutant and wild-type PhoN at their respective pH optima.

Acid Phosphatase↗
PubMed2004

Bioactivation of the heterocyclic aromatic amine 2-amino-3-methyl-9H-pyrido [2,3-b]indole (MeAalphaC) in recombinant test systems expressing human xenobiotic-metabolizing enzymes.

2-Amino-3-methyl-9H-pyrido[2,3-b]indole (MeAalphaC) and some metabolites were investigated for mutagenicity in mammalian cell lines and bacterial strains engineered for the expression of human enzymes. MeAalphaC induced gene mutations (studied at the hprt locus) in Chinese hamster V79-derived cells co-expressing cytochrome (CYP) 1A2 and sulphotransferase (SULT) 1A1 even at a concentration of 30 nM, but was inactive in cells co-expressing CYP1A2 and N-acetyltransferase (NAT) 1 or 2. MeAalphaC, tested in the presence of rat liver post-mitochondrial fraction, showed strongly enhanced mutagenicity in a Salmonella typhimurium strain expressing human SULT1A1 compared with the control (recipient) strain TA1538/1,8-DNP (deficient in endogenous acetyltransferase). Mutagenicity was also enhanced, although to a lesser extent, when NAT2 was expressed in the latter strain. The metabolite, 2-hydroxylamino-3-methyl-9H-pyrido[2,3-b]indole (N-OH-MeAalphaC) was a direct mutagen to strains TA1538 and TA1538/ 1,8-DNP. This mutagenicity was strongly enhanced in corresponding strains expressing SULT1A1. A moderate enhancement was observed when SULT1A2, SULT1B1, SULT1C2 or NAT2 were expressed in strain TA1538. The remaining enzymes studied (SULT1A3, 1C1, 1E1, 2A1, 2B1a, 2B1b, 4A1 and NAT1) did not indicate any activation of N-OH-MeAalphaC. Preliminary mutagenicity experiments in SULT-expressing S.typhimurium strains were conducted with other hydroxylated metabolites of MeAalphaC. The phenols, 6- and 7-hydroxy-MeAalphaC, were inactive under the conditions studied. The benzylic alcohol, 2-amino-3-hydroxymethyl-9H-pyrido[2,3-b]indole, was mutagenic in a strain expressing SULT1A1, but its activity was much weaker than that of N-OH-MeAalphaC. Thus, N-hydroxylation (e.g. mediated by CYP1A2) and sulpho conjugation (primarily mediated by SULT1A1) was the dominating activation pathway of MeAalphaC in model systems engineered for human enzymes. Some other SULT forms as well as NAT2 were also capable of activating N-OH-MeAalphaC, although with much lower efficiency than SULT1A1. Another minor activation pathway involved benzylic hydroxylation followed by sulpho conjugation by SULT1A1.

Animals↗
PubMed1998

Studies of brush border enzymes, basement membrane components, and electrophysiology of tissue-engineered neointestine.

BACKGROUND/PURPOSE: Previous studies have shown that intestinal crypt cell transplantation using biodegradable scaffolds can generate stratified epithelium reminiscent of embryonic gut. The authors propose to tissue engineer small intestine on biodegradable scaffolds by transplanting intestinal epithelial organoid units, which maintain the epithelial mesenchymal cell-cell interaction necessary for epithelial survival, proliferation, and differentiation. METHODS: Intestinal epithelial organoid units were isolated from neonatal Lewis rats by enzyme digestion and differential sedimentation. Organoid units were seeded on to tubular scaffolds made of nonwoven polyglycolic acid (PGA) sprayed with 5% polylactic acid (PLA). Polymers either were coated (28 constructs) or noncoated (33 constructs) with collagen type I. A total of 61 organoid unit polymer constructs were implanted into 61 animals. Animals were killed and constructs harvested at 2, 6, 7, 8, 9, 10, 12, and 14 weeks. RESULTS: Histological analysis showed formation of neomucosa characterized by columnar epithelium with goblet, and paneth cells were evident in 47 of the 61 constructs. The outer walls were composed of fibrovascular tissue, degradable polymer, extracellular matrix, and smooth muscle-like cells. Immunofluorescent microscopy showed apical staining of brush border enzymes, sucrase and lactase, and basolateral staining for laminin, indicating the establishment of cell polarity. Electrophysiology of Ussing-chambered neomucosa and adult ileal mucosa exhibited similar transepithelial resistance. CONCLUSION: These results suggest that intestinal crypt cells heterotopically transplanted as epithelial organoid units on PGA-PLA tubular scaffolds can survive, reorganize, and regenerate complex composite tissue resembling small intestine demonstrating organ morphogenesis, cytodifferentiation, and phenotypic maturation.

Animals↗
PubMed2002

Transmutation of human glutathione transferase A2-2 with peroxidase activity into an efficient steroid isomerase.

A major goal in protein engineering is the tailor-making of enzymes for specified chemical reactions. Successful attempts have frequently been based on directed molecular evolution involving libraries of random mutants in which variants with desired properties were identified. For the engineering of enzymes with novel functions, it would be of great value if the necessary changes of the active site could be predicted and implemented. Such attempts based on the comparison of similar structures with different substrate selectivities have previously met with limited success. However, the present work shows that the knowledge-based redesign restricted to substrate-binding residues in human glutathione transferase A2-2 can introduce high steroid double-bond isomerase activity into the enzyme originally characterized by glutathione peroxidase activity. Both the catalytic center activity (k(cat)) and catalytic efficiency (k(cat)/K(m)) match the values of the naturally evolved glutathione transferase A3-3, the most active steroid isomerase known in human tissues. The substrate selectivity of the mutated glutathione transferase was changed 7000-fold by five point mutations. This example demonstrates the functional plasticity of the glutathione transferase scaffold as well as the potential of rational active-site directed mutagenesis as a complement to DNA shuffling and other stochastic methods for the redesign of proteins with novel functions.

Binding Sites↗
PubMed1996

Furilisin: a variant of subtilisin BPN' engineered for cleaving tribasic substrates.

The serine protease, subtilisin BPN', was engineered to cleave proteins after tribasic sequences in a manner that resembles the substrate specificity of furin, one of the mammalian subtilisin homologs that processes prohormones. As a starting point we used a double mutant of subtilisin BPN' (N62D/ G166D) that showed substantial preference for cleaving after sequences having consecutive dibasic residues (namely, at the P1 and P2 substrate positions) [Ballinger et al. (1995) Biochemistry 34, 13312-13319]. Additional specificity for basic residues was engineered at the P4 position by introducing subtilisin-to-furin substitutions at three hydrophobic residues that composed the S4 subsite (Y104, I107, and L126). Initial attempts to incorporate a Y104D or I107E mutation or the Y104D/I107E double mutation into the dibasic specific enzyme failed to generate the processed enzyme. The problem was traced to the inability of the mutant prosubtilisins to process themselves and fold correctly. Replacing the natural processing site sequence (AHAY) with a good furin substrate sequence (RHKR) resulted in expression of the triple subtilisin mutant (N62D/Y104D/G166D) we call "furilisin". Furilisin hydrolyzes synthetic tribasic substrates (succinyl-RAKR-pNA or succinyl-KAKR-pNA) with high catalytic efficiency (kcat/K(m) > 3 x 10(5) M-1 s-1) and discriminates in favor of Arg versus Ala at the P4 position by a factor of 360. The overall specificity change versus the wild-type enzyme was dramatic. For example, succinyl-RAKR-pNA was cleaved approximately 60000 times faster than succinyl-AAPF-pNA, a good substrate for wild-type subtilisin. Similarly, furilisin was inhibited (K1* = 29 nM) by a variant of the turkey ovomucoid third domain inhibitor that contained an engineered furin substrate site (RCKR decreases) [Lu et al. (1993) J. Biol. Chem. 268, 14583-14585] and not by one having a good wild-type subtilisin substrate sequence (ACTL decreases). Interestingly, the extreme changes in substrate specificity resulted from substantial synergy between the engineered subsites. These studies provide a basic example of how to manipulate substrate specificity in a modular fashion, thereby creating an engineered-enzyme that may be useful as a protein processing tool.

Amino Acid Sequence↗