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PubMed2005

[Directed evolution of D-lactonohydrolase by error prone PCR and DNA shuffling].

D-lactonohydrolase is useful in the procedure of resolution of racemic pantolactone to produce D-pantolactone, but the activity and stability under low pH of the wild type enzyme is not satisfactory enough to be applied to industrial production. The expected properties of wild type enzyme were enhanced by directed evolution. According to the formation of products and pH indicators, a screening system was designed. After three sequential error prone PCR and one round DNA shuffling followed by screening, Mut E-861, the best mutant with improved activity and stability under low pH situation was obtained. Gene analysis of the Mut E-861 mutant indicated that the mutant enzyme had A352C, G721A mutations and a silent mutation of position 1038. Moreover, the activity and stability of Mut E-861 were determined. The results showed that the activity of this mutant was 5.5-fold higher than that of wild type, and the stability under low pH was improved at no expense of D-lactonohydrolase activity. After incubated at pH 6.0 and pH 5.0 the activity of D-lactonohydrolase could be retained 75% to 50%, however, compared with 40% to 20% for wild type.

Carboxylic Ester Hydrolases↗

PubMed1996

Common origin of arthropod tyrosinase, arthropod hemocyanin, insect hexamerin, and dipteran arylphorin receptor.

Dipteran arylphorin receptors, insect hexamerins, cheliceratan and crustacean hemocyanins, and crustacean and insect tyrosinases display significant sequence similarities. We have undertaken a systematic comparison of primary and secondary structures of these proteins. On the basis of multiple sequence alignments the phylogeny of these proteins was investigated. Hexamerin subunits, hemocyanin subunits, and tyrosinases share extensive similarities throughout the entire amino acid sequence. Our studies suggest the origin of arthropod hemocyanins from ancient tyrosinase-like proteins. Insect hexamerins likely evolved from hemocyanins of ancient crustaceans, supporting the proposed sister-group position of these subphyla. Arylphorin receptors, responsible for incorporation of hexamerins into the larval fat body of diptera, are related to hexamerins, hemocyanins, and tyrosinase. The receptor sequences display extensive similarities to the first and third domains of hemocyanins and hexamerins. In the middle region only limited amino acid conservation was observed. Elements important for hexamer formation are deleted in the receptors. Phylogenetic analysis indicated that dipteran arylphorin receptors diverged from ancient hexamerins, probably early in insect evolution.

Amino Acid Sequence↗

PubMed2001

Quantifying the ion selectivity of the Ca2+ site in photosystem II: evidence for direct involvement of Ca2+ in O2 formation.

Calcium is an essential cofactor in the oxygen-evolving complex (OEC) of photosystem II (PSII). The removal of Ca2+ or its substitution by any metal ion except Sr2+ inhibits oxygen evolution. We used steady-state enzyme kinetics to measure the rate of O2 evolution in PSII samples treated with an extensive series of mono-, di-, and trivalent metal ions in order to determine the basis for the affinity of metal ions for the Ca2+-binding site. Our results show that the Ca2+-binding site in PSII behaves very similarly to the Ca2+-binding sites in other proteins, and we discuss the implications this has for the structure of the site in PSII. Activity measurements as a function of time show that the binding site achieves equilibrium in 4 h for all of the PSII samples investigated. The binding affinities of the metal ions are modulated by the 17 and 23 kDa extrinsic polypeptides; their removal decreases the free energy of binding of the metal ions by 2.5 kcal/mol, but does not significantly change the time required to reach equilibrium. Monovalent ions are effectively excluded from the Ca2+-binding site, exhibiting no inhibition of O2 evolution. Di- and trivalent metal ions with ionic radii similar to that of Ca2+ (0.99 A) bind competitively with Ca2+ and have the highest binding affinity, while smaller metal ions bind more weakly and much larger ones do not bind competitively. This is consistent with a size-selective Ca2+-binding site that has a rigid array of coordinating ligands. Despite the large number of metal ions that competitively replace Ca2+ in the OEC, only Sr2+ is capable of partially restoring activity. Comparing the physical characteristics of the metal ions studied, we identify the pK(a) of the aqua ion as the factor that determines the functional competence of the metal ion. This suggests that Ca2+ is directly involved in the chemistry of water oxidation and is not only a structural cofactor in the OEC. We propose that the role of Ca2+ is to act as a Lewis acid, binding a substrate water molecule and tuning its reactivity.

Binding, Competitive↗

PubMed2001

Packing is a key selection factor in the evolution of protein hydrophobic cores.

The energy derived from optimized van der Waals interactions in closely packed, folded proteins has been proposed to be of similar energetic magnitude to hydrophobicity in stabilizing the native state. If packing is this energetically important, it should influence the evolution of protein core sequences. To test this hypothesis, the occurrence of various amino acid side chains in the major hydrophobic core of staphylococcal nuclease and 42 homologous proteins was determined. Most such positions in this protein family are usually isoleucine, leucine, or valine. Previously we have constructed and measured the stabilities of 12 single, 44 double, 64 triple, and 32 quadruple mutants, representing all possible permutations of these three side chains at two overlapping sets of four positions in the core of staphylococcal nuclease. The stabilities and interaction energies of those mutants with various combinations of the most common, or consensus, sequence were compared to the stabilities of all other mutants. Mutants which had the consensus side-chain combinations were not necessarily the most stable, but usually were found to have the best interaction energies. In other words, these proteins were far more stable than would be predicted from simply summing the observed energetic effects of the component single mutations, apparently reflecting particularly favorable packing interactions that are possible for the most common side chains. An additional 12 mutants which tested possible alternate explanations of the results were constructed. The stabilities and interaction energies of these mutants also support the conclusion that packing is a crucial determinant guiding the sequence evolution of protein cores.

Amino Acid Sequence↗

PubMed2003

A compelling experimental test of the hypothesis that enzymes have evolved to enhance quantum mechanical tunneling in hydrogen transfer reactions: the beta-neopentylcobalamin system combined with prior adocobalamin data.

An intriguing but controversial hypothesis has appeared that "The optimization of enzyme catalysis may entail the evolutionary implementation of chemical strategies that increase the probability of tunneling and thereby accelerate the reaction rate" (Kohen, A.; Klinman, J. P. Acc. Chem. Res. 1998, 31, 397). Restated, enzymes may have evolved to enhance quantum mechanical tunneling by coupling to protein low nu modes that squeeze the reacting centers together in, for example, their H(*) atom abstraction reactions. Such a putative "protein squeezing" mechanism would enhance hydrogen quantum mechanical tunneling by reducing the barrier width. An alternative hypothesis is that enzymes do not enhance tunneling, but simply exploit the same amount of tunneling present in their enzyme-free solution reactions, if those reactions occur. A third, conceivable hypothesis is that enzymes might even inadvertently decrease the amount of tunneling as an undesired result of increasing the barrier width while reducing the barrier height. Testing these hypotheses experimentally requires the extremely rare event of being able to measure the amount of tunneling both in the enzyme system and in a very similar if not identical reaction in enzyme-free solution. This has been accomplished experimentally in only one prior case, our recent study of AdoCbl (coenzyme B(12)) and 8-Meo-AdoCbl undergoing enzyme-like H(*) abstraction reactions (Doll, K. M.; Bender, B. R.; Finke, R. G. to J. Am. Chem. Soc. 2003, in press). The data there reveal no change in the level of tunneling within or outside of the enzyme in comparison to the best literature data for an AdoCbl-dependent enzyme, methylmalonyl-CoA mutase. However, that first system suffers from two limitations: the measurement of the KIE (kinetic isotope effect) data in a nonenzymic 80-110 degrees C temperature range; and lower precision data than desired due to the HPLC-MS method required for one of the KIE analyses. These limitations have now been overcome by the synthesis, then thermolysis and KIE study vs temperature of the H(*) abstraction reaction of beta-neopentylcobalamin (beta-NpCbl) in ethylene glycol-d(0) and ethylene glycol-d(4). This is the first experimental test of Klinman's hypothesis using KIE data obtained at enzyme-relevant temperatures. The key data obtained are as follows: deuterium KIEs of 23.1 +/- 3.0 at 40 degrees C to 39.0 +/- 2.3 at 10 degrees C; an activation energy difference E(D) - E(H) of 3.1 +/- 0.3 kcal mol(-)(1); and a pre-exponential factor ratio A(H)/A(D) of 0.14 +/- 0.07. Moreover, our now three sets of data (NpCbl; AdoCbl; 8-MeOAdoCbl) are shown to lie on the same ln KIE vs 1/T linear plot yielding a set of enzyme-temperature-relevant, high-precision KIE, E(D) - E(H), and A(H)/A(D) data over a relatively large, 110 degrees C temperature range. Significantly, the enzyme-free solution KIE, E(D) - E(H), and A(H)/A(D) are identical within experimental error to those for methylmalonyl-CoA mutase. This finding leads to the conclusion that there is no enzymic enhancement of the tunneling in at least this B(12)-dependent enzyme. This B(12) enzyme does, however, exploit the same (unchanged) level of tunneling measured for the nonenzymic, Ado(*) solution H(*) abstraction reaction. A discussion is presented of the still open question of if this first experimental finding, of "no enzymic enhancement of tunneling" in one B(12)-dependent enzymic system, is likely to prove more general or not.

Algorithms↗

PubMed1989

Reduction of cyclopropene by NifV- and wild-type nitrogenases from Klebsiella pneumoniae.

The nitrogenase from wild-type Klebsiella pneumoniae reduces cyclopropene to cyclopropane and propene in the ratio 1:2 at pH 7.5. We show in this paper that the nitrogenase from a nifV mutant of K. pneumoniae also reduces cyclopropene to cyclopropane and propene, but the ratio of products is now 1:1.4. However, both nitrogenases exhibit the same Km for cyclopropene (2.1 x 10(4) +/- 0.2 x 10(4) Pa), considerably more than the Km for the analogous reaction with Azotobacter vinelandii nitrogenase under the same conditions (5.1 x 10(3) Pa). Analysis of the data shows that the different product ratio arises from the slower production of propene compared with cyclopropane by the mutant nitrogenase. During turnover, both nitrogenases use a large proportion of the electron flux for H2 production. CO inhibits the reduction of cyclopropene by both K. pneumoniae proteins, but the mutant nitrogenase exhibits 50% inhibition at approx. 10 Pa, whereas the corresponding value for the wild-type nitrogenase is approx. 110 Pa. However, H2 evolution by the mutant enzyme is much less affected than is cyclopropene reduction. CO inhibition of cyclopropene reduction by the nitrogenases coincides with a relative increase in H2 evolution, so that in the wild-type (but not the mutant) the electron flux is approximately maintained. The cyclopropane/propene production ratios are little affected by the presence of CO within the pressure ranges studied at least up to 50% inhibition.

Binding Sites↗

PubMed2001

The phosphoglucose isomerase from the hyperthermophilic archaeon Pyrococcus furiosus is a unique glycolytic enzyme that belongs to the cupin superfamily.

Pyrococcus furiosus uses a variant of the Embden-Meyerhof pathway during growth on sugars. All but one of the genes that encode the glycolytic enzymes of P. furiosus have previously been identified, either by homology searching of its genome or by reversed genetics. We here report the isolation of the missing link of the pyrococcal glycolysis, the phosphoglucose isomerase (PGI), which was purified to homogeneity from P. furiosus and biochemically characterized. The P. furiosus PGI, a dimer of identical 23.5-kDa subunits, catalyzes the reversible isomerization of glucose 6-phosphate to fructose 6-phosphate, with K(m) values of 1.99 and 0.63 mm, respectively. An optimum pH of 7.0 has been determined in both directions, and at its optimum temperature of 90 degrees C the enzyme has a half-life of 2.4 h. The N-terminal sequence was used for the identification of the pgiA gene in the P. furiosus genome. The pgiA transcription start site has been determined, and a monocistronic messenger was detected in P. furiosus during growth on maltose and pyruvate. The pgiA gene was functionally expressed in Escherichia coli BL21(DE3). The deduced amino acid sequence of this first archaeal PGI revealed that it is not related to its bacterial and eukaryal counterparts. In contrast, this archaeal PGI shares similarity with the cupin superfamily that consists of a variety of proteins that are generally involved in sugar metabolism in both prokaryotes and eukaryotes. As for the P. furiosus PGI, distinct phylogenetic origins have previously been reported for other enzymes from the pyrococcal glycolytic pathway. Apparently, convergent evolution by recruitment of several unique enzymes has resulted in the unique Pyrococcus glycolysis.

Amino Acid Sequence↗

PubMed2000

Evolutionary implications of the mosaic pyrimidine-biosynthetic pathway in eukaryotes.

The de-novo pyrimidine biosynthetic pathway involves six enzymes, in order from the first to the sixth step, carbamoyl-phosphate synthetase II (CPS II) comprising glutamine amidotransferase (GAT) and carbamoyl-phosphate synthetase (CPS) domains or subunits, aspartate carbamoyltransferase (ACT), dihydroorotase (DHO), dihydroorotate dehydrogenase (DHOD), orotate phosphoribosyltransferase (OPRT), and orotidine-5'-monophosphate decarboxylase (OMPDC). In contrast with reports on molecular evolution of the individual enzymes, we attempted to draw an evolutionary picture of the whole pathway using the protein phylogeny. We demonstrate highly mosaic organizations of the pyrimidine biosynthetic pathway in eukaryotes. During evolution of the eukaryotic pathway, plants and fungi (or their ancestors) in particular may have secondarily acquired the characteristic enzymes. This is consistent with the fact that the organization of plant enzymes is highly chimeric: (1) two subunits of CPS II, GAT and CPS, cluster with a clade including cyanobacteria and red algal chloroplasts, (2) ACT not with a cyanobacterium, Synechocystis spp., irrespective of its putative signal sequence targeting into chloroplasts, and (3) DHO with a clade of proteobacteria. In fungi, DHO and OPRT cluster respectively with the corresponding proteobacterial counterparts. The phylogenetic analyses of DHOD and OMPDC also support the implications of the mosaic pyrimidine biosynthetic pathway in eukaryotes. The potential importance of the horizontal gene transfer(s) and endosymbiosis in establishing the mosaic pathway is discussed.

Amino Acid Sequence↗

PubMed2001

The fidelity of the translation of the genetic code.

Aminoacyl-tRNA synthetases play a central role in maintaining accuracy during the translation of the genetic code. To achieve this challenging task they have to discriminate against amino acids that are very closely related not only in structure but also in chemical nature. A 'double-sieve' editing model was proposed in the late seventies to explain how two closely related amino acids may be discriminated. However, a clear understanding of this mechanism required structural information on synthetases that are faced with such a problem of amino acid discrimination. The first structural basis for the editing model came recently from the crystal structure of isoleucyl-tRNA synthetase, a class I synthetase, which has to discriminate against valine. The structure showed the presence of two catalytic sites in the same enzyme, one for activation, a coarse sieve which binds both isoleucine and valine, and another for editing, a fine sieve which binds only valine and rejects isoleucine. Another structure of the enzyme in complex with tRNA showed that the tRNA is responsible for the translocation of the misactivated amino-acid substrate from the catalytic site to the editing site. These studies were mainly focused on class I synthetases and the situation was not clear about how class II enzymes discriminate against similar amino acids. The recent structural and enzymatic studies on threonyl-tRNA synthetase, a class II enzyme, reveal how this challenging task is achieved by using a unique zinc ion in the active site as well as by employing a separate domain for specific editing activity. These studies led us to propose a model which emphasizes the mirror symmetrical approach of the two classes of enzymes and highlights that tRNA is the key player in the evolution of these class of enzymes.

Amino Acids↗

PubMed2004

Bicarbonate is a native cofactor for assembly of the manganese cluster of the photosynthetic water oxidizing complex. Kinetics of reconstitution of O2 evolution by photoactivation.

Assembly of the inorganic core (Mn(4)O(x)Ca(1)Cl(y)) of the water oxidizing enzyme of oxygenic photosynthesis generates O(2) evolution capacity via the photodriven binding and photooxidation of the free inorganic cofactors within the cofactor-depleted enzyme (apo-WOC-PSII) by a process called photoactivation. Using in vitro photoactivation of spinach PSII membranes, we identify a new lower affinity site for bicarbonate interaction in the WOC. Bicarbonate addition causes a 300% stimulation of the rate and a 50% increase in yield of photoassembled PSII centers when using Mn(2+) and Ca(2+) concentrations that are 10-50-fold larger range than previously examined. Maintenance of a fixed Mn(2+)/Ca(2+) ratio (1:500) produces the fastest rates and highest yields of photoactivation, which has implications for intracellular cofactor homeostasis. A two-step (biexponential) model is shown to accurately fit the assembly kinetics over a 200-fold range of Mn(2+) concentrations. The first step, the binding and photooxidation of Mn(2+) to Mn(3+), is specifically stimulated via formation of a ternary complex between Mn(2+), bicarbonate, and apo-WOC-PSII, having a proposed stoichiometry of [Mn(2+)(HCO(3)(-))]. This low-affinity bicarbonate complex is thermodynamically easier to oxidize than the aqua precursor, [Mn(2+)(OH(2))]. The photooxidized intermediate, [Mn(3+)(HCO(3)(-))], is longer lived and increases the photoactivation yield by suppressing irreversible photodamage to the cofactor-free apo-WOC-PSII (photoinhibition). Bicarbonate does not affect the second (rate-limiting) dark step of photoactivation, attributed to a protein conformational change. Together with the previously characterized high-affinity site, these results reveal that bicarbonate is a multifunctional "native" cofactor important for photoactivation and photoprotection of the WOC-PSII complex.

Apoenzymes↗

PubMedDate not supplied

Clinical and etiopathogenetic profile of heart failure in the First Medical Clinic of Cluj-Napoca. A study of 5 years.

The authors analysed the main etiopathogenetic conditions and the clinical and evolutional profile of 1586 patients with heart failure (HF), admitted to the First Medical Clinic of Cluj-Napoca between 1990 and 1994. Ischemic heart disease was found in 1,236 patients (78%), followed by chronic cor pulmonale, valvulopathies and congenital heart diseases. Among the precipitating and/or aggravating factors of HF, the most important were infections in 434 patients (33.10%), and arrhythmias, especially atrial fibrillation, in 332 patients (25.39%). In decreased order of frequency there were also failure to observe prescribed therapy, uncontrolled arterial hypertension, anemias, dyselectrolytemias, dysproteinemias. HF had a chronic evolution in 1,450 patients (91.40%), and an acute one in 136 (8.6%). In conditions of complex therapy including cardiotonics, diuretics, plus, more recently, conversion enzyme inhibitors, the clinical evolution was favourable in 1,432 patients (90.20%), which had a lower functional class on discharge from hospital.

Acute Disease↗

PubMed1989

Evolution and regulation of the gene encoding superoxide dismutase from the archaebacterium Halobacterium cutirubrum.

The gene encoding the manganese-containing superoxide dismutase (SOD) of Halobacterium cutirubrum was isolated and characterized. The gene and 5'- and 3'-untranslated regions were located on a genomic DNA fragment of 1127 nucleotides. The deduced amino acid sequence is 200 residues long and has 39-42% identity with manganese-containing SODs of eubacteria and mitochondria. This homology may be due to either lateral transfer of the gene between eubacteria and archaebacteria or to high amino acid sequence conservation in the enzyme during the separate evolution of eubacteria and archaebacteria. Transcription of the gene initiates only about three nucleotides upstream of the translation initiation codon. The 5' end of the transcript does not contain a purine-rich Shine-Dalgarno sequence, and the promoter region does not contain consensus sequences found in other archaebacterial promoters. Termination of transcription occurs at 5 consecutive thymine residues that are preceded by a GC-rich region. The gene is basally expressed in anaerobically grown cells but is also inducible by paraquat, a generator of oxygen radicals. The same transcription initiation site is used in both types of expression, suggesting that one promoter is responsible for both basal and regulated expression. In addition to the single copy of the authentic SOD gene, the genome of H. cutirubrum contains a sequence that is very closely related to but does not code for the previously purified SOD of this organism.

Amino Acid Sequence↗

PubMed2002

Primary structure, unique enzymatic properties, and molecular evolution of pepsinogen B and pepsin B.

Purification of pepsinogen B from dog stomach was achieved. Activation of pepsinogen B to pepsin B is likely to proceed through a one-step pathway although the rate is very slow. Pepsin B hydrolyzes various peptides including beta-endorphin, insulin B chain, dynorphin A, and neurokinin A, with high specificity for the cleavage of the Phe-X bonds. The stability of pepsin B in alkaline pH is noteworthy, presumably due to its less acidic character. The complete primary structure of pepsinogen B was clarified for the first time through the molecular cloning of the respective cDNA. Molecular evolutional analyses show that pepsinogen B is not included in other known pepsinogen groups and constitutes an independent cluster in the consensus tree. Pepsinogen B might be a sister group of pepsinogen C and the divergence of these two zymogens seems to be the latest event of pepsinogen evolution.

Animals↗

PubMed2005

Directed evolution strategies for improved enzymatic performance.

The engineering of enzymes with altered activity, specificity and stability, using directed evolution techniques that mimic evolution on a laboratory timescale, is now well established. However, the general acceptance of these methods as a route to new biocatalysts for organic synthesis requires further improvement of the methods for both ease-of-use and also for obtaining more significant changes in enzyme properties than is currently possible. Recent advances in library design, and methods of random mutagenesis, combined with new screening and selection tools, continue to push forward the potential of directed evolution. For example, protein engineers are now beginning to apply the vast body of knowledge and understanding of protein structure and function, to the design of focussed directed evolution libraries, with striking results compared to the previously favoured random mutagenesis and recombination of entire genes. Significant progress in computational design techniques which mimic the experimental process of library screening is also now enabling searches of much greater regions of sequence-space for those catalytic reactions that are broadly understood and, therefore, possible to model. Biocatalysis for organic synthesis frequently makes use of whole-cells, in addition to isolated enzymes, either for a single reaction or for transformations via entire metabolic pathways. As many new whole-cell biocatalysts are being developed by metabolic engineering, the potential of directed evolution to improve these initial designs is also beginning to be realised.

Journal Article↗

PubMed1987

Recruitment of lysozyme as a major enzyme in the mouse gut: duplication, divergence, and regulatory evolution.

Two major types of lysozyme c (M and P) occur in the mouse genus, Mus, and have been purified from an inbred laboratory strain (C58/J) of M. domesticus. They differ in physical, catalytic, and antigenic properties as well as by amino acid replacements at 6 of 49 positions in the amino-terminal sequence. Comparisons with four other mammalian lysozymes c of known sequence suggest that M and P are related by a gene duplication that took place before the divergence of the rat and mouse lineages. M lysozyme is present in most tissues; achieves its highest concentration in the kidney, lung, and spleen; and corresponds to the lysozyme partially sequenced before from another strain of M. domesticus. In M. domesticus and several related species, P lysozyme was detected chiefly in the small intestine, where it is probably produced mainly by Paneth cells. A survey of M and P levels in 22 species of muroid rodents (from Mus and six other genera) of known phylogenetic relationships suggests that a mutation that derepressed the P enzyme arose about 4 million years ago in the ancestor of the housemouse group of species. Additional regulatory shifts affecting M and P levels have taken place along lineages leading to other muroid species. Our survey of 187 individuals of wild house mice and their closest allies reveals a correlation between latitude of origin and level of intestinal lysozyme.

Amino Acid Sequence↗

PubMed1995

Protease evolution in Streptomyces griseus. Discovery of a novel dimeric enzymes.

This report describes the cloning and sequencing of a novel protease gene derived from Streptomyces griseus. Also described is the heterologous expression of the gene in Bacillus subtilis and characterization of the gene product. The sprD gene encodes a prepro mature protease of 392 amino acids tentatively named S. griseus protease D (SGPD). A significant component of the enzyme preregion was found to be homologous with the mitochondrial import signal of hsp60. The sprD gene was subcloned into an Escherichia coli/B. subtilis shuttle vector system such that the pro mature portion of SGPD was fused in frame with the promoter, ribosome binding site, and signal sequences of subtilisin. The gene fusion was subsequently expressed in B. subtilis DB104, and active protease was purified. SGPD has a high degree of sequence homology to previously described S. griseus proteases A, B, C, and E and the alpha-lytic protease of Lysobacter enzymogenes, but unlike all previously characterized members of the chymotrypsin superfamily, the recombinant SGPD forms a stable alpha 2 dimer. The amino acid sequence of the protein in the region of the specificity pocket is similar to that of S. griseus proteases A, B, and C. The purified enzyme was found to have a primary specificity for large aliphatic or aromatic amino acids. Nucleotide sequence data were used to construct a phylogenetic tree using a method of maximum parsimony which reflects the relationships and potentially the lineage of the chymotrypsin-like proteases of S. griseus.

Amino Acid Sequence↗

PubMed2004

High-activity barley alpha-amylase by directed evolution.

Barley alpha-amylase isozyme 2 was cloned into and constitutively secreted by Saccharomyces cervisiae. The gene coding for the wild-type enzyme was subjected to directed evolution. Libraries of mutants were screened by halo formation on starch agar plates, followed by high-throughput liquid assay using dye-labeled starch as the substrate. The concentration of recombinant enzyme in the culture supernatant was determined by immunodetection, and used for the calculation of specific activity. After three rounds of directed evolution, one mutant (Mu322) showed 1000 times the total activity and 20 times the specific activity of the wild-type enzyme produced by the same yeast expression system. Comparison of the amino acid sequence of this mutant with the wild type revealed five substitutions: Q44H, R303K and F325Y in domain A, and T94A and R128Q in domain B. Two of these mutations. Q44H and R303K, result in amino acids highly conserved in cereal alpha-amylases. R303K and F325Y are located in the raw starch-binding fragment of the enzyme molecule.

Amino Acid Sequence↗

PubMed1995

Evolutionary origins of apoB mRNA editing: catalysis by a cytidine deaminase that has acquired a novel RNA-binding motif at its active site.

The site-specific C to U editing of apolipoprotein B100 (apoB100) mRNA requires a 27 kDa protein (p27) with homology to cytidine deaminase. Here, we show that p27 is a zinc-containing deaminase, which operates catalytically like the E. coli enzyme that acts on monomeric substrate. In contrast with the bacterial enzyme that does not bind RNA, p27 interacts with its polymeric apoB mRNA substrate at AU sequences adjacent to the editing site. This interaction is necessary for editing. RNA binding is mediated through amino acid residues involved in zinc coordination, in proton shuttling, and in forming the alpha beta alpha structure that encompasses the active site. However, certain mutations that inactivate the enzyme do not affect RNA binding. Thus, RNA binding does not require a catalytically active site. The acquisition of polymeric substrate binding provides a route for the evolution of this editing enzyme from one that acts on monomeric substrates.

APOBEC-1 Deaminase↗