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PubMed1997

A decade of protein engineering on ribonuclease T1--atomic dissection of the enzyme-substrate interactions.

During the last decade, protein engineering has been used to identify the residues that contribute to the ribonuclease-T1-catalyzed transesterification. His40, Glu58 and His92 accelerate the associative nucleophilic displacement at the phosphate atom by the entering 2'-oxygen downstream guanosines in a highly cooperative manner. Glu58, assisted by the protonated His40 imidazole, abstracts a proton from the 2'-oxygen, while His92 protonates the leaving group. Tyr38, Arg77 and Phe100 further stabilize the transition state of the reaction. A functionally independent subsite, including Asn36 and Asn98, contributes to chemical turnover by aligning the substrate relative to the catalytic side chains upon binding of the leaving group. An invariant structural motive, involving residues 42-46, renders ribonuclease T1 guanine specific through a series of intermolar hydrogen bonds. Tyr42 contributes significantly to guanine binding through a parallel face-to-face stacking interaction. Tyr45, often referred to as the lid of the guanine-binding site, does not contribute to the binding of the base.

Guanine↗

PubMed1997

Microbial ribulose 1,5-bisphosphate carboxylase/oxygenase: a molecule for phylogenetic and enzymological investigation.

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) catalyzes the key reaction of the Calvin reductive pentose phosphate cycle and as such is responsible for life as we know it. This enzyme has been intensively studied for decades. Evidence that RubisCO phylogenies are incongruent with those derived from other macromolecules has been accumulating and recent discoveries have driven home this point. Here we review findings regarding RubisCO phylogeny and discuss these in the context of the important biochemical and structural features of the enzyme. The implications for the engineering of improved RubisCO enzymes are considered.

Cyanobacteria↗

PubMed2002

Application potency of engineered G159 mutants on P1 substrate pocket of subtilisin YaB as improved meat tenderizers.

A serine protease, subtilisin YaB, produced by alkalophilic Bacillus YaB, shows promises as a potent meat tenderizer, because its substrate specificity is for small amino acids, which are found at high levels in meat connective tissue proteins. Substrate specificity engineering of the substrate binding pockets was used to generate more suitable meat-tenderizing mutants, G124A, G124V, G159A, and G159S, derived from recombinant wild subtilisin YaB and expressed in Bacillus subtilis DB104. The characteristics of these recombinant enzymes were studied to evaluate their usefulness as improved meat tenderizers. The proteolytic activities of recombinant subtilisin YaB, engineered subtilisin YaBs, and commercially available papain, bromelain, collagenase, and elastase were compared using elastin, collagen, casein, and myofibrillar proteins as substrates. Hydrolysis of beef proteins was evaluated using the myofibrillar fragmentation index and collagen solubility. The results demonstrated that recombinant mutant G159A was the most improved meat tenderizer and can be used in the meat pH range of 5.5-6.0 and the temperature range of 10-50 degrees C. Contrary to the result obtained from artificial substrate, mutant enzymes engineered on G124 residues did not exhibit better tenderizing ability when elastin, collagen, or meat was used as substrate, suggesting the necessity of evaluation by real substrate before protein-engineered enzymes are applied commercially.

Animals↗

PubMed1990

Site directed mutagenesis: a tool for enzyme mechanism dissection.

Protein engineering has become the principle means of examining the active site of an enzyme to identify and quantify the roles of specific residues in ligand binding, specificity and catalysis. Site-specific mutagenesis has extended our knowledge gained from X-ray crystallography, and has provided striking proof that the intricate active-site geometry is supported by the remainder of the protein infrastructure for maximum catalytic efficiency.

Animals↗

PubMed2006

Engineering isoflavone metabolism with an artificial bifunctional enzyme.

Plant secondary metabolism has been a focus of research in recent years due to its significant roles in plant defense and in human medicine and nutrition. A protein engineering strategy was designed to more effectively manipulate plant secondary metabolite (isoflavonoid) biosynthesis. A bifunctional isoflavone synthase/chalcone isomerase (IFS/CHI) enzyme was constructed by in-frame gene fusion, and expressed in yeast and tobacco. The fusion protein was targeted to the endoplasmic reticulum (ER) membrane and the individual enzymatic functions of its component fragments were retained when assayed in yeast. Petals and young leaves of IFS/CHI transgenic tobacco plants produced higher levels of the isoflavone genistein and genistein glycosides as a ratio of total flavonoids produced than did plants transformed with IFS alone. Thus, through a combined molecular modeling, in vitro protein engineering and in planta metabolic engineering approach, it was possible to increase the potential for accumulation of isoflavonoid compounds in non-legume plants. Construction of bifunctional enzymes will simplify the transformation of plants with multiple pathway genes, and such enzymes may find broad uses for enzyme (e.g., cytochrome P450 family) and biochemical pathway engineering.

Artificial Gene Fusion↗

PubMed2005

Medical bioremediation: prospects for the application of microbial catabolic diversity to aging and several major age-related diseases.

Several major diseases of old age, including atherosclerosis, macular degeneration and neurodegenerative diseases are associated with the intracellular accumulation of substances that impair cellular function and viability. Moreover, the accumulation of lipofuscin, a substance that may have similarly deleterious effects, is one of the most universal markers of aging in postmitotic cells. Reversing this accumulation may thus be valuable, but has proven challenging, doubtless because substances resistant to cellular catabolism are inherently hard to degrade. We suggest a radically new approach: augmenting humans' natural catabolic machinery with microbial enzymes. Many recalcitrant organic molecules are naturally degraded in the soil. Since the soil in certain environments - graveyards, for example - is enriched in human remains but does not accumulate these substances, it presumably harbours microbes that degrade them. The enzymes responsible could be identified and engineered to metabolise these substances in vivo. Here, we survey a range of such substances, their putative roles in age-related diseases and the possible benefits of their removal. We discuss how microbes capable of degrading them can be isolated, characterised and their relevant enzymes engineered for this purpose and ways to avoid potential side-effects.

Aging↗

PubMed1993

Designer enzyme and cell applications in industry and in environmental monitoring.

Renewed world interest in enzyme biotechnological industries now derives from the expectation that many new biocatalysts will be created by genetic engineering associated with protein engineering designer techniques, or by chemical modification of existing enzymes by use of protein tailoring methods. The biocatalysts produced are mainly enzymes, abzymes (catalytic antibodies) and synzymes (synthetic analogues or mimics), and these will be used in industry, synthesis, therapy: and in bioanalysis of components of foodstuffs, and the environment including water, air and soil. The biocatalysts, including whole cells, are firstly incorporated into a particular bioreactor form by use of enzyme engineering techniques such as immobilization, and are then used, as appropriate, to modify their substrates. Improved processing or enhanced products are thereby achieved in the case of manufacturing industry: or monitoring signals are generated, often in the form of a measurable change in current flow, in the case of environmental biosensors. Designer enzymes and cells can be made now for identified applications where the presently available biocatalysts are inadequate, incompatible or uncompetitive.

Biotechnology↗

PubMed1999

Rational design of a scytalone dehydratase-like enzyme using a structurally homologous protein scaffold.

The generation of enzymes to catalyze specific reactions is one of the more challenging problems facing protein engineers. Structural similarities between the enzyme scytalone dehydratase with nuclear transport factor 2 (NTF2) suggested the potential for NTF2 to be re-engineered into a scytalone dehydratase-like enzyme. We introduced four key catalytic residues into NTF2 to create a scytalone dehydratase-like active site. A C-terminal helix found in scytalone dehydratase but absent in NTF2 also was added. Mutant NTF2 proteins were tested for catalytic activity by using a spectroscopic assay. One of the engineered enzymes exhibited catalytic activity with minimal kcat and Km values of 0.125 min-1 and 800 microM, respectively. This level of catalytic activity represents minimally a 150-fold improvement in activity over the background rate for substrate dehydration and a dramatic step forward from the catalytically inert parent NTF2. This work represents one of the few examples of converting a protein scaffold into an enzyme, outside those arising from the induction of catalytic activity into antibodies.

Amino Acid Sequence↗

PubMed2002

[Study on fermentation condition of alkaline protease gene engineering strain and the purification and characterization of recombinant enzyme].

In a 5L fermentor the production conditions of alkaline protease gene engineering strain BA071 were investigated. The maximum activity of alkaline protease reached 24,480 u/mL in 40 hours of fermentation by combination of enhancing aeration and changing the agitation rate. The fast purification method of recombinant protease was conducted with FPLC (Fast Protein Liquid Chromatography). The crude enzyme, treated with ammonium sulfate fractionation and decolored with DEAE-A-50 and polyethylene glycol concentration, was purified with CM-Sephadex-C-50 and Sephadex-G-75. The purified enzyme appears homologous on SDS-PAGE. The purity of enzyme was increased 76.2 times. SDS-PAGE analysis showed that the molecular weights of expressed recombinant products were about 28 kD. The optimal reaction pH and temperature of recombinant enzyme were at pH11 and 60 degrees C, respectively. The recombinant enzyme exhibited high temperature tolerance and was stable at a wide range of pH. Ca2+, MG2+ can enhance the stability of the recombinant enzyme. While the protease activity of the enzyme was strongly inhibited by Hg2+, Ag+, PMFS [symbol: see text] DFP, and was not affected by SDS and Urea.

Bacillus↗

PubMed2006

Role of Na+ and K+ in enzyme function.

Metal complexation is a key mediator or modifier of enzyme structure and function. In addition to divalent and polyvalent metals, group IA metals Na+ and K+ play important and specific roles that assist function of biological macromolecules. We examine the diversity of monovalent cation (M+)-activated enzymes by first comparing coordination in small molecules followed by a discussion of theoretical and practical aspects. Select examples of enzymes that utilize M+ as a cofactor (type I) or allosteric effector (type II) illustrate the structural basis of activation by Na+ and K+, along with unexpected connections with ion transporters. Kinetic expressions are derived for the analysis of type I and type II activation. In conclusion, we address evolutionary implications of Na+ binding in the trypsin-like proteases of vertebrate blood coagulation. From this analysis, M+ complexation has the potential to be an efficient regulator of enzyme catalysis and stability and offers novel strategies for protein engineering to improve enzyme function.

Animals↗

PubMed1998

Engineering of a stable mutant malic enzyme by introducing an extra ion-pair to the protein.

A double mutant (R9E/M17K) of pigeon liver malic enzyme with glutamate and lysine replaced for arginine and methionine at positions 9 and 17, respectively, was found to be much more stable in urea and thermal denaturation, but was enzymatically less active than the wild-type enzyme (WT). Unfolding of the enzyme by urea produced a large red shifting of the protein fluorescence maximum from 320 to 360 nm, which was completely reversible upon dilution. Analysis of the denaturation curves monitored by enzyme activity lost suggested that a putative intermediate was involved in the denaturation process. The half unfolding urea concentration, measured by fluorescence spectral changes, increased from 2.24 M for WT to 3.13 M for R9E/M17K. The melting temperature increased by approximately 10 degrees C for R9E/M17K compared with that for WT. Kinetic analysis of the thermal inactivation at 58 degrees C also conformed to a three-state model with the rate constant for the intermediate state of R9E/M17K (k2 = 0.03 min(-1)) being much smaller than the WT value (k2 = 2.39 min(-1)). Results obtained from single mutants indicated that the decreasing enzyme activity of R9E/M17K was exclusively due to R9 mutation, which increased the K(mMn) and K(mMal) by at least one order of magnitude compared with WT. Consequently, a decrease occurred in the specificity constant [k(cat)/(K(mMm)K(mNADP)K(mMal))] for the R9 mutants at least four orders of magnitude smaller than the WT. M17K has similar properties to the WT, while R9E is more labile than the WT enzyme. The above results indicate that the extra stability gained by the double mutant possibly occurs through the introduction of an extra ion-pair between E9 and K17, which freezes the double mutant in the putative intermediate state. Examination of the N-terminal amino acid sequence of pigeon liver malic enzyme reveals that position 15 is also a lysine residue. Since the R9E mutant, which has an extra Glu9-Lys15 ion-pair, is less stable than the WT, we conclude that the contribution to malic enzyme stability is specific for the Glu9-Lys17 ion-pair.

Amino Acid Sequence↗

PubMed1991

Protein engineering to change thermal stability for food enzymes.

In this review we have briefly indicated how the present state of knowledge allows proteins to be mutated to increase or decrease stability. We have discussed experiments on both model proteins and those of relevance to the food industry, and show how hydrophobic forces are a major driving force for folding as well as having a major role in thermostability. We have also indicated the large contribution that hydrogen bonding, electrostatic interactions and, in a less well predicted way, disulphide bridges make to thermostability.

Enzyme Stability↗

PubMed2004

Reliable high-throughput screening with Pichia pastoris by limiting yeast cell death phenomena.

Comparative screening of gene expression libraries employing the potent industrial host Pichia pastoris for improving recombinant eukaryotic enzymes by protein engineering was an unsolved task. We simplified the protocol for protein expression by P. pastoris and scaled it down to 0.5-ml cultures. Optimising standard growth conditions and procedures, programmed cell death and necrosis of P. pastoris in microscale cultures were diminished. Uniform cell growth in 96-deep-well plates now allows for high-throughput protein expression and screening for improved enzyme variants. Furthermore, the change from one host for protein engineering to another host for enzyme production becomes dispensable, and this accelerates the protein breeding cycles and makes predictions for large-scale production more accurate.

Apoptosis↗

PubMed2006

Improvement of the fungal enzyme pyranose 2-oxidase using protein engineering.

Native pyranose 2-oxidase (P2Ox) was purified from Peniophora sp. and characterized. To improve its catalytic efficiencies and stabilities by protein engineering, we cloned and expressed the P2Ox gene in Escherichia coli and received active, fully flavinylated recombinant P2OxA. Selenomethionine-labeled P2OxA was used for X-ray analysis and the resulting crystal structure enabled the rational design using variant P2OxA1 with the substitution E542K as template. Besides increased thermal and pH stabilities this variant showed improved catalytic efficiencies (k(cat)/K(m)) for the main substrates. A new variant, P2OxA2H, with an additional substitution T158A and a C-terminal His(6)-tag exhibited significantly decreased apparent K(m) values for D-glucose (0.47 mM), l-sorbose (1.79 mM), and D-xylose (1.35 mM). Compared to native P2Ox, the catalytic efficiencies were substantially improved for D-glucose (230-fold), L-sorbose (874-fold), and D-xylose (1751-fold). This P2Ox variant was used for the bioconversion of L-sorbose under O(2)-saturation in a molar scale. The structure-activity relationships of the amino acid substitutions were analyzed by modelling of the mutated P2Ox structures. Molecular docking calculations of various carbohydrates into the crystal structure of P2OxA and the analysis of the protein-ligand interactions in the docked complexes enabled us to explain the substrate specificity of the enzyme by a conserved hydrogen bond pattern which is formed between the protein and all substrates.

Amino Acid Sequence↗

PubMed1994

Engineering the quaternary structure of an enzyme: construction and analysis of a monomeric form of malate dehydrogenase from Escherichia coli.

The citric acid cycle enzyme, malate dehydrogenase (MDH), is a dimer of identical subunits. In the crystal structures of 2 prokaryotic and 2 eukaryotic forms, the subunit interface is conformationally homologous. To determine whether or not the quaternary structure of MDH is linked to the catalytic activity, mutant forms of the enzyme from Escherichia coli have been constructed. Utilizing the high-resolution structure of E. coli MDH, the dimer interface was analyzed critically for side chains that were spatially constricted and needed for electrostatic interactions. Two such residues were found, D45 and S226. At their nearest point in the homodimer, they are in different subunits, hydrogen bond across the interface, and do not interact with any catalytic residues. Each residue was mutated to a tyrosine, which should disrupt the interface because of its large size. All mutants were cloned and purified to homogeneity from an mdh- E. coli strain (BHB111). Gel filtration of the mutants show that D45Y and D45Y/S226Y are both monomers, whereas the S226Y mutant remains a dimer. The monomeric D45Y and D45Y/S226Y mutants have 14,000- and 17,500-fold less specific activity, respectively, than the native enzyme. The dimeric S226Y has only 1.4-fold less specific activity. All forms crystallized, indicating they were not random coils. Data have been collected to 2.8 A resolution for the D45Y mutant. The mutant is not isomorphous with the native protein and work is underway to solve the structure by molecular replacement.

Amino Acid Sequence↗

PubMed2004

Enzymatic bioremediation: from enzyme discovery to applications.

1. Enzymatic bioremediation is potentially a rapid method of removing environmental pesticide residues. Applications include the treatment of residues resulting from agricultural production and processing industries, such as the treatment of irrigation waters, surface-contaminated fruit and vegetables and spent dip liquors. 2. A specific application for some organophosphate-degrading enzymes involves detoxification of nerve agent stockpiles. Effective and affordable remediation requires highly specialized enzymes, so protein engineering techniques are being used to improve properties of various source enzymes to enhance catalytic rates, stability and substrate range. 3. Trials with an optimized organophosphate-degrading enzyme have shown the feasibility of such technology in various applications. 4. The enzymes developed for environmental remediation for specific pesticide classes also have applications as antidotes for high-dose pesticide poisonings and as prophylaxis for people at risk of high pesticide doses.

Animals↗

PubMed1996

Modelling and engineering of enzyme/substrate interactions in subtilisin-like enzymes of unknown 3-dimensional structure.

Homology modelling was used to predict enzyme-substrate interactions in three entirely different subtilisin-like enzymes of unknown three-dimensional structure. i.e. (a) cell-envelope proteinase of Lactococcus lactis, (b) putative leader peptidase for pre-nisin from L. lactis, and (c) human furin. Models were based on known three-dimensional structures of subtilisins and thermitase in complex with inhibitors. Detailed analysis of interactions of the P1-P4 residues of model substrates with the S1-S4 binding sites in each enzyme suggest that electrostatic interactions at all four binding sites can contribute to binding and hence to specificity. In particular, one or more negative charges in the S1 or S4 pockets can lead to a high selectivity for Arg residues in the substrate. Many of the predicted interactions have been confirmed by engineering of either enzyme, substrate or both.

Amino Acid Sequence↗

PubMed2006

[Key enzymes in starch synthesis in plants].

Starch, the most common form of stored carbon in plants, is both the major food source for mankind and important raw material for many industries. It is composed of two types of alpha-1,4-linked glucan polymer: essentially unbranched amylose and regularly branched amylopectin, and synthesized in photosynthetic and non-photosynthetic organs. Starch is synthesized via four committed enzyme steps: ADP-Glc pyrophosphorylase, which synthesizes sugar nucleotide precursors; starch synthase, which extends the alpha-1,4-linked glucan chains using ADP-Glc; starch-branching enzymes, which introduce alpha-1,6 branch points to form amylopectin; and starch debranching enzymes, which hydrolyze alpha-1,6 branches in glucans. In this paper, recent advances in biochemical characterizations and gene engineering concerning these enzymes were reviewed, and the achievements in gene engineering involved in manipulation of starch amount and quality were also cited.

1,4-alpha-Glucan Branching Enzyme↗