UCL biochemical engineering.
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Biochemistry↗
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Signal amplification by allosteric catalysis.
In this article we unify a series of recent studies on bio- and chemosensors under a single signaling strategy: signal amplification by allosteric catalysis (SAAC). The SAAC strategy mimics biological signal transduction processes, where molecular recognition between an external signal and a protein receptor is allosterically transduced into catalytically amplified chemical information (usually second messengers). Several recent biosensing and chemosensing studies apply this nature-inspired strategy by using engineered allosteric enzymes, ribozymes, or regulatable organic catalysts. The factors pertinent to achieving high sensitivity and specificity in SAAC strategies are analyzed. The authors believe that these early studies from a variety of research groups have opened up a new venue for the development of sensing technologies where molecular recognition and catalysis can be coupled for practical purposes.
Allosteric Regulation↗
Covalent tethering of the dimer interface annuls aggregation in thymidylate synthase.
Thymidylate synthase (TS), a dimeric enzyme, forms large soluble aggregates at concentrations of urea (3.3-5M), well below that required for complete denaturation, as established by fluorescence and size-exclusion chromatography. In contrast to the wild-type enzyme, an engineered mutant of TS (T155C/E188C/C244T), TSMox, in which two subunits are crosslinked by disulfide bridges between residues 155-188' and 188-155' does not show this behavior. Aggregation behavior is restored upon disulfide bond reduction in the mutant protein, indicating the involvement of interface segments in forming soluble associated species. Intermolecular disulfide crosslinking has been used as a probe to investigate the formation of larger non-native aggregates. The studies argue for the formation of large multimeric species via a sticky patch of polypeptide from the dimer interface region that becomes exposed on partial unfolding. Covalent reinforcement of relatively fragile protein-protein interfaces may be a useful strategy in minimizing aggregation of non-native structures in multimeric proteins.
Bacterial Proteins↗
Use of a biomimetic peptide in the design of a competitive binding assay for biotin and biotin analogues.
A competitive binding assay for biotin, biocytin, and desthiobiotin utilizing a genetically engineered enzyme-ligand conjugate is described herein. This assay is unique in that the enzyme-ligand conjugate consists of the streptavidin binding peptide Strep-tag II, which mimics the binding of biotin to streptavidin, rather than biotin itself. This allows for the construction of a well-defined, oligosubstituted enzyme-ligand conjugate for which the site of attachment of the ligand on the enzyme is known precisely. The assay has detection limits of 5 x 10(-8) M for biotin, 1 x 10(-7) M for biocytin, and 2 x 10(-6) M for desthiobiotin, and it serves as a model system in that it demonstrates the feasibility of using enzyme-ligand conjugates in which a peptide mimic of the analyte ligand is genetically fused to the enzyme. This avoids the problems associated with covalent attachment of the ligand to the enzyme, such as multiple substitution of the ligand and variability of the site of attachment. To our knowledge, this is the first example of using an enzyme-peptide mimic conjugate to detect a nonpeptide analyte.
Amino Acid Sequence↗
Enzymatic properties of newly found green turtle egg white ribonuclease.
Egg white ribonuclease was first found in green turtle eggs. The general properties were studied on substrate specificity, the optimum pH and temperature, and the effect of pH and temperature on the RNase activity. The enzyme studied was specific for poly (C) and degraded poly (U) at the lower rate and had the pH optimum at 7.0 and the optimum temperature at 40 degrees C. It was stable at alkaline range (pH 8.0-10.0) and up to 60 degrees C in pH 9.0 for 1 h, and unstable at acidic side for all temperatures. All of the properties studied showed similarity to RNase A. However, the optimum pH, broad range of optimum temperature and pH stability were different from RNase A. To evaluate the relationship of the structure and enzymatic properties, the 3D-structure of this enzyme was engineered by program MODELLER using two RNases (2BWL and 2BLZ) as starting models. The differences found in activity might be affected from the structure of micro environmental changing caused by amino acids deletion and substitution on the molecule.
Amino Acid Sequence↗
Biosynthesis of lovastatin analogs with a broadly specific acyltransferase.
The natural product lovastatin and its semisynthetic, more effective derivative, simvastatin, are important drugs for the treatment of hypercholesterolemia. Here, we report the biochemical characterization of a dedicated acyltransferase, LovD, encoded in the lovastatin biosynthetic pathway. We demonstrate that LovD has broad substrate specificity towards the acyl carrier, the acyl substrate, and the decalin acyl acceptor. LovD can efficiently catalyze the acyl transfer from coenzyme A thioesters or N-acetylcysteamine (SNAC) thioesters to monacolin J. When alpha-dimethylbutyryl-SNAC was used as the acyl donor, LovD was able to convert monacolin J and 6-hydroxyl-6-desmethylmonacolin J into simvastatin and huvastatin, respectively. Using the Escherichia coli LovD overexpression strain as a whole-cell biocatalyst, preparative amounts of simvastatin were synthesized in a single fermentation step. Our results demonstrate LovD is an attractive enzyme for engineered biosynthesis of pharmaceutically important cholesterol-lowering drugs.
Acyl Coenzyme A↗
In-vitro protein evolution by ribosome display and mRNA display.
In-vitro display technologies combine two important advantages for identifying and optimizing ligands by evolutionary strategies. First, by obviating the need to transform cells in order to generate and select libraries, they allow a much higher library diversity. Second, by including PCR as an integral step in the procedure, they make PCR-based mutagenesis strategies convenient. The resulting iteration between diversification and selection allows true Darwinian protein evolution to occur in vitro. We describe two such selection methods, ribosome display and mRNA display. In ribosome display, the translated protein remains connected to the ribosome and to its encoding mRNA; the resulting ternary complex is used for selection. In mRNA display, mRNA is first translated and then covalently bonded to the protein it encodes, using puromycin as an adaptor molecule. The covalent mRNA-protein adduct is purified from the ribosome and used for selection. Successful examples of high-affinity, specific target-binding molecules selected by in-vitro display methods include peptides, antibodies, enzymes, and engineered scaffolds, such as fibronectin type III domains and synthetic ankyrins, which can mimic antibody function.
Animals↗
Investigation of toxic metabolites during drug development.
Adverse drug reactions (ADRs) are a significant human health problem. Any organ system can be affected, including the liver, skin and kidney. Drug-induced liver injury is the most frequent reason for the withdrawal of an approved drug from the market, and it also accounts for up to 50% of cases of acute liver failure. The clinical picture is often diverse, even for the same drug. Mild, asymptomatic effects occur at a relatively high frequency with a number of drugs. Idiosyncratic toxicity is rare but potentially life-threatening. Many serious ADRs that occur in man are unpredictable from routine pathology and clinical chemistry in laboratory animals and are therefore poorly understood. The drug metabolist can determine the propensity of a novel chemical entity to either accumulate in the hepatocyte or undergo bioactivation in numerous model systems, from expressed enzymes, genetically engineered cells to whole animals. Bioactivation can be measured using trapping experiments with model nucleophiles or by measurement of non-specific covalent binding. The chemistry of the process is defined and the medicinal chemist can address the issue by seeking a metabolically stable pharmacophore to replace the potential toxicophore. However, we require a more fundamental understanding of the role of drug chemistry and biochemistry in ADRs. This requires knowledge of the ultimate toxin, signalling in cell defense and the sequence of molecular events, which ultimately lead to cell and tissue damage. It is imperative that such studies have a clinical level, but then translated into laboratory-based molecular studies. This will provide a deeper understanding of potential toxicophores for drug design and define candidate genes for pharmacogenomic approaches to individualized medicines.
Animals↗
Unique peptide modifications involved in the biosynthesis of lantibiotics.
The lantibiotics are a unique class of bacterium-derived peptide antibiotics, all of which contain the rare amino acid lanthionine, as well as a number of other nonprotein amino acids. Unlike most other peptide antibiotics, lantibiotics are produced on the ribosome as a prepeptide, and a series of post-translational modifications converts this precursor into the biologically active peptide. A complex set of gene products involved in lantibiotic biosynthesis have been identified, including the genes for specific amino acid modifications, as well as other ancillary biosynthetic functions. In the future, it should be possible to use some of these enzymes to engineer novel, non-protein amino acids into other proteins of biotechnological interest and importance.
Alanine↗
Immobilization of P450 monooxygenase and chloroplast for use in light-driven bioreactors.
P450 monooxygenases exhibit great potential for application to bioreactors for the decomposition of various hydrophobic chemicals including pollutant compounds. P450-containing microsomes were immobilized in spinach chloroplasts for use in light-driven bioreactors. We tested three methods (entrapment, adsorption and cross-linking) to immobilize chloroplasts and yeast microsomes containing a genetically engineered fusion enzyme between rat P450 1A1 and yeast P450 reductase. Entrapment in agarose gave the best activity for the conversion of 7-ethoxycoumarin to 7-hydroxycoumarin under illumination of 6200 lx. We then tested three light-driven bioreactors (two column-type and one batch-type reactors developed) using the immobilized gels. A two-phase column-type reactor with separately immobilized microsomes and chloroplasts showed a higher conversion rate than a reactor with co-immobilization of both components. The reactor showed a turnover rate of 6.32 mol product/mol P450/min after a 40-min run, and 2.49 after a 180-min run. These turnover rates are higher than the values reported by others using immobilized microsomal P450s.
Journal Article↗
Covalent reinforcement of a fragile region in the dimeric enzyme thymidylate synthase stabilizes the protein against chaotrope-induced unfolding.
Urea and guanidinium chloride induced unfolding of thymidylate synthase, a dimeric enzyme, and engineered interface mutants have been monitored by circular dichroism, fluorescence, and size-exclusion chromatography. Equilibrium unfolding studies show biphasic transitions, with a plateau between 3.5 and 5 M urea, when monitored by far-UV CD and fluorescence energy transfer employing an (aminoethylamino) naphthalenesulfonyl (AEDANS) label at the active site residue, Cys198. AEDANS was also specifically incorporated at position Cys155 in the mutant protein T155C. Direct excitation of this extrinsic fluorophore in the wild type protein (labeled at Cys198) and mutant T155C (labeled at Cys155) showed remarkable differences in the unfolding profiles. C155 AEDANS has a transition centered at 3.5 M urea, which is in contrast to Cys 198 AEDANS (5.5 M urea). Unfolding studies monitored by following intrinsic fluorescence of Trp residues which are located in a small structural domain suggest that this region of the protein is intrinsically fragile. The stable equilibrium intermediate is identified to be an ensemble of partially unfolded aggregated species by gel filtration studies. The chaotrope-induced denaturation of TS appears to proceed through a partially unfolded intermediate that is stabilized by aggregation. Dissociation and loss of structure occur concomitantly at high denaturant concentrations. Introduction of two symmetrically positioned disulfide bridges across the dimer interface in the triple mutant T155C/E188C/C244T (TSMox) stabilized the protein against denaturant-induced unfolding. Aggregate formation was completely abolished in the mutant TSMox, which also enhanced the overall structural stability of the protein. Structural reinforcement of the fragile interface in thymidylate synthase results in dramatic stabilization toward chaotrope-induced unfolding.
Binding Sites↗
Biomimetic synthesis of a H2 catalyst using a protein cage architecture.
A biomimetic approach has been used to develop an artificial hydrogenase that catalyses the efficient reduction of protons producing hydrogen gas. Analogous to the unique biological metal clusters found in hydrogenase enzymes, the engineered active sites are small, well-defined Pt clusters deposited on the interior of a heat shock protein cage architecture with stoichiometries of 150 to 1000 Pt per protein cage. The proton reduction reaction is driven by visible light through a coupled reaction with Ru(bpy)3(2+) and methyl viologen as an electron-transfer mediator. Hydrogen production rates are comparable to those of hydrogenase on a per protein basis and exceed production rates of other reported Pt-based catalysts. These results demonstrate the utility of a biomimetic approach toward addressing the needs of hydrogen production.
Archaeal Proteins↗
A synthetic peptide ligase.
The preparation of synthetic molecules showing the remarkable efficiencies characteristic of natural biopolymer catalysts remains a formidable challenge for chemical biology. Although significant advances have been made in the understanding of protein structure and function, the de novo construction of such systems remains elusive. Re-engineered natural enzymes and catalytic antibodies, possessing tailored binding pockets with appropriately positioned functional groups, have been successful in catalysing a number of chemical transformations, sometimes with impressive efficiencies. But efforts to produce wholly synthetic catalytic peptides have typically resulted in compounds with questionable structural stability, let alone reactivity. Here we describe a 33-residue synthetic peptide, based on the coiled-coil structural motif, which efficiently catalyses the condensation of two shorter peptide fragments with high sequence- and diastereoselectivity. Depending on the substrates used, we observe rate enhancements of tenfold to 4,100-fold over the background, with catalytic efficiencies in excess of 10(4). These results augur well for the rational design of functional peptides.
Amino Acid Sequence↗
Gene delivery from the E3 region of replicating human adenovirus: evaluation of the ADP region.
Genetically modified replication-selective human adenoviruses are currently undergoing testing in the clinical setting as anticancer agents. Coupling the lytic function of these viruses with virus-mediated transgene delivery represents a powerful extension of this treatment. We have designed a unique system for gene delivery from the replicating virus. It takes advantage of the endogenous gene expression control sequences (promoter, splicing, polyadenylation signals) to efficiently and predictably deliver transgenes from the non-essential E3 transcription unit while still maintaining the expression of the remaining E3 genes in the multi-gene transcription unit. In this article, we engineered restriction enzyme sites into the virus genome selectively to delete the ADP gene and replace it with the therapeutic transgenes CD and TNFalpha. We demonstrate that: (1) transgene expression from this region mirrors the substituted ADP gene; (2) the loss of ADP in these viruses results in infected cells with extended viability and protein synthesis when compared with a wild-type Ad5 infected cell; and (3) expression of surrounding E3 genes can be maintained in such a system. The potential advantages of delivering transgenes from the ADP region of the replicating adenovirus are discussed.
Adenosine Diphosphate↗
Semisynthesis of cytochrome c analogues. The effect of modifying the conserved residues 38 and 39.
We have used chemical and enzymic protein engineering techniques to create analogues of the semisynthetic two-fragment complex (1-37).(38-104) of mitochondrial cytochrome c. This complex, unlike the natural product of specific tryptic cleavage, (1-38).(39-104), from which it is prepared, quite closely resembles the parent protein in functional characteristics and is thus a suitable substrate for modifications designed to study structure-function relations. We have replaced the invariant Arg-38 and the conserved Lys-39 with a range of alternative amino acids and have studied the effects on the principal functional parameters. The hydrogen-bonding capacity of Arg-38 is crucial to the stabilization of the bottom omega-loop, while the positive charge of Lys-39 helps maintain the high redox potential by electrostatic effects at the haem iron.
Amino Acids↗
Assembly of aryl-capped siderophores by modular peptide synthetases and polyketide synthases.
Bacterial siderophores assist pathogens in iron acquisition inside their hosts. They are often essential for achieving a successful infection, and their biosynthesis represents an attractive antibiotic target. Recently, several siderophore biosynthetic loci have been identified, and in vitro studies have advanced our knowledge of the biosynthesis of aryl-capped peptide and peptide-polyketide siderophores from Mycobacterium spp., Pseudomonas spp., Yersinia spp. and other bacteria. These studies also provided insights into the assembly of related siderophores and many secondary metabolites of medical relevance. Assembly of aryl-capped peptide and peptide-polyketide siderophores involves non-ribosomal peptide synthetase, polyketide synthase and non-ribosomal-peptide polyketide hybrid subunits. Analysis of these subunits suggests that their domains and modules are functionally and structurally independent. It appears that nature has selected a set of functional domains and modules that can be rearranged in different order and combinations to biosynthesize different products. Although much remains to be learned about modular synthetases and synthases, it is already possible to conceive strategies to engineer these enzymes to generate novel products.
Bacteria↗
Exploring the O-GlcNAc proteome: direct identification of O-GlcNAc-modified proteins from the brain.
The covalent modification of intracellular proteins by O-linked beta-N-acetylglucosamine (O-GlcNAc) is emerging as a crucial regulatory posttranslational modification akin to phosphorylation. Numerous studies point to the significance of O-GlcNAc in cellular processes such as nutrient sensing, protein degradation, and gene expression. Despite its importance, the breadth and functional roles of O-GlcNAc are only beginning to be elucidated. Advances in our understanding will require the development of new strategies for the detection and study of O-GlcNAc-modified proteins in vivo. Herein we report the direct, high-throughput analysis of O-GlcNAc-glycosylated proteins from the mammalian brain. The proteins were identified by using a chemoenzymatic approach that exploits an engineered galactosyltransferase enzyme to selectively label O-GlcNAc proteins with a ketone-biotin tag. The tag permits enrichment of low-abundance O-GlcNAc species from complex mixtures and localization of the modification to short amino acid sequences. Using this approach, we discovered 25 O-GlcNAc-glycosylated proteins from the brain, including regulatory proteins associated with gene expression, neuronal signaling, and synaptic plasticity. The functional diversity represented by this set of proteins suggests an expanded role for O-GlcNAc in regulating neuronal function. Moreover, the chemoenzymatic strategy described here should prove valuable for identifying O-GlcNAc-modified proteins in various tissues and facilitate studies of the physiological significance of O-GlcNAc across the proteome.
Acetylglucosamine↗
Synthesis, bacterial expression, and mutagenesis of the gene coding for mammalian cytochrome b5.
We have totally synthesized a gene that codes for rat hepatic cytochrome b5. The 5' flanking region was designed for efficient expression of this gene in Escherichia coli by incorporating an optimum ribosome binding site and spacer region. Both a soluble form, analogous to the protease-treated microsomal protein, as well as the complete cytochrome with hydrophobic membrane anchor, was constructed and expressed. Transformants with the gene for the soluble protein overproduce authentic cytochrome b5 to a level of 8% of the total cell protein. The complete cytochrome is expressed to a lesser extent with most of the protein found in the cell membrane fraction. This represents complete synthesis and bacterial expression of a mammalian metalloprotein gene. Cytochrome b5 is normally a six-coordinate low spin heme protein with histidine-39 and histidine-63 as axial ligands. We have replaced histidine-63 with a methionine residue by cassette mutagenesis, utilizing specific restriction enzyme sites engineered into the synthetic gene. The resultant protein has histidine-39 as sole axial ligand and is five-coordinate high spin in the ferric resting state, as indicated by optical and electron spin resonance spectroscopy. The ability to generate mutant cytochrome b5 in high yield is a crucial step in understanding heme protein folding, protein-protein recognition and binding, and biological electron transfer processes.
Animals↗