Studies on the substrate specificity of Escherichia coli galactokinase

In vitro glycorandomization (IVG) technology is dependent upon the ability to rapidly synthesize sugar phosphates. Compared with chemical synthesis, enzymatic (kinase) routes to sugar phosphates would be attractive for this application. This work focuses upon the development of a high-throughput colorimetric galactokinase (GalK) assay and its application toward probing the substrate specificity and kinetic parameters of Escherichia coli GalK. The demonstrated dinitrosalicylic assay should also be generally applicable to a variety of sugar-processing enzymes. [reaction: see text]     

Directed evolution of a gatekeeper domain in nonribosomal peptide synthesis

Modular natural products are biosynthesized by series of enzymes that activate, assemble, and process a nascent chain of building blocks. Adenylation domains are gatekeepers in nonribosomal peptide biosynthesis, providing the entry point for assembly of typical peptide-based natural products. We report the directed evolution of an adenylation domain based on a strategy of using a weak, promiscuous activity as a springboard for reprogramming the biosynthetic assembly line. Randomization of residues invoked in a "specificity-conferring code" and selection for a non-native substrate lead to mutant G2.1, favoring smaller amino acids with a specificity change of 10(5): a 170-fold improvement for L-alanine corresponds to a 10(3)-fold decrease for its original substrate (L-phenylalanine). These results establish directed evolution as a method to change gatekeeper domain specificity and suggest that adaptation of modules in combinatorial biosynthesis is achievable with few mutations during evolution.     

Engineered urdamycin glycosyltransferases are broadened and altered in substrate specificity

Combinatorial biosynthesis is a promising technique used to provide modified natural products for drug development. To enzymatically bridge the gap between what is possible in aglycon biosynthesis and sugar derivatization, glycosyltransferases are the tools of choice. To overcome limitations set by their intrinsic specificities, we have genetically engineered the protein regions governing nucleotide sugar and acceptor substrate specificities of two urdamycin deoxysugar glycosyltransferases, UrdGT1b and UrdGT1c. Targeted amino acid exchanges reduced the number of amino acids potentially dictating substrate specificity to ten. Subsequently, a gene library was created such that only codons of these ten amino acids from both parental genes were independently combined. Library members displayed parental and/or a novel specificity, with the latter being responsible for the biosynthesis of urdamycin P that carries a branched saccharide side chain hitherto unknown for urdamycins.     

Studying enzyme binding specificity in acetylcholinesterase using a combined molecular dynamics and multiple docking approach

A combined molecular dynamics simulation and multiple ligand docking approach is applied to study the binding specificity of acetylcholinesterase (AChE) with its natural substrate acetylcholine (ACh), a family of substrate analogues, and choline. Calculated docking energies are well correlated to experimental k(cat)/K(M) values, as well as to experimental binding affinities of a related series of TMTFA inhibitors. The "esteratic" and "anionic" subsites are found to act together to achieve substrate binding specificity. We find that the presence of ACh in the active site of AChE not only stabilizes the setup of the catalytic triad but also tightens both subsites to achieve better binding. The docking energy gained from this induced fit is 0.7 kcal/mol for ACh. For the binding of the substrate tailgroup to the anionic subsite, both the size and the positive charge of the tailgroup are important. The removal of the positive charge leads to a weaker binding of 1 kcal/mol loss in docking energy. Substituting each tail methyl group with hydrogen results in both an incremental loss in docking energy and also a decrease in the percentage of structures docked in the active site correctly set up for catalysis.     

Two sequence elements of glycosyltransferases involved in urdamycin biosynthesis are responsible for substrate specificity and enzymatic activity.

BACKGROUND: Two deoxysugar glycosyltransferases (GTs), UrdGT1b and UrdGT1c, involved in urdamycin biosynthesis share 91% identical amino acids. However, the two GTs show different specificities for both nucleotide sugar and acceptor substrate. Generally, it is proposed that GTs are two-domain proteins with a nucleotide binding domain and an acceptor substrate site with the catalytic center in an interface cleft between these domains. Our work aimed at finding out the region responsible for determination of substrate specificities of these two urdamycin GTs. RESULTS: A series of 10 chimeric GT genes were constructed consisting of differently sized and positioned portions of urdGT1b and urdGT1c. Gene expression experiments in host strains Streptomyces fradiae Ax and XTC show that nine of 10 chimeric GTs are still functional, with either UrdGT1b- or UrdGT1c-like activity. A 31 amino acid region (aa 52-82) located close to the N-terminus of these enzymes, which differs in 18 residues, was identified to control both sugar donor and acceptor substrate specificity. Only one chimeric gene product of the 10 was not functional. Targeted stepwise alterations of glycine 226 (G226R, G226S, G226SR) were made to reintroduce residues conserved among streptomycete GTs. Alterations G226S and G226R restored a weak activity, whereas G226SR showed an activity comparable with other functional chimeras. CONCLUSIONS: A nucleotide sugar binding motif is present in the C-terminal moiety of UrdGT1b and UrdGT1c from S. fradiae. We could demonstrate that it is an N-terminal section that determines specificity for the nucleotide sugar and also the acceptor substrate. This finding directs the way towards engineering this class of streptomycete enzymes for antibiotic derivatization applications. Amino acids 226 and 227, located outside the putative substrate binding site, might be part of a larger protein structure, perhaps a solvent channel to the catalytic center. Therefore, they could play a role in substrate accessibility to it.     

A designed synthetic analogue of 4-OT is specific for a non-natural substrate

The substrate specificity of 4-oxalocrotonate tautomerase (4-OT) is characterized by electrostatic interactions between positively charged arginine (Arg) side chains on the enzyme and the dianionic substrate, 4-oxalocrotonate. To generate specific hydrogen-bonding interactions with a monoanionic substrate analogue, we have introduced a urea functional group into the active site by replacing arginine side chains with isosteric citrulline (Cit) residues. This design was based on the complementarity between the urea functionality of citrulline and the uncharged amide function of the substrate, as opposed to the guanidinium-carboxylate electrostatic interaction between the wild-type enzyme and the natural substrate. Indeed, the synthetic (Arg39Cit)4-OT analogue catalyzed the tautomerization of the non-natural monoamide-monoacid substrate while it was a poor catalyst for the natural diacid substrate. The specificity of (Arg39Cit)4-OT for the monoamide-monoacid substrate relative to that of the diacid substrate was found to be 740-fold greater than that of the wild-type enzyme for tautomerization of the non-natural substrate as compared with the natural one. The role of electrostatic interactions in the tautomerization of the monoamide-monoacid substrate was probed in detail with several other Arg to Cit analogues of this enzyme. This study has demonstrated that chemical manipulation of the functional groups within the active site of an enzyme can modify its catalytic activity and substrate specificity in a predictable way, suggesting that the incorporation of noncoded amino acids into proteins has great promise for the development of new enzymatic mechanisms and new binding interactions.     

Novel RNA catalysts for the Michael reaction

BACKGROUND: In vitro selected ribozymes with nucleotide synthase, peptide and carbon-carbon bond forming activity provide insight into possible scenarios on how chemical transformations may have been catalyzed before protein enzymes had evolved. Metabolic pathways based on ribozymes may have existed at an early stage of evolution. RESULTS: We have isolated a novel ribozyme that mediates Michael-adduct formation at a Michael-acceptor substrate, similar to the rate-limiting step of the mechanistic sequence of thymidylate synthase. The kinetic characterization of this catalyst revealed a rate enhancement by a factor of approximately 10(5). The ribozyme shows substrate specificity and can act as an intermolecular catalyst which transfers the Michael-donor substrate onto an external 20-mer RNA oligonucleotide containing the Michael-acceptor system. CONCLUSION: The ribozyme described here is the first example of a catalytic RNA with Michael-adduct forming activity which represents a key mechanistic step in metabolic pathways and other biochemical reactions. Therefore, previously unforeseen RNA-evolution pathways can be considered, for example the formation of dTMP from dUMP. The substrate specificity of this ribozyme may also render it useful in organic syntheses.     

A hyaluronidase supercatalyst for the enzymatic polymerization to synthesize glycosaminoglycans

Hyaluronidase (HAase) catalyzes multiple enzymatic polymerizations with controlling regio- and stereoselectivity perfectly. This behavior, that is, the single enzyme being effective for multireactions and retaining the enzyme catalytic specificity, is not usual, and hence, HAase is a supercatalyst. Various sugar oxazoline monomers prepared based on the concept "transition-state analogue substrate" were successfully polymerized and copolymerized with HAase catalysis, yielding natural and unnatural glycosaminoglycans.     

Genotyping single-nucleotide polymorphisms of human genes involved in organophosphate detoxification by high-resolution melting

Paraoxonase-1 (PON1) and butyrylcholinesterase (BCHE) are natural bioscavengers of organophosphate acetylcholinesterase inhibitors in the human body, which can determine individual sensitivity to organophosphate toxicity. Interindividual differences in activity of PON1 (catalytic bioscavenger) and substrate specificity are strongly associated with the substitution of two amino acids: Leu/Met (L/M) at position 55 (rs854560) and Gln/Arg (Q/R) at position 192 (rs662). In the case of BCHE (stoichiometric bioscavenger) substitution, Ala/Thr (A/T) at position 539 produces the so-called “K-variant” of the enzyme (rs1803274). Threonine allele is often co-inherited with an atypical BCHE allele (rs1799807). The atypical variant of BCHE displays a lower affinity for cholinesterase inhibitors. Genotyping rs662 and rs1803274 single-nucleotide polymorphisms (SNP) by high-resolution melting (HRM) is facilitated by the nucleotide substitution A>G (G>A), which resulted in a changed number of hydrogen bonds in the PCR product and, consequently, shifted T _m. In the case of rs854560, genotyping is complicated by the nucleotide substitution T>A, which has no significant effect on the T _m of the PCR product. An addition of a small quantity of LL homozygote DNA into the reaction mixture before PCR discriminates the three genotypes by the melt curves due to different amounts of heteroduplexes formed in the LM and MM samples. HRM analysis can be applied for genotyping human rs854560, rs662, and rs1803274 SNPs.

Adaptability at a protein-DNA interface: re-engineering the engrailed homeodomain to recognize an unnatural nucleotide

We explore the adaptability at a protein-DNA interface using phage display to re-engineer a homeodomain to specifically recognize a chemically synthesized, unnatural nucleotide. The engineered homeodomain preferentially binds to the modified DNA with affinity and specificity similar to those of natural homeodomains.     

The specificity-conferring code of adenylation domains in nonribosomal peptide synthetases.

BACKGROUND: Many pharmacologically important peptides are synthesized nonribosomally by multimodular peptide synthetases (NRPSs). These enzyme templates consist of iterated modules that, in their number and organization, determine the primary structure of the corresponding peptide products. At the core of each module is an adenylation domain that recognizes the cognate substrate and activates it as its aminoacyl adenylate. Recently, the crystal structure of the phenylalanine-activating adenylation domain PheA was solved with phenylalanine and AMP, illustrating the structural basis for substrate recognition. RESULTS: By comparing the residues that line the phenylalanine-binding pocket in PheA with the corresponding moieties in other adenylation domains, general rules for deducing substrate specificity were developed. We tested these in silico 'rules' by mutating specificity-conferring residues within PheA. The substrate specificity of most mutants was altered or relaxed. Generalization of the selectivity determinants also allowed the targeted specificity switch of an aspartate-activating adenylation domain, the crystal structure of which has not yet been solved, by introducing a single mutation. CONCLUSIONS: In silico studies and structure-function mutagenesis have defined general rules for the structural basis of substrate recognition in adenylation domains of NRPSs. These rules can be used to rationally alter the specificity of adenylation domains and to predict from the primary sequence the specificity of biochemically uncharacterized adenylation domains. Such efforts could enhance the structural diversity of peptide antibiotics such as penicillins, cyclosporins and vancomycins by allowing synthesis of 'unnatural' natural products.     

Screening for protease substrate by polyvalent phage display

Proteases are key regulators of many physiological and pathological processes [1,2], and are recognized as important and tractable drug candidates. Consequently, knowledge of protease substrate recognition and specificity promotes identification of biologically relevant substrates, helps elucidating a protease's biological function, and the design of specific inhibitors. Traditional methods for establishing substrate recognition profiles involve the identification of the scissile bond within a given protein substrate by proteomic methods such as Edman degradation. Then, synthetic peptide variants of this sequence can be screened in an iterative fashion to arrive at more optimized substrates. Even though it can be fruitful, this iterative strategy is biased toward the original substrate sequence and it is also tremendously cumbersome. Furthermore, it is not amenable to high throughput analysis. In 1993, Matthew & Wells presented a method for the use of monovalent "substrate phage" libraries for discovering peptide substrates for proteases, in which more than 10(7) potential substrates can be tested concurrently [3]. A library of fusion proteins was constructed containing randomized substrate sequences placed between a binding domain and the gene III coat protein of the filamentous phage, M13, which displays the fusion protein and packages the gene coding for it inside. Each fusion protein was displayed as a single copy on filamentous phagemid particles (substrate phage). This method allows one to rapidly survey the substrate recognition and specificity of individual or closely related members of proteases. Over the past decade, substrate phage screening has shown terrific utility in rapidly determining protease specificity and characterization of substrate recognition profile of proteases. In some cases, the structural insights of the catalytic domain were obtained from comparison of substrate specificity among closely related family of proteases [4-6]. The number of proteases (from various classes) characterized by this approach testifies to its power. Since the initial development of substrate phage library, different versions of the substrate phage cloning vectors have been constructed to further improve the utility of substrate phage display. This review will provide an overview of the construction of substrate phage display libraries, screening of substrate phage libraries, examples of application, summary and future directions.     

Determination of enzyme/substrate specificity constants using a multiple substrate ESI-MS assay

The traditional method used to investigate the reaction specificity of an enzyme with different substrates is to perform individual kinetic measurements. In this case, a series of varied concentrations are required to study each substrate and a non-regression analysis program is used several times to obtain all the specificity constants for comparison. To avoid the large amount of experimental materials, long analysis time, and redundant data processing procedures involved in the traditional method, we have developed a novel strategy for rapid determination of enzyme substrate specificity using one reaction system containing multiple competing substrates. In this multiplex assay method, the electrospray ionization mass spectrometry (ESI-MS) technique was used for simultaneous quantification of multiple products and a steady-state kinetics model was established for efficient specificity constant calculation. The system investigated was the bacterial sulfotransferase NodH (NodST), which is a host specific nod gene product that catalyzes the sulfate group transfer from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to natural Nod factors or synthetic chitooligosaccharides. Herein, the reaction specificity of NodST for four chitooligosaccharide acceptor substrates of different chain length (chitobiose, chitotriose, chitotetraose, and chitopentaose) was determined by both individual kinetic measurements and the new multiplex ESI-MS assay. The results obtained from the two methods were compared and found to be consistent. The multiplex ESI-MS assay is an accurate and valid method for substrate specificity evaluation, in which multiple substrates can be evaluated in one assay.     

Molecular beacons with oxidized bases report on substrate specificity of DNA oxoguanine glycosylases

DNA glycosylase enzymes recognize and remove structurally distinct modified forms of DNA bases, thereby repairing genomic DNA from chemically induced damage or erasing epigenetic marks. However, these enzymes are often promiscuous, and advanced tools are needed to evaluate and engineer their substrate specificity. Thus, in the present study, we developed a new strategy to rapidly profile the substrate specificity of 8-oxoguanine glycosylases, which cleave biologically relevant oxidized forms of guanine. We monitored the enzymatic excision of fluorophore-labeled oligonucleotides containing synthetic modifications 8-oxoG and FapyG, or G. Using this molecular beacon approach, we identified several hOGG1 mutants with higher specificity for FapyG than 8-oxoG. This approach and the newly synthesized probes will be useful for the characterization of glycosylase substrate specificity and damage excision mechanisms, as well as for evaluating engineered enzymes with altered reactivities.

A three-color fluorescent molecular beacon assay for rapid profiling of substrate specificity of hOGG1 variants, and for engineering proteins to map genomic modifications.     

Single-molecule Förster resonance energy transfer reveals an innate fidelity checkpoint in DNA polymerase I

Enzymatic reactions typically involve complex dynamics during substrate binding, conformational rearrangement, chemistry, and product release. The noncovalent steps provide kinetic checkpoints that contribute to the overall specificity of enzymatic reactions. DNA polymerases perform DNA replication with outstanding fidelity by actively rejecting noncognate nucleotide substrates early in the reaction pathway. Substrates are delivered to the active site by a flexible fingers subdomain of the enzyme, as it converts from an open to a closed conformation. The conformational dynamics of the fingers subdomain might also play a role in nucleotide selection, although the precise role is currently unknown. Using single-molecule F?rster resonance energy transfer, we observed individual Escherichia coli DNA polymerase I (Klenow fragment) molecules performing substrate selection. We discovered that the fingers subdomain actually samples through three distinct conformations--open, closed, and a previously unrecognized intermediate conformation. We measured the overall dissociation rate of the polymerase-DNA complex and the distribution among the various conformational states in the absence and presence of nucleotide substrates, which were either correct or incorrect. Correct substrates promote rapid progression of the polymerase to the catalytically competent closed conformation, whereas incorrect nucleotides block the enzyme in the intermediate conformation and induce rapid dissociation from DNA. Remarkably, incorrect nucleotide substrates also promote partitioning of DNA to the spatially separated 3'-5' exonuclease domain, providing an additional mechanism to prevent misincorporation at the polymerase active site. These results reveal the existence of an early innate fidelity checkpoint, rejecting incorrect nucleotide substrates before the enzyme encloses the nascent base pair.     

On the generation of novel anticancer drugs by recombinant DNA technology: the use of combinatorial biosynthesis to produce novel drugs

Chemotherapeutic drugs for cancer treatment have been traditionally originated by the isolation of natural products from different environmental niches, by chemical synthesis or by a combination of both approaches thus generating semisynthetic drugs. In the last years, a number of gene clusters from several antitumor biosynthetic pathways, mainly produced by actinomycetes and belonging to the polyketides family, are being characterized. Genetic manipulation of these antitumor biosynthetic pathways will offer in the near future an alternative for the generation of novel antitumor derivatives and thus complementing current methods for obtaining novel anticancer drugs. Novel antitumor derivatives have been produced by targetted gene disruption and heterologous expression of single (or a few) gene(s) in another hosts or by combining genes from different, but structurally related, biosynthetic pathways ("combinatorial biosynthesis"). These strategies take advantage from the "relaxed substrate specificity" that characterize secondary metabolism enzymes.     

Targeting the Substrate Binding Site of E. coli Nitrile Reductase QueF by Modeling,Substrate and Enzyme Engineering

Nitrile reductase QueF catalyzes the reduction of 2‐amino‐5‐cyanopyrrolo[2,3‐d]pyrimidin‐4‐one (preQ₀) to 2‐amino‐5‐aminomethylpyrrolo[2,3‐d]pyrimidin‐4‐one (preQ₁) in the biosynthetic pathway of the hypermodified nucleoside queuosine. It is the only enzyme known to catalyze a reduction of a nitrile to its corresponding primary amine and could therefore expand the toolbox of biocatalytic reactions of nitriles. To evaluate this new oxidoreductase for application in biocatalytic reactions, investigation of its substrate scope is prerequisite. We report here an investigation of the active site binding properties and the substrate scope of nitrile reductase QueF from Escherichia coli. Screenings with simple nitrile structures revealed high substrate specificity. Consequently, binding interactions of the substrate to the active site were identified based on a new homology model of E. coli QueF and modeled complex structures of the natural and non‐natural substrates. Various structural analogues of the natural substrate preQ₀ were synthesized and screened with wild‐type QueF from E. coli and several active site mutants. Two amino acid residues Cys190 and Asp197 were shown to play an essential role in the catalytic mechanism. Three non‐natural substrates were identified and compared to the natural substrate regarding their specific activities by using wild‐type and mutant nitrile reductase.     

A deoxyribozyme that forms a three-helix-junction complex with its RNA substrates and has general RNA branch-forming activity

We recently used in vitro selection to identify 7S11, a deoxyribozyme that synthesizes 2',5'-branched RNA. The 7S11 DNA enzyme mediates the nucleophilic attack of an adenosine 2'-hydroxyl group at a 5'-triphosphate, forming 2',5'-branched RNA in a reaction that resembles the first step of in vivo RNA splicing. Here, we describe 7S11 characterization experiments that have two important implications for nucleic acid chemistry and biochemistry. First, on the basis of a comprehensive analysis of its substrate sequence requirements, 7S11 is shown to be generally applicable for the synthesis of a wide range of 2',5'-branched RNAs. Strict substrate sequence requirements are found at the two RNA nucleotides that directly form the branched linkage, and these requirements correspond to those nucleotides found most commonly at these two positions in natural spliced RNAs. Outside of these two nucleotides, most substrate sequences are tolerated with useful ligation activity, although rates and yields vary. Because chemical synthesis approaches to branched RNA are extremely limited in scope, the deoxyribozyme-based route using 7S11 will enable many experiments that require branched RNA. Second, comprehensive nucleotide covariation experiments demonstrate that 7S11 and its RNA substrates adopt a three-helix-junction structure in which the branch-site nucleotide is located at the intersection of the three helices. Because many natural ribozymes have multi-helix junctions, 7S11 is an interesting model system for catalytic nucleic acids.     

Structure-activity correlation between natural glutathione peroxidase (GPx) and mimics: a biomimetic concept for the design and synthesis of more efficient GPx mimics

Among the organoselenium compounds that mimic the action of the natural enzyme glutathione peroxidase (GPx), there are certain basic differences in the activity, substrate specificity and mechanism. These differences arise mainly from the nature of the substituents near the reaction center, and stability and reactivity of the intermediates. As an attempt to draw some general concepts for the development of new mimics, a structure - activity correlation between natural GPx and some existing mimics is described.     

Aminoacyl-SNACs as small-molecule substrates for the condensation domains of nonribosomal peptide synthetases

BACKGROUND: Nonribosomal peptide synthetases (NRPSs) are large multidomain proteins that catalyze the formation of a wide range of biologically active natural products. These megasynthetases contain condensation (C) domains that catalyze peptide bond formation and chain elongation. The natural substrates for C domains are biosynthetic intermediates that are covalently tethered to thiolation (T) domains within the synthetase by thioester linkages. Characterizing C domain substrate specificity is important for the engineered biosynthesis of new compounds. RESULTS: We synthesized a series of aminoacyl-N-acetylcysteamine thioesters (aminoacyl-SNACs) and show that they are small-molecule substrates for NRPS C domains. Comparison of rates of peptide bond formation catalyzed by the C domain from enterobactin synthetase with various aminoacyl-SNACs as downstream (acceptor) substrates revealed high selectivity for the natural substrate analog L-Ser-SNAC. Comparing L- and D-Phe-SNACs as upstream (donor) substrates for the first C domain from tyrocidine synthetase revealed clear D- versus L-selectivity. CONCLUSIONS: Aminoacyl-SNACs are substrates for NRPS C domains and are useful for characterizing the substrate specificity of C domain-catalyzed peptide bond formation.