Pyridoxal-5′-phosphate-dependent catalytic antibodies

Cofactors—i.e., metal ions and coenzymes—extend the catalytic scope of enzymes and might have been among the first biological catalysts. They may be expected to efficiently extend the catalytic potential of antibodies. Monoclonal antibodies (MAbs) against N^α-phosphopyridoxyl-l-lysine were screened for 1) binding of 5′-phosphopyridoxyl amino acids, 2) binding of the planar Schiff base of pyridoxal-5′-phosphate (PLP) and amino acids, the first intermediate of all PLP-dependent reactions, and 3), catalysis of the PLP-dependent α, β-elimination reaction with β-chloro-D/L-alanine. Antibody 15A9 fulfilled all criteria and was also found to catalyze the cofactor-dependent transamination reaction of hydrophobic D-amino acids and oxo acids (k′ _cat=0.42 min⁻¹ with D-alanine at 25°C). No other reactions with either D- or L-amino acids were detected. PLP markedly contributes to catalytic effecacy—it is a 10⁴ times more efficient acceptor of the amino group than pyruvate. The antibody ensures reaction specificity, stereospecificity, and substrate specificity, and further accelerates the transamination reaction (k′ _cat(Ab)/k′ _cat(PLP)=5×10³). The successive screening steps simulate the selection criteria that might have been operative in the evolution of protein-assisted psyridoxal catalysis.     

Metal ion inhibition of nonenzymatic pyridoxal phosphate catalyzed decarboxylation and transamination.

Nonenzymatic pyridoxal phosphate (PLP) catalyzed decarboxylations and transaminations have been revisited experimentally. Metal ions are known to catalyze a variety of PLP-dependent reactions in solution, including transamination. It is demonstrated here that the rate accelerations previously observed are due solely to enhancement of Schiff base formation under subsaturating conditions. A variety of metal ions were tested for their effects on the reactivity of the 2-methyl-2-aminomalonate Schiff bases. All were found to have either no effect or a small inhibitory one. The effects of Al(3+) were studied in detail with the Schiff bases of 2-methyl-2-aminomalonate, 2-aminoisobutyrate, alanine, and ethylamine. The decarboxylation of 2-methyl-2-aminomalonate is unaffected by metalation with Al(3+), while the decarboxylation of 2-aminoisobutyrate is inhibited 125-fold. The transamination reaction of ethylamine is 75-fold slower than that of alanine. Ethylamine transamination is inhibited 4-fold by Al(3+) metalation, while alanine transamination is inhibited only 1.3-fold. Metal ion inhibition of Schiff base reactivity suggests a simple explanation for the lack of known PLP dependent enzymes that make direct mechanistic use of metal ions. A comparison of enzyme catalyzed, PLP catalyzed, and uncatalyzed reactions shows that PLP dependent decarboxylases are among the best known biological rate enhancers: decarboxylation occurs 10(18)-fold faster on the enzyme surface than it does free in solution. PLP itself provides the lion's share of the catalytic efficiency of the holoenzyme: at pH 8, free PLP catalyzes 2-aminoisobutyrate decarboxylation by approximately 10(10)-fold, with the enzyme contributing an additional approximately 10(8)-fold.     

Exploring the pyridoxal 5'-phosphate-dependent enzymes

Pyridoxal 5'-phosphate (PLP)-dependent enzymes represent about 4% of the enzymes classified by the Enzyme Commission. The versatility of PLP in carrying out a large variety of reactions exploiting the electron sink effect of the pyridine ring, the conformational changes accompanying the chemical steps and stabilizing distinct catalytic intermediates, and the spectral properties of the different coenzyme-substrate derivatives signaling the reaction progress, are some of the features that have attracted our interest to investigate the structure-dynamics-function relationships of PLP-dependent enzymes. To this goal, an integrated approach combining biochemical, biophysical, computational, and molecular biology methods was used. The extensive work carried out on two enzymes, tryptophan synthase and O-acetylserine sulfhydrylase, is presented and discussed as representative of other PLP-dependent enzymes we have investigated. Finally, perspectives of PLP-dependent enzymes functional genomics and drug targeting highlight the continuous novelty of an "old" class of enzymes.     

Theoretical studies on pyridoxal 5'-phosphate-dependent transamination of alpha-amino acids

Density functional methods have been applied to investigate the irreversible transamination between glyoxylic acid and pyridoxamine analog and the catalytic mechanism for the critical [1,3] proton transfer step in aspartate aminotransferase (AATase). The results indicate that the catalytic effect of pyridoxal 5'-phosphate (PLP) may be attributed to its ability to stabilize related transition states through structural resonance. Additionally, the PLP hydroxyl group and the carboxylic group of the amino acid can shuttle proton, thereby lowering the barrier. The rate-limiting step is the tautomeric conversion of the aldimine to ketimine by [1,3] proton transfer, with a barrier of 36.3 kcal/mol in water solvent. A quantum chemical model consisting 142 atoms was constructed based on the crystal structure of the native AATase complex with the product L-glutamate. The electron-withdrawing stabilization by various residues, involving Arg386, Tyr225, Asp222, Asn194, and peptide backbone, enhances the carbon acidity of 4'-C of PLP and Calpha of amino acid. The calculations support the proposed proton transfer mechanism in which Lys258 acts as a base to shuttle a proton from the 4'-C of PLP to Calpha of amino acid. The first step (proton transfer from 4'-C to lysine) is shown to be the rate-limiting step. Furthermore, we provided an explanation for the reversibility and specificity of the transamination in AATase.     

A New Pyridoxal Derivative for Transamination of N‐Terminus of Proteins

A new pyridoxal‐5‐phosphate (PLP) derivative FHMDP was developed for the transamination of different peptides with three most hindered amino acid residues (Leu, Ile, Val) as their N‐terminus. Compared to the previously reported reactions of PLP derivatives, the N‐terminus transamination could be accomplished efficiently with the new compound.     

Role of the pyridine nitrogen in pyridoxal 5'-phosphate catalysis: activity of three classes of PLP enzymes reconstituted with deazapyridoxal 5'-phosphate

Pyridoxal 5'-phosphate (PLP; vitamin B(6))-catalyzed reactions have been well studied, both on enzymes and in solution, due to the variety of important reactions this cofactor catalyzes in nitrogen metabolism. Three functional groups are central to PLP catalysis: the C4' aldehyde, the O3' phenol, and the N1 pyridine nitrogen. In the literature, the pyridine nitrogen has traditionally been assumed to be protonated in enzyme active sites, with the protonated pyridine ring providing resonance stabilization of carbanionic intermediates. This assumption is certainly correct for some PLP enzymes, but the structures of other active sites are incompatible with protonation of N1, and, consequently, these enzymes are expected to use PLP in the N1-unprotonated form. For example, aspartate aminotransferase protonates the pyridine nitrogen for catalysis of transamination, while both alanine racemase and O-acetylserine sulfhydrylase are expected to maintain N1 in the unprotonated, formally neutral state for catalysis of racemization and β-elimination. Herein, kinetic results for these three enzymes reconstituted with 1-deazapyridoxal 5'-phosphate, an isosteric analogue of PLP lacking the pyridine nitrogen, are compared to those for the PLP enzyme forms. They demonstrate that the pyridine nitrogen is vital to the 1,3-prototropic shift central to transamination, but not to reactions catalyzed by alanine racemase or O-acetylserine sulfhydrylase. Not all PLP enzymes require the electrophilicity of a protonated pyridine ring to enable formation of carbanionic intermediates. It is proposed that modulation of cofactor electrophilicity plays a central role in controlling reaction specificity in PLP enzymes.     

Molecular evolution of B<Subscript>6</Subscript> enzymes: Binding of pyridoxal-5'-phosphate and Lys41Arg substitution turn ribonuclease A into a model B<Subscript>6</Subscript> protoenzyme

Background  

The pyridoxal-5'-phosphate (PLP)-dependent or vitamin B₆-dependent enzymes that catalyze manifold reactions in the metabolism of amino acids belong to no fewer than four evolutionarily independent protein families. The multiple evolutionary origin and the essential mechanistic role of PLP in these enzymes argue for the cofactor having arrived on the evolutionary scene before the emergence of the respective apoenzymes and having played a dominant role in the molecular evolution of the B₆ enzyme families. Here we report on an attempt to re-enact the emergence of a PLP-dependent protoenzyme. The starting protein was pancreatic ribonuclease A (RNase), in which active-site Lys41 or Lys7 readily form a covalent adduct with PLP.     

Optimization of a biomimetic transamination reaction

For a range of protein substrates, N-terminal transamination offers a convenient way to install a reactive ketone or aldehyde functional group at a single location. We report herein the effects of the identity of N-terminal residues on the product distribution generated upon reaction with pyridoxal 5'-phosphate (PLP). This study was accomplished through the combination of solid-phase peptide synthesis with detailed liquid chromatography-mass spectrometry analysis. Many N-terminal amino acids provided high yields of the desired transaminated products, but some residues (His, Trp, Lys, and Pro) generated adducts with PLP itself. N-terminal Cys and Ser residues were observed to undergo beta-elimination in addition to transamination, and the transamination product of N-terminal Gln was resistant to subsequent oxime formation attempts. The information generated through the screening of peptide substrates was successfully applied to a protein target, changing an initially unreactive terminus into one that could be modified in high (70%) yield. Thus, these studies have increased our predictive power for the reaction, both in terms of improving conversion and suppressing reaction byproducts. An initial set of guidelines that may be used to increase the applicability of this reaction to specific proteins of interest is provided.     

Kinetic isotope effects of L-Dopa decarboxylase

A mixed centroid path integral and free energy perturbation method (PI-FEP/UM) has been used to investigate the primary carbon and secondary hydrogen kinetic isotope effects (KIEs) in the amino acid decarboxylation of L-Dopa catalyzed by the enzyme L-Dopa decarboxylase (DDC) along with the corresponding uncatalyzed reaction in water. DDC is a pyridoxal 5'-phosphate (PLP) dependent enzyme. The cofactor undergoes an internal proton transfer between the zwitterionic protonated Schiff base configuration and the neutral hydroxyimine tautomer. It was found that the cofactor PLP makes significant contributions to lowering the decarboxylation barrier, while the enzyme active site provides further stabilization of the transition state. Interestingly, the O-protonated configuration is preferred both in the Michaelis complex and at the decarboxylation transition state. The computed kinetic isotope effects (KIE) on the carboxylate C-13 are consistent with that observed on decarboxylation reactions of other PLP-dependent enzymes, whereas the KIEs on the α carbon and secondary proton, which can easily be validated experimentally, may be used as a possible identification for the active form of the PLP tautomer in the active site of DDC.     

The Uncommon Active Site of D-Amino Acid Transaminase from Haliscomenobacter hydrossis: Biochemical and Structural Insights into the New Enzyme

Among industrially important pyridoxal-5’-phosphate (PLP)-dependent transaminases of fold type IV D-amino acid transaminases are the least studied. However, the development of cascade enzymatic processes, including the synthesis of D-amino acids, renewed interest in their study. Here, we describe the identification, biochemical and structural characterization of a new D-amino acid transaminase from Haliscomenobacter hydrossis (Halhy). The new enzyme is strictly specific towards D-amino acids and their keto analogs; it demonstrates one of the highest rates of transamination between D-glutamate and pyruvate. We obtained the crystal structure of the Halhy in the holo form with the protonated Schiff base formed by the K143 and the PLP. Structural analysis revealed a novel set of the active site residues that differ from the key residues forming the active sites of the previously studied D-amino acids transaminases. The active site of Halhy includes three arginine residues, one of which is unique among studied transaminases. We identified critical residues for the Halhy catalytic activity and suggested functions of the arginine residues based on the comparative structural analysis, mutagenesis, and molecular modeling simulations. We suggested a strong positive charge in the O-pocket and the unshaped P-pocket as a structural code for the D-amino acid specificity among transaminases of PLP fold type IV. Characteristics of Halhy complement our knowledge of the structural basis of substrate specificity of D-amino acid transaminases and the sequence-structure-function relationships in these enzymes.     

Catalytic antibodies for complex reactions

Success in generating catalytic antibodies as enzyme mimics lies in the strategic design of the transition-state analog (TSA) for the reaction of interest, and careful development of screening processes for the selection of antibodies that are catalysts. Typically, the choice of TSA structure is straightforward, and the criterion for selection in screening is often binding of the TSA to the antibody in a microtiter-plate assay. This article emphasizes the problems of TSA design in complex reactions and the importance of selecting antibodies on the basis of catalysis as well as binding to the TSA. The target reaction is the derivatization of primary amines with naphthalene-2,3-dicarboxaldehyde (NDA) in the presence of cyanideion. The desired outcome is selective catalysis of formation of the fluorescent derivative in preference to nonfluorescent side-products. In the study, TSA design was directed toward the reaction branch leading to the fluorescent product. Here, we describe a microtiter plate-based assay that is capable of detecting antibodies showing catalytic activity atan early stage. Of the antibodies selected, 36% showed no appreciable binding to any of the substrates tested, but did show catalytic activity in deriving one or more of the amino acids screened. In contrast, only two out of 77 clones that showed binding did not show catalysis. Thus, in this complex system, observation of binding is a good predictor of the presence of catalytic activity, and failure to observe binding is a poor predictor of the absence of catalytic activity.     

Identification of highly reactive sequences for PLP-mediated bioconjugation using a combinatorial peptide library

Chemical reactions that facilitate the attachment of synthetic groups to proteins are useful tools for the field of chemical biology and enable the incorporation of proteins into new materials. We have previously reported a pyridoxal 5'-phosphate (PLP)-mediated reaction that site-specifically oxidizes the N-terminal amine of a protein to afford a ketone. This unique functional group can then be used to attach a reagent of choice through oxime formation. Since its initial report, we have found that the N-terminal sequence of the protein can significantly influence the overall success of this strategy. To obtain short sequences that lead to optimal conversion levels, an efficient method for the evaluation of all possible N-terminal amino acid combinations was needed. This was achieved by developing a generalizable combinatorial peptide library screening platform suitable for the identification of sequences that display high levels of reactivity toward a desired bioconjugation reaction. In the context of N-terminal transamination, a highly reactive alanine-lysine motif emerged, which was confirmed to promote the modification of peptide substrates with PLP. This sequence was also tested on two protein substrates, leading to substantial increases in reactivity relative to their wild-type termini. This readily encodable tripeptide thus appears to provide a significant improvement in the reliability with which the PLP-mediated bioconjugation reaction can be used. This study also provides an important first example of how synthetic peptide libraries can accelerate the discovery and optimization of protein bioconjugation strategies.     

Catalytic antibodies for complex reactions: hapten design and the importance of screening for catalysis in the generation of catalytic antibodies for the NDA/CN reaction

Success in generating catalytic antibodies as enzyme mimics lies in the strategic design of the transition-state analog (TSA) for the reaction of interest, and careful development of screening processes for the selection of antibodies that are catalysts. Typically, the choice of TSA structure is straightforward, and the criterion for selection in screening is often binding of the TSA to the antibody in a microtiter-plate assay. This article emphasizes the problems of TSA design in complex reactions and the importance of selecting antibodies on the basis of catalysis as well as binding to the TSA. The target reaction is the derivatization of primary amines with naphthalene-2,3-dicarboxaldehyde (NDA) in the presence of cyanide ion. The desired outcome is selective catalysis of formation of the fluorescent derivative in preference to nonfluorescent side-products. In the study, TSA design was directed toward the reaction branch leading to the fluorescent product. Here, we describe a microtiter plate-based assay that is capable of detecting antibodies showing catalytic activity at an early stage. Of the antibodies selected, 36% showed no appreciable binding to any of the substrates tested, but did show catalytic activity in derivatizing one or more of the amino acids screened. In contrast, only two out of 77 clones that showed binding did not show catalysis. Thus, in this complex system, observation of binding is a good predictor of the presence of catalytic activity, and failure to observe binding is a poor predictor of the absence of catalytic activity.     

Enantioselective biomimetic transamination of α-keto acids catalyzed by H4-naphthalene-derived axially chiral biaryl pyridoxamines

Asymmetric biomimetic transamination is a highly attractive method for synthesis of chemically and biologically important chiral amino acids and chiral amines. Development of chiral pyridoxamines/pyridoxals is the key for the reaction. New axially chiral biaryl pyridoxamines based on H₄-naphathene skeleton have been developed. The pyridoxamines display good enantioselectivity and high catalytic activity in asymmetric biomimetic transamination of α-keto acids, affording various optically active unnatural amino acids in 61–98% yields with up to 91% ee’s.     

A QM/MM simulation study of transamination reaction at the active site of aspartate aminotransferase: Free energy landscape and proton transfer pathways

Transaminase is a key enzyme for amino acid metabolism, which reversibly catalyzes the transamination reaction with the help of PLP (pyridoxal 5^' -phosphate) as its cofactor. Here we have investigated the mechanism and free energy landscape of the transamination reaction involving the aspartate transaminase (AspTase) enzyme and aspartate-PLP (Asp-PLP) complex using QM/MM simulation and metadynamics methods. The reaction is found to follow a stepwise mechanism where the active site residue Lys258 acts as a base to shuttle a proton from α -carbon (CA) to imine carbon (C4A) of the PLP-Asp Schiff base. In the first step, the Lys258 abstracts the CA proton of the substrate leading to the formation of a carbanionic intermediate which is followed by the reprotonation of the Asp-PLP Schiff base at C4A atom by Lys258. It is found that the free energy barrier for the proton abstraction by Lys258 and that for the reprotonation are 17.85 and 3.57 kcal/mol, respectively. The carbanionic intermediate is 7.14 kcal/mol higher in energy than the reactant. Hence, the first step acts as the rate limiting step. The present calculations also show that the Lys258 residue undergoes a conformational change after the first step of transamination reaction and becomes proximal to C4A atom of the Asp-PLP Schiff base to favor the second step. The active site residues Tyr70* and Gly38 anchor the Lys258 in proper position and orientation during the first step of the reaction and stabilize the positive charge over Lys258 generated at the intermediate step.     

Biomimetic Chiral Pyridoxal and Pyridoxamine Catalysts

Vitamin B₆ serves as universal co‐enzymes in biological systems. However, its catalytic power has not been applied into the area of asymmetric catalysis. Based on the core structure of vitamin B₆, we have developed several types of pyridoxal and pyridoxamine catalysts with different structural skeletons and different electronic properties. With these pyridoxals and pyridoxamines as catalysts, we have realized biomimetic asymmetric transamination of α‐keto acids and biomimetic asymmetric Mannich reaction of glycinate, respectively, to give various chiral α‐amino acids and α,β‐diamino acids in good yields with excellent diastero‐ and/or enantioselectivities. Both of the reactions have perfectly mimicked the corresponding biological transformations.     

From cofactor to enzymes. The molecular evolution of pyridoxal-5'-phosphate-dependent enzymes

The pyridoxal-5'-phosphate (vitamin B(6))-dependent enzymes that act on amino acid substrates have multiple evolutionary origins. Thus, the common mechanistic features of B(6) enzymes are not accidental historical traits but reflect evolutionary or chemical necessities. The B(6) enzymes belong to four independent evolutionary lineages of paralogous proteins, of which the alpha family (with aspartate aminotransferase as the prototype enzyme) is by far the largest and most diverse. The considerably smaller beta family (tryptophan synthase beta as the prototype enzyme) is structurally and functionally more homogenous. Both the D-alanine aminotransferase family and the alanine racemase family consist of only a few enzymes. The primordial pyridoxal-5'-phosphate-dependent protein catalysts apparently first diverged into reaction-specific protoenzymes, which then diverged further by specializing for substrate specificity. Aminotransferases as well as amino acid decarboxylases are found in two different evolutionary lineages, providing examples of convergent enzyme evolution. The functional specialization of most B(6) enzymes seems to have already occurred in the universal ancestor cell before the divergence of eukaryotes, archebacteria, and eubacteria 1500 million years ago. Pyridoxal-5'-phosphate must have emerged very early in biological evolution; conceivably, metal ions and organic cofactors were the first biological catalysts. To simulate particular steps of molecular evolution, both the substrate and reaction specificity of existent B(6) enzymes were changed by substitution of active-site residues, and monoclonal pyridoxal-5'-phosphate-dependent catalytic antibodies were produced with selection criteria that might have been operative in the evolution of protein-assisted pyridoxal catalysis.     

Pyridoxamine,a scavenger agent of carbohydrates

Pyridoxamine has been found to inhibit protein glycation and to avoid the formation of advanced glycation end‐products (AGEs). One of the mechanisms by which pyridoxamine can inhibit glycation involves the scavenger of carbonyl groups with glycation capacity. In this work, we conducted a kinetic study of the reactions of pyridoxamine with various carbohydrates under physiological pH and temperature. The reactions involving hexoses were found to give a tricyclic compound ( 5 ) in addition to pyridoxal and pyridoxine. Such a tricyclic compound inhibits the Amadori rearrangement and the formation of other carbonyl compounds with glycating properties. The reactions involving pentoses gave compound 7 and pyridoxal—by transamination of the Schiff base. The transamination reaction enhances the inhibitory action of pyridoxamine. The formation rate constants for the Schiff base, k₃, were found to be similar to those for the reactions of D ‐glucose with amino acids, which suggests competition between pyridoxamine and terminal amino residues in proteins for glycating sites in sugars. These constants are dependent on the electrophilic character of the carbonyl carbon in the carbohydrate. © 2007 Wiley Periodicals, Inc. 39: 154–167, 2007     

Enzymatic racemization of amines catalyzed by enantiocomplementary ω-transaminases

A strategy for the biocatalytic racemization of primary α-chiral amines was developed by employing a pair of stereocomplementary PLP-dependent ω-transaminases. The interconversion of amine enantiomers proceeded through reversible transamination by a prochiral ketone intermediate, either catalyzed by a pair of stereocomplementary ω-transaminases or by a single enzyme possessing low stereoselectivity. To tune the system, the type and concentration of a nonchiral amino acceptor proved to be crucial. Finally, racemization could be achieved by the cross-transamination of two different amines without a requirement for an external amino acceptor. Several synthetically and industrially important amines could be enzymatically racemized under mild reaction conditions.     

Microwave-Assisted Esterification of Diverse Carboxylic Acids and Chiral Amino Acids

A facile and efficient synthetic method of esters from their corresponding carboxylic acids and amino acids is described. The esterification reaction of carboxylic acids and amino acids could be greatly accelerated under microwave irradiation because the reactions described in this article took place in only 5 min with almost quantitative yields, and distinct acidity of catalytic acids was well tolerated. Unlike the racemation problem in microwave-assisted N-acylation reactions, the esters of chiral amino acids could be achieved with retention of configuration under this condition.