Pyridoxamine-amino acid chimeras in semisynthetic aminotransferase mimics (original) (raw)
Related papers
Protein Engineering Design and Selection, 2002
Two artificial transaminases were assembled by linking a pyridoxamine derivative within an engineered fatty acid binding protein. The goal of mimicking a native transamination site by stabilizing a cationic pyridoxamine ring system was approached using two different strategies. First, the scaffold of intestinal fatty acid binding protein (IFABP) was tailored by molecular modeling and site-directed mutagenesis to position a carboxylate group close to the pyridine nitrogen of the cofactor. When these IFABP mutants (IFABP-V60C/L38K/E93E and -V60C/E51K/E93E) proved to be unstable, a second approach was explored. By N-methylation of the pyridoxamine, a cationic cofactor was created and tethered to Cys60 of IFABP-V60C/L38K and -V60C/E51K; this latter strategy had the effect of permanently installing a positive charge on the cofactor. These chemogenetic assemblies catalyze the transamination between α-ketoglutarate and various amino acids with enantioselectivities of up to 96% ee. The pH profile of the initial rates is bell shaped and similar to native aminotransferases. The k cat values and the turnover numbers for these new constructs are the highest achieved to date in our system. This success was only made possible by the unique flexibility of the underlying enzyme design concept employed, which permits full control of both the protein scaffold and the catalytically active group. Keywords: aminotransferase/enzyme design/fatty acid binding protein/pyridoxamine/semisynthetic enzyme
Biochemistry, 1998
Using a high-performance liquid chromatography-based method for the determination of pyridoxo cofactors, we detected a new intermediate closely related to the inactivation by D-alanine; its formation occurred at the same rate as the inactivation and upon reactivation it reverted to PLP. Conditions were found under which it was characterized by ultraviolet-visible spectral analysis and mass spectroscopy; it is a pyridoxamine phosphate-like compound with a C 2 fragment derived from the substrate attached to the C′-4 of the pyridinium ring and it has a molecular mass of 306 consistent with this structure. In the presence of D-serine, slow accumulation of a quinonoid intermediate is also related to inactivation. The inactivation can be prevented by salts, which possibly stabilize the protonated aldimine coenzyme complex. The reduced cofactor, nicotinamide adenine dinucleotide, prevents D-aspartate-induced inactivation. Both of these events also are related to formation of the novel intermediate. D-Amino acid transaminase catalyzes the transamination of D-amino acids, which are important constituents of the peptidoglycan layer of the bacterial cell wall (1). Transaminases for D-or L-amino acids contain pyridoxal 5′-phosphate (PLP) 1 as cofactor, which is tightly bound both by noncovalent interactions and also covalently to a lysine residue in the active site (2-4). Recently, the crystal structure of D-amino acid transaminase has revealed the various interactions of PLP in the active site (5). The transamination proceeds via a two-step mechanism, in which D-alanine is first bound to the PLP and is converted via a quinonoid intermediate to pyridoxamine phosphate (PMP) with a concomitant release of pyruvate. In the second step, R-ketoglutarate binds to the PMP and, upon formation
Non-Canonical Amino Acid-Based Engineering of (R)-Amine Transaminase
Frontiers in Chemistry, 2022
Non-canonical amino acids (ncAAs) have been utilized as an invaluable tool for modulating the active site of the enzymes, probing the complex enzyme mechanisms, improving catalytic activity, and designing new to nature enzymes. Here, we report site-specific incorporation of p-benzoyl phenylalanine (pBpA) to engineer (R)-amine transaminase previously created from d-amino acid aminotransferase scaffold. Replacement of the single Phe88 residue at the active site with pBpA exhibits a significant 15-fold and 8-fold enhancement in activity for 1-phenylpropan-1-amine and benzaldehyde, respectively. Reshaping of the enzyme’s active site afforded an another variant F86A/F88pBpA, with 30% higher thermostability at 55°C without affecting parent enzyme activity. Moreover, various racemic amines were successfully resolved by transaminase variants into (S)-amines with excellent conversions (∼50%) and enantiomeric excess (>99%) using pyruvate as an amino acceptor. Additionally, kinetic resoluti...
Synthesis of pyridoxamine 5′-phosphate using an MBA:pyruvate transaminase as biocatalyst
Journal of Molecular Catalysis B: Enzymatic, 2009
Transaminases (TAs) have useful applications as biocatalysts because of their capability of introducing amino groups into ketones and keto acids with high enantioselectivity, regioselectivity and broad substrate specificity. In this study we have shown that purified His-tagged omega-TA CV2025 from Chromobacterium violaceum is capable of complete conversion of pyridoxal 5 -phosphate (PLP) to pyridoxamine 5 -phosphate (PMP) in the presence of (S)-␣-methylbenzylamine (MBA) as the amine donor. Conversions of 5 mM PLP with at least 0.8 mg/ml CV2025 TA (5.8 U/ml) were complete within 24 h. The fastest completion was achieved with an enzyme concentration of 3 mg/ml (22 U/ml): Within 4 h 5 mM PLP/MBA were converted to 100% and 10 mM PLP/MBA to 70%. PLP amination was only partially inhibited in the presence of 0.5 mM gabaculine, whereas the MBA:pyruvate transamination was shown to be inhibited completely. PMP formation of comparable efficiency could not be achieved with equivalent units of porcine ␣-TA. This represents the first example of a PLP-converting TA with an attributed gene and the first demonstration of quantitative biocatalytic PMP synthesis.
Journal of Biological Chemistry, 2006
Antibody 15A9, raised with 5-phosphopyridoxyl (PPL)-N ⑀acetyl-L-lysine as hapten, catalyzes the reversible transamination of hydrophobic D-amino acids with pyridoxal 5-phosphate (PLP). The crystal structures of the complexes of Fab 15A9 with PPL-L-alanine, PPL-D-alanine, and the hapten were determined at 2.3, 2.3, and 2.5 Å resolution, respectively, and served for modeling the complexes with the corresponding planar imine adducts. The conformation of the PLP-amino acid adduct and its interactions with 15A9 are similar to those occurring in PLPdependent enzymes, except that the amino acid substrate is only weakly bound, and, due to the immunization and selection strategy, the lysine residue that covalently binds PLP in these enzymes is missing. However, the N-acetyl-L-lysine moiety of the hapten appears to have selected for aromatic residues in hypervariable loop H3 (Trp-H100e and Tyr-H100b), which, together with Lys-H96, create an anion-binding environment in the active site. The structural situation and mutagenesis experiments indicate that two catalytic residues facilitate the transamination reaction of the PLP-D-alanine aldimine. The space vacated by the absent L-lysine side chain of the hapten can be filled, in both PLP-alanine aldimine complexes, by mobile Tyr-H100b. This group can stabilize a hydroxide ion, which, however, abstracts the C␣ proton only from D-alanine. Together with the absence of any residue capable of deprotonating C␣ of L-alanine, Tyr-H100b thus underlies the enantiomeric selectivity of 15A9. The reprotonation of C4 of PLP, the rate-limiting step of 15A9-catalyzed transamination, is most likely performed by a water molecule that, assisted by Lys-H96, produces a hydroxide ion stabilized by the anion-binding environment. * This work was supported by the Centre National de la Recherche Scientifique, Groupement de Recherche 897, by the Stiftung fü r Wissenschaftliche Forschung an der Universität Zü rich, and the Stiftung fü r medizinische Forschung und Entwicklung.
Design of peptide-acridine mimics of ribonuclease activity
Proceedings of the National Academy of Sciences, 1992
A series of peptide-acridine conjugates was desned and synthesized, based on three features of the proposed catalytic mechanism of RNase A: 2'-proton abstraction by His-12, proton donation to the leaving 5'-oxygen by His-119, and stabilization of the pentacoordinated phosphorous transition state by Lys-41. The substrate binding capability of RNase A was mimicked by the intercalator, acridine. Lysine served as a linker between acridine and the catalytic tripeptide. Cleavage of target RNA was monitored by agarose gel electrophoresis and by gel-permeation chromatography. The carboxyl-amidated conjugates HGHK(Acr)-NH2,
Protein Science, 1995
Of the major amino acid side chains that anchor pyridoxal 5"phosphate at the coenzyme binding site of bacterial D-amino acid transaminase, two have been substituted using site-directed mutagenesis. Thus, Ser-180 was changed to an Ala (S180A) with little effect on enzyme activity, but replacement of Tyr-31 by Gln (Y31Q) led to 99% loss of activity. Titration of SH groups of the native Y31Q enzyme with DTNB proceeded much faster and to a greater extent than the corresponding titration for the native wild-type and S180A mutant enzymes. The stability of each mutant to denaturing agents such as urea or guanidine was similar, Le., in their PLP forms, S180A and Y31Q lost 50% of their activities at a 5-15% lower concentration of urea or guanidine than did the wild-type enzyme. Upon removal of denaturing agent, significant activity was restored in the absence of added pyridoxal 5'-phosphate, but addition of thiols was required. In spite of its low activity, Y31Q was able to form the PMP form of the enzyme just as readily as the wild-type and the S180A enzymes in the presence of normal D-amino acid substrates. However, 0-chloro-D-alanine was a much better substrate and inactivator of the Y31Q enzyme than it was for the wild-type or S180A enzymes, most likely because the Y31Q mutant formed the pyridoxamine 5-phosphate form more rapidly than the other two enzymes. The stereochemical fidelity of the Y31Q recombinant mutant enzyme was much less than that of the S180A and wild-type enzymes because racemase activity, i.e., conversion of L-alanine to D-alanine, was higher than for the wild-type or S180A mutant enzymes, perhaps because the coenzyme has more flexibility in this mutant enzyme.
A peptide template as an allosteric supramolecular catalyst for the cleavage of phosphate esters
Proceedings of the National Academy of Sciences, 2002
The heptapeptide H-Iva-Api-Iva-ATANP-Iva-Api-Iva-NHCH3 (P1a), where Iva is (S)-isovaline, Api is 4-amino-4-carboxypiperidine, and ATANP is (S)-2-amino-3-[1-(1,4,7-triazacyclononane)]propanoic acid, has been synthesized. Its conformation in aqueous solution is essentially that of a 310-helix. By connecting three copies of P1a to a functionalized Tris(2-aminoethyl)amine (Tren) platform a new peptide template, [T(P1)3], was obtained. This molecule is able to bind up to four metal ions (Cu II or Zn II ): one in the Tren subsite and three in the azacyclononane subunits. The binding of the metals to the Tren platform induces a change from an open to a closed conformation in which the three short, helical peptides are aligned in a parallel manner with the azacyclonane units pointing inward within the pseudocavity they define. T(P1) 3 shows a peculiar behavior in the transphosphorylation of phosphate esters; the tetrazinc complex is a catalyst of the cleavage of 2-hydroxypropylp-nitrophenyl phosphate (HPNP), whereas the free ligand is a catalyst of the cleavage of an oligomeric RNA sequence with selectivity for pyrimidine bases. In the case of HPNP, Zn II acts as a positive allosteric effector by enhancing the catalytic efficiency of the system. In the case of the polyanionic RNA substrate, Zn II switches off the activity, thus behaving as a negative allosteric regulator. It is suggested that the opposite behavior of the catalyst induced by Zn II is associated with the change of conformation of the Tren platform, and consequently of the relative spatial disposition of the three linked peptides, that occurs after binding of the metal ion.
Journal of the American Chemical Society, 1998
Hydrazinophenylalanyl-tRNA Phe , an aminoacyl-tRNA derivative containing the unnatural amino acid (S)-R-hydrazinophenylalanine, was prepared in an effort to examine the stereochemical requirements of the A-site of the ribosome during in vitro protein synthesis. The (S)-R-hydrazinophenylalanine moiety was of interest because it contains two nucleophilic centers, the secondary nitrogen attached to C R , which is normally acylated during the course of peptide bond formation, and the sterically less hindered primary nitrogen. To determine the position of acylation, (S)-R-hydrazinophenylalanyl-tRNA Phe was tested in an Escherichia coli in vitro protein biosynthesizing system lacking elongation factor G, such that only dipeptide products were formed. The dipeptide product mixture was analyzed by HPLC in direct comparison with authentic synthetic standards. The dipeptide assay utilizing (S)-R-hydrazinophenylalanyl-tRNA Phe as the A-site tRNA established that the analogue functioned well as an acceptor tRNA; HPLC analysis of the products showed that both dipeptides were formed in approximately equal amounts. When attached to a suppressor tRNA transcript, (S)-R-hydrazinophenylalanine was also incorporated into position 27 of dihydrofolate reductase in an E. coli protein synthesizing system by readthrough of a nonsense codon. This finding expands the currently accepted model of peptide bond formation at the ribosome and adds to the repertoire of peptide-like products shown to form at the peptidyltransferase center of the ribosome.