János Rétey

Phenylalanine Ammonia‐Lyase: The Use of Its Broad Substrate Specificity for Mechanistic Investigations and Biocatalysis—Synthesis of L‐Arylalanines

Several fluoro- and chlorophenylalanines were found to be good substrates of phenylalanine ammonia-lyase (PAL/EC 4.3.1.5) from parsley. The enantiomerically pure L-amino acids were obtained in good yields by reaction of the corresponding cinnamic acids with 5M ammonia solution (buffered to pH 10) in the presence of PAL. The kinetic constants for nine different fluoro- and chlorophenylalanines do not provide a rigorous proof for but are consistent with the previously proposed mechanism comprising an electrophilic attack of the methylidene-imidazolone cofactor of PAL at the aromatic nucleus as a first chemical step. In the resulting Friedel-Crafts-type sigma complex the beta-protons are activated for abstraction and consequently the pro-S is abstracted by an enzymic base. Results from semi-empirical calculations combined with a proposed partial active site model showed a correlation between the experimental kinetic constants and the change in polarization of the pro-S Cbeta-H bond and heat of formation of the sigma complexes, thus making the electrophilic attack at the neutral aromatic ring plausible. Furthermore, while 5-pyrimidinylalanine was found to be a moderately good substrate of PAL, 2-pyrimidinylalanine was an inhibitor.

Ethylmalonyl-CoA mutase from Rhodobacter sphaeroides defines a new subclade of coenzyme B12-dependent acyl-CoA mutases

Coenzyme B12-dependent mutases are radical enzymes that catalyze reversible carbon skeleton rearrangement reactions. Here we describe Rhodobacter sphaeroides ethylmalonyl-CoA mutase (Ecm), a novel member of the family of coenzyme B12-dependent acyl-CoA mutases, that operates in the recently discovered ethylmalonyl-CoA pathway for acetate assimilation. Ecm is involved in the central reaction sequence of this novel pathway and catalyzes the transformation of ethylmalonyl-CoA to methylsuccinyl-CoA in combination with a second enzyme that was further identified as promiscuous ethylmalonyl-CoA/methylmalonyl-CoA epimerase. In contrast to the epimerase, Ecm is highly specific for its substrate, ethylmalonyl-CoA, and accepts methylmalonyl-CoA only at 0.2% relative activity. Sequence analysis revealed that Ecm is distinct from (2R)-methylmalonyl-CoA mutase as well as isobutyryl-CoA mutase and defines a new subfamily of coenzyme B12-dependent acyl-CoA mutases. In combination with molecular modeling, two signature sequences were identified that presumably contribute to the substrate specificity of these enzymes.

Discovery and role of methylidene imidazolone, a highly electrophilic prosthetic group.

The elimination of ammonia from alpha-amino acids is a chemically difficult process. While the non-acidic beta-proton has to be abstracted, the much more acidic ammonium protons must remain untouched to maintain the leaving group ability of this positively charged group. Histidine and phenylalanine ammonia-lyases (HAL and PAL) possess a catalytically essential electrophilic group which has been believed to be dehydroalanine for 30 years. Recently, the X-ray structure of HAL has been solved. The electron density was not consistent with dehydroalanine but showed the presence of methylidene imidazolone (MIO) instead. The high electrophilicity of this prosthetic group as well as the geometry at the active site support a previously proposed mechanism involving a Friedel-Crafts-type attack at the aromatic ring of the substrate. Further biochemical evidence for this unprecedented electrophile-assisted ammonia elimination is also presented. Although no X-ray structure of PAL has been published as yet, spectrophotometrical evidence for the presence of MIO has been provided. Finally, a chemical model for the PAL reaction is described.

Serine‐202 is the putative precursor of the active site dehydroalanine of phenylalanine ammonia lyase Site‐directed mutagenesis studies on the enzyme from parsley (Petroselinum crispum L.)

To investigate the possible role of serine as a precursor of dehydroalanine at the active site of phenylalanine ammonia lyase, two serines, conserved in all known PAL and histidase sequences, were changed to alanine by site‐directed mutagenesis. The resulting mutant genes were subcloned into the expression vector pT7.7 and the gene products were assayed for PAL activity. Mutant PALMutS209A showed the same catalytic property as wild‐type PAL, whereas mutant PALMutS202A was devoid of catalytic activity, indicating that serine‐202 is the most likely precursor of the active site dehydroalanine.

Structural and functional comparison of HemN to other radical SAM enzymes.

Abstract Radical SAM enzymes have only recently been recognized as an ancient family sharing an unusual radical-based reaction mechanism. This late appreciation is due to the extreme oxygen sensitivity of most radical SAM enzymes, making their characterization particularly arduous. Nevertheless, realization that the novel apposition of the established cofactors S-adenosylmethionine and [4Fe-4S] cluster creates an explosive source of catalytic radicals, the appreciation of the sheer size of this previously neglected family, and the rapid succession of three successfully solved crystal structures within a year have ensured that this family has belatedly been noted. In this review, we report the characterization of two enzymes: the established radical SAM enzyme, HemN or oxygen-independent coproporphyrinogen III oxidase from Escherichia coli, and littorine mutase, a presumed radical SAM enzyme, responsible for the conversion of littorine to hyoscyamine in plants. The enzymes are compared to other radical SAM enzymes and in particular the three reported crystal structures from this family, HemN, biotin synthase and MoaA, are discussed.

Binding of cob(II)alamin to the adenosylcobalamin-dependent ribonucleotide reductase from Lactobacillus leichmannii: Identification of dimethylbenzimidazole as the axial ligand

The ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii catalyzes the reduction of nucleoside 5′-triphosphates to 2′-deoxynucleoside 5′-triphosphates and uses coenzyme B12, adenosylcobalamin (AdoCbl), as a cofactor. Use of a mechanism-based inhibitor, 2′-deoxy-2′-methylenecytidine 5′-triphosphate, and isotopically labeled RTPR and AdoCbl in conjunction with EPR spectroscopy has allowed identification of the lower axial ligand of cob(II)alamin when bound to RTPR. In common with the AdoCbl-dependent enzymes catalyzing irreversible heteroatom migrations and in contrast to the enzymes catalyzing reversible carbon skeleton rearrangements, the dimethylbenzimidazole moiety of the cofactor is not displaced by a protein histidine upon binding to RTPR.

The essential tyrosine-containing loop conformation and the role of the C-terminal multi-helix region in eukaryotic phenylalanine ammonia-lyases

Besides the post‐translationally cyclizing catalytic Ala‐Ser‐Gly triad, Tyr110 and its equivalents are of the most conserved residues in the active site of phenylalanine ammonia‐lyase (PAL, EC 4.3.1.5), histidine ammonia‐lyase (HAL, EC 4.3.1.3) and other related enzymes. The Tyr110Phe mutation results in the most pronounced inactivation of PAL indicating the importance of this residue. The recently published X‐ray structures of PAL revealed that the Tyr110‐loop was either missing (for Rhodospridium toruloides) or far from the active site (for Petroselinum crispum). In bacterial HAL (∼500 amino acids) and plant and fungal PALs (∼710 amino acids), a core PAL/HAL domain (∼480 amino acids) with ≥ 30% sequence identity along the different species is common. In plant and fungal PAL a ∼100‐residue long C‐terminal multi‐helix domain is present. The ancestor bacterial HAL is thermostable and, in all of its known X‐ray structures, a Tyr83‐loop‐in arrangement has been found. Based on the HAL structures, a Tyr110‐loop‐in conformation of the P. crispum PAL structure was constructed by partial homology modeling, and the static and dynamic behavior of the loop‐in/loop‐out structures were compared. To study the role of the C‐terminal multi‐helix domain, Tyr‐loop‐in/loop‐out model structures of two bacterial PALs (Streptomyces maritimus, 523 amino acids and Photorhabdus luminescens, 532 amino acids) lacking this C‐terminal domain were also built. Molecular dynamics studies indicated that the Tyr‐loop‐in conformation was more rigid without the C‐terminal multi‐helix domain. On this basis it is hypothesized that a role of this C‐terminal extension is to decrease the lifetime of eukaryotic PAL by destabilization, which might be important for the rapid responses in the regulation of phenylpropanoid biosynthesis.

Methylidene-imidazolone (MIO) from histidine and phenylalanine ammonia-lyase.

Publisher Summary This chapter discusses the history, discovery, structure, reactivity, and catalytic role of the Methylidene-Imidazolone (MIO) group in two ammonia-lyase reactions. Enzymes, however, take advantage also of electrophilic catalysis. In addition to the use of protons as electrophile, they often use cofactors, such as pyridoxal phosphate, the classical electrophilic cocatalyst. Alternatively, the catalytically essential electrophilic group may be generated by post-translational modification of an amino acid side chain. Two such modifications of the serine side chain are known: (1) Dehydration coupled with the removal of an N-terminal peptide portion leads to an N-terminal pyruvyl group (Snell, 1986). (2) Cyclization of the internal tripeptide AlaSerGly concomitant with the elimination of two molecules of water. The latter leads to the highly electrophilic 4-methylidene- imidazol-5-one (MIO) prosthetic group (Schwede et al., 1999). Although the pyruvyl group is involved in similar catalytic processes as pyridoxal phosphate and may be its primitive precursor in evolution (Snell, 1986), the MIO group participates in chemically unique ammonia eliminations catalyzed by the enzymes histidine and phenylalanine ammonia-lyases.

The role and source of 5'-deoxyadenosyl radical in a carbon skeleton rearrangement catalyzed by a plant enzyme.

The last step in the biosynthesis of tropane alkaloids is the carbon skeleton rearrangement of littorine to hyoscyamine. The reaction is catalyzed by a cell‐free extract prepared from cultured hairy roots of Datura stramonium. Adenosylmethionine stimulated the rearrangement 10–20‐fold and showed saturation kinetics with an apparent K m of 25 μM. It is proposed that S‐adenosylmethionine is the source of a 5′‐deoxyadenosyl radical which initiates the rearrangement in a similar manner as it does in analogous rearrangements catalyzed by coenzyme B12‐dependent enzymes. Possible roles of S‐adenosylmethionine as a radical source in higher plants are discussed.

Synthesis of characteristic lipopeptides of the human N-Ras protein and their evaluation as possible inhibitors of protein farnesyl transferase

Lipopeptides carrying a farnesyl thioether or a palmitic acid thioester and a farnesyl thioether were prepared from S-farnesyl cysteine methyl ester by N-terminal extension of the peptide chain employing the base labile Fmoc blocking group of the palladium(0) sensitive Aloc urethane. By means of this technique a lipohexapeptide representing the completely functionalized, i.e. palmitoylated and farnesylated C-terminus of the human N-Ras protein, was prepared. If acid labile blocking functions like the Boc group were used, upon deprotection an undesired addition of the acid to the double bonds of the farnesyl residue occurred. Therefore, acid labile blocking groups should not be employed in the synthesis of farnesylated lipopeptides. The lipopeptide methyl esters which carry only a farnesyl group do not inhibit protein farnesyl transferase, whereas palmitoylated peptides are weak inhibitors of this enzyme.