Histidases Research Articles

Systematic Analysis of the MIO-forming Residues of Aromatic Ammonia Lyases.

Aromatic ammonia lyases (AALs) and tyrosine/phenylalanine ammonia mutases (TAM/PAM) are 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO)-dependent enzymes. Usually, the MIO moiety is autocatalytically formed from the tripeptide Ala-Ser-Gly (ASG) and acts as an electrophile during the enzymatic reaction. However, the MIO-forming residues (ASG) have some diversity in this enzyme class. In this work, a systematic investigation on the variety of MIO-forming residues was carried out using in-depth sequence analyses. Several protein clusters of AAL-like enzymes with unusual MIO-forming residues such as ACG, TSG, SSG, and CSG were identified, including two novel histidine ammonia lyases and one PAM with CSG and TSG residues, respectively, as well as three novel ergothioneine trimethylammonia lyases without MIO motif. The mutagenesis of common MIO-groups confirmed the function of these MIO variants, which provides good starting points for future functional prediction and mutagenesis research of AALs.

Microreactor equipped with naturally acid-resistant histidine ammonia lyase from an extremophile.

Extremophile enzymes are useful in biotechnology and biomedicine due to their abilities to withstand harsh environments. The abilities of histidine ammonia lyases from different extremophiles to preserve their catalytic activities after exposure to acid were assessed. Thermoplasma acidophilum histidine ammonia lyase was identified as an enzyme with a promising catalytic profile following acid treatment. The fusion of this enzyme with the maltose-binding protein or co-incubation with the chaperone HdeA further helped Thermoplasma acidophilum histidine ammonia lyase to withstand acid treatments down to pH 2.8. The assembly of a microreactor by encapsulation of MBP-Thermoplasma acidophilum histidine ammonia lyase into a photocrosslinked poly(vinyl alcohol) hydrogel allowed the enzyme to recover over 50% of its enzymatic activity following exposure to simulated gastric and intestinal fluids. Our results show that using engineered proteins obtained from extremophiles in combination with polymer-based encapsulation can advance the oral formulations of biologicals.

Structure and expression of two phenylalanine ammonia-lyase genes of the basidiomycete mushroom Tricholoma matsutake

We cloned cDNAs of TmPAL1 and TmPAL2, which encode phenylalanine ammonia-lyases (PALs), and their corresponding genomic DNAs from the basidiomycete mushroom Tricholoma matsutake NBRC 30605. TmPAL1 and TmPAL2 contain several active sites, including the phenylalanine and histidine ammonia-lyases signature pattern. TmPAL1 and TmPAL2 are interrupted by seven and 11 introns, respectively, seven of which are located at the same positions. Transcript levels of TmPAL1 tended to be high in the pileus, stipe, and base of mature fruiting bodies, while transcript levels of TmPAL2 were highest in the gills. Furthermore, PAL activity and methyl cinnamate content were highest in the gills.

Insights into the Mechanistic Pathway of the Pantoea agglomerans Phenylalanine Aminomutase

The biosynthesis of the hybrid peptide-polyketide antibiotic andrimid in Pantoea agglomerans requires the function of a phenylalanine aminomutase, AdmH (designated PaPAM), which converts (S)-a-phenylalanine to (S)-b-phenylalanine. PaPAM is a member of the class I lyase-like family that includes phenylalanine and tyrosine aminomutases (PAMs and TAMs , respectively), phenylalanine ammonia lyases (PALs), tyrosine ammonia lyases (TALs), and histidine ammonia lyases (HALs). PALs, TALs, and HALs produce aryl acrylates from the corresponding aminoacid substrate by the elimination of ammonia. The transformations that are performed by this family of enzymes are, in part, catalyzed by a 4-methylidene-1H-imidazol-5(4H)-one (MIO) prosthetic group. This cofactor is formed post-translationally from a tandem of active site residues, typically Ala-Ser-Gly, and is believed to function as an electrophile through its a/b-unsaturated keto functional group (Scheme 1). Two mechanisms for these transformations have been proposed; in the first, the amino group of the amino acid substrate acts as a nucleophile and attacks the methylidene of MIO through conjugate addition (earlier reports suggested that MIO was a dehydroalanyl moiety). Ammonia is subsequently expelled from the N-alkylated substrate through an a/b-elimination process, which results in the formation of an acrylate reaction intermediate that is released as such in the ammonia lyase reaction. Alternatively, the acrylate remains in the active site for amino group rebound to form the b-amino acid product in the aminomutase reaction. A second proposed mechanism suggests that p-electrons at the ortho-carbon atom of the phenyl ring of the substrate attack MIO, which acts as a Lewis acid, by Friedel–Crafts-like activation. The second process has been principally assigned to ammonia lyase reactions that yield unsaturated products by a/b-elimination, 13] but has also been implicated in the aminomutase reactions. The structures of several enzymes of the MIO-dependent family were characterized in earlier reports. The structure of Rhodobacter sphaeroides tyrosine ammonia lyase (RsTAL) in complex with the competitive inhibitor 2-aminoindan-2phosphonic acid, which is covalently bound by its amino group to MIO in the active site (Protein Data Bank (PDB) 2O7E) was determined. Yet, based on this structure, one of the MIO-based mechanisms was not suggested for the Scheme 1. Two proposed mechanisms for the conversion of substrate to product in a generic aminomutase: a) amino-group alkylation pathway and b) Friedel–Crafts aryl-alkylation pathway. An ammonia lyase reaction terminates at trans-cinnamate or trans-coumarate (X=H (phenylalanine) or OH (tyrosine), respectively).

Discovery of Two Cyanobacterial Phenylalanine Ammonia Lyases: Kinetic and Structural Characterization,

Phenylalanine ammonia lyase (PAL) catalyzes the deamination of phenylalanine to cinnamate and ammonia. While PALs are common in terrestrial plants where they catalyze the first committed step in the formation of phenylpropanoids, only a few prokaryotic PALs have been identified to date. Here we describe for the first time PALs from cyanobacteria, in particular, Anabaena variabilis ATCC 29413 and Nostoc punctiforme ATCC 29133, identified by screening the genome sequences of these organisms for members of the aromatic amino acid ammonia lyase family. Both PAL genes associate with secondary metabolite biosynthetic gene clusters as observed for other eubacterial PAL genes. In comparison to eukaryotic homologues, the cyanobacterial PALs are 20% smaller in size but share similar substrate selectivity and kinetic activity toward L-phenylalanine over L-tyrosine. Structure elucidation by protein X-ray crystallography confirmed that the two cyanobacterial PALs are similar in tertiary and quatenary structure to plant and yeast PALs as well as the mechanistically related histidine ammonia lyases.

Discovery of a Substrate Selectivity Switch in Tyrosine Ammonia-Lyase, a Member of the Aromatic Amino Acid Lyase Family

Tyrosine ammonia-lyase (TAL) is a recently described member of the aromatic amino acid lyase family, which also includes phenylalanine (PAL) and histidine ammonia-lyases (HAL). TAL is highly selective for L-tyrosine, and synthesizes 4-coumaric acid as a protein cofactor or antibiotic precursor in microorganisms. In this report, we identify a single active site residue important for substrate selection in this enzyme family. Replacing the active site residue His89 with Phe in TAL completely switched its substrate selectivity from tyrosine to phenylalanine, thereby converting it into a highly active PAL. When a corresponding mutation was made in PAL, the enzyme lost PAL activity and gained TAL activity. The discovered substrate selectivity switch is a rare example of a complete alteration of substrate specificity by a single point mutation. We also show that the identity of the amino acid at the switch position can serve as a guide to predict substrate specificities of annotated aromatic amino acid lyases in genome sequences.

Structural Determinants and Modulation of Substrate Specificity in Phenylalanine-Tyrosine Ammonia-Lyases

Aromatic amino acid ammonia-lyases catalyze the deamination of L-His, L-Phe, and L-Tyr, yielding ammonia plus aryl acids bearing an alpha,beta-unsaturated propenoic acid. We report crystallographic analyses of unliganded Rhodobacter sphaeroides tyrosine ammonia-lyase (RsTAL) and RsTAL bound to p-coumarate and caffeate. His 89 of RsTAL forms a hydrogen bond with the p-hydroxyl moieties of coumarate and caffeate. His 89 is conserved in TALs but replaced in phenylalanine ammonia-lyases (PALs) and histidine ammonia-lyases (HALs). Substitution of His 89 by Phe, a characteristic residue of PALs, yields a mutant with a switch in kinetic preference from L-Tyr to L-Phe. Structures of the H89F mutant in complex with the PAL product, cinnamate, or the PAL-specific inhibitor, 2-aminoindan-2-phosphonate (AIP), support the role of position 89 as a specificity determinant in the family of aromatic amino acid ammonia-lyases and aminomutases responsible for beta-amino acid biosynthesis.

Friedel—Crafts‐Type Mechanism for the Enzymatic Elimination of Ammonia from Histidine and Phenylalanine

AbstractFor Abstract see ChemInform Abstract in Full Text.

Friedel–Crafts‐Type Mechanism for the Enzymatic Elimination of Ammonia from Histidine and Phenylalanine

The surprisingly high catalytic activity and selectivity of enzymes stem from their ability to both accelerate the target reaction and suppress competitive reaction pathways that may even be dominant in the absence of enzymes. For example, histidine and phenylalanine ammonia-lyases (HAL and PAL) trigger the abstraction of the nonacidic beta protons of these amino acids while leaving the much more acidic ammonium hydrogen atoms untouched. Both ammonia-lyases have a catalytically important electrophilic group, which was believed to be dehydroalanine for 30 years but has now been revealed by X-ray crystallography and UV spectroscopy to be a highly electrophilic 5-methylene-3,5-dihydroimidazol-4-one (MIO) group. Experiments suggest that the reaction is initiated by the electrophilic attack of MIO on the aromatic ring of the substrate. This incomplete Friedel-Crafts-type reaction leads to the activation of a beta proton and its stereospecific abstraction, followed by the elimination of ammonia and regeneration of the MIO group. The plausibility of such a mechanism is supported by a synthetic model. The application of the PAL reaction in the biocatalytic synthesis of enantiomerically pure alpha-amino beta-aryl propionates from aryl acrylates is also discussed.

Kinetic Analysis of the Reactions Catalyzed by Histidine and Phenylalanine Ammonia Lyases

Although both the structures and the reactions of histidine and phenylalanine ammonia lyases (HAL and PAL) are very similar, the former shows a primary kinetic deuterium (D) isotope effect, while the latter does not. In the HAL reaction, the release of ammonia is partially rate-determining and is slower than the release of the product (E)-urocanate (4), whereas in the PAL reaction, the release of (E)-cinnamate (2) is the rate-limiting step. With (2S,3S)-[3-(2)H1]phenylalanine (1a), we determined the kinetic D isotope effects with the PAL mutants Q487A, Y350F, L137 H, and the double mutant L137 H/Q487E. The kH/kD values for the former two were of the same magnitude as with wild-type PAL (1.20+/-0.07), while the exchange of L137 to H almost doubled the effect (kH/kD=2.32+/-0.01). We conclude that L137 is part of the hydrophobic pocket harboring the phenyl group of the substrate/product and is responsible for its strong binding. The stability of the HAL ammonia complex was demonstrated 40 years ago. Here, we show that, in contrast to the former assumption, ammonia in the complex is not covalently bound to the prosthetic electrophile, 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO; 5). We carried out experiments with a mutant enzyme lacking MIO and exhibiting ca. 10(3) times less activity. Nevertheless, the enzyme-ammonia complex was formed, and the mutant behaved upon addition of (E)-[14C]urocanate (4a) like wild-type HAL. We conclude, therefore, that ammonia is bound in the complex by Coulomb forces as ammonium ion and can be released only after (E)-urocanate (4).

Mechanistic Investigation of Phenylalanine Ammonia Lyase by Using N‐Methylated Phenylalanines

AbstractN‐Methyl‐L‐phenylalanine (5), N‐methyl‐4‐nitro‐L‐phenylalanine (6), and N,N‐dimethyl‐4‐nitro‐L‐phenylalanine (7⋅H+) were investigated as substrates or inhibitors of phenylalanine ammonia lyase from Petroselinum crispum. Whereas the former was a reluctant substrate (Km =6.6 mM, kcat =0.22 s−1), no reverse reaction could be detected by using methylamine and (E)‐cinnamate (2). The Km value for ammonia in the reverse reaction by using (E)‐cinnamate (2) was determined to be 4.4 and 2.6M at pH 8.8 and 10, respectively. The N‐methylated 4‐nitro‐L‐phenylalanines 6 and 7 showed only strong inhibitory effects (Ki =130 nM and 8 nM, resp.). These and former results are discussed in terms of the mechanism of action of phenyalalanine and histidine ammonia lyases.

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.

ChemInform Abstract: Mimicking the Reaction of Phenylalanine Ammonia Lyase by a Synthetic Model.

Phenylalanine and histidine ammonia lyases (PAL and HAL) catalyze the reversible conversion of α-amino acids to the corresponding acrylic acids by elimination of ammonia. The prosthetic group 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) at the active site of both enzymes supposedly undergoes an electrophilic attack at the aromatic nucleus in the first step of the mechanism of action. Since no chemical analogy existed for such an electrophile-assisted elimination, we synthesized model compounds, some portion of which mimicked the essentials of the substrate phenylalanine and another portion the electrophilic Michael acceptor in a sterically appropriate distance. The first model, (±)-rel-(1R,2S,3S)-3-[1-methylidene-2-oxo-2-(pyrrolidin-1-yl)ethyl]-2-phenylcyclohexanamine (7) did not react under Friedel-Crafts conditions in the expected way (Scheme 2). The second model compound (±)-2-rel-(1R,2S,3S)-3-(dimethylamino)-2-(3-methoxyphenyl)cyclohexyl]prop-2-enal (12) with a more nucleophilic methoxyphenyl and a more electrophilic α,β-unsaturated carbonyl moiety, underwent an intramolecular Friedel-Crafts-type substitution, but no elimination of the dimethylamino group (Scheme 4). The third model compound, (±)-γ-[(dimethylamino)methyl]-3-methoxy-2,4,6-trimethyl-α-methylidenebenzenebutanal (25) eliminated dimethylamine upon treatment with Lewis acids and subsequent hydrolysis of the intermediate (Scheme 6). When the 3-methoxy-2,4,6-trimethylphenyl moiety of 25 was replaced by the 2,4,6-trimethyl-3-nitrophenyl group, no elimination product could be observed (Scheme 7).

Mimicking the Reaction of Phenylalanine Ammonia Lyase by a Synthetic Model

Phenylalanine and histidine ammonia lyases (PAL and HAL) catalyze the reversible conversion of α-amino acids to the corresponding acrylic acids by elimination of ammonia. The prosthetic group 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) at the active site of both enzymes supposedly undergoes an electrophilic attack at the aromatic nucleus in the first step of the mechanism of action. Since no chemical analogy existed for such an electrophile-assisted elimination, we synthesized model compounds, some portion of which mimicked the essentials of the substrate phenylalanine and another portion the electrophilic Michael acceptor in a sterically appropriate distance. The first model, (±)-rel-(1R,2S,3S)-3-[1-methylidene-2-oxo-2-(pyrrolidin-1-yl)ethyl]-2-phenylcyclohexanamine (7) did not react under Friedel-Crafts conditions in the expected way (Scheme 2). The second model compound (±)-2-rel-(1R,2S,3S)-3-(dimethylamino)-2-(3-methoxyphenyl)cyclohexyl]prop-2-enal (12) with a more nucleophilic methoxyphenyl and a more electrophilic α,β-unsaturated carbonyl moiety, underwent an intramolecular Friedel-Crafts-type substitution, but no elimination of the dimethylamino group (Scheme 4). The third model compound, (±)-γ-[(dimethylamino)methyl]-3-methoxy-2,4,6-trimethyl-α-methylidenebenzenebutanal (25) eliminated dimethylamine upon treatment with Lewis acids and subsequent hydrolysis of the intermediate (Scheme 6). When the 3-methoxy-2,4,6-trimethylphenyl moiety of 25 was replaced by the 2,4,6-trimethyl-3-nitrophenyl group, no elimination product could be observed (Scheme 7).

Spectroscopic Evidence for a 4-Methylidene Imidazol-5-one in Histidine and Phenylalanine Ammonia-Lyases We thank Prof. G. E. Schulz and Dipl.-Chem. M. Baedeker (University of Freiburg) for providing us with the new PAL expression system and Dr. M. Stieger (Hoffmann-La Roche, Basel, Switzerland) for vector pREP4-GroESL carrying the HSP-60 system. D.R. and D.M. thank the Land of Baden-Württemberg for a scholarship for

HAL and PAL are closely related enzymes: They both contain a recently discovered highly electrophilic prosthetic group (shown in the picture) at the active site. This observation adds weight to the previously published hypothesis that the reactions catalyzed by HAL and PAL (histidine and phenylalanine ammonia-lyases) have analogous mechanisms.

Phosphonic Analogues of Phenylalanine and Histidine as Strong Inhibitors of Phenylalanine and Histidine Ammonia-Lyases

Phosphonic Analogues of Phenylalanine and Histidine as Strong Inhibitors of Phenylalanine and Histidine Ammonia-Lyases

Phosphonic Analogues of Phenylalanine and Histidine as Strong Inhibitors of Phenylalanine and Histidine Ammonia-Lyases

Abstract Syntheses of phosphonic analogues of phenylalanine (5) and histidine (7) as well as their biological activities as phenylalanine and histidine ammonialyases inhibitors have been discussed.

The effect of histidine ammonia-lyase on some murine tumours

The histidine ammonia-lyase from bacterial strain CAMR 5315 was partially purified to assess its effect on the growth of murine tumours. This strain was selected as the source after an extensive screening programme for histidine ammonia-lyases. The enzyme was partially purified by ammonium sulphate fractionation, chromatography on DEAE-cellulose and Sephadex G-150. The enzyme reduced circulating l-histidine levels in Wistar rats and in mice persisted with a half-life of 6–7 h. Neither LDH virus nor chemical modification with ethylacetimidate increased the half-life as observed with l-asparaginase and l-glutaminase. The enzyme was tested in mice against Ehrlich carcinoma, L5178Y lymphoblastic leukaemia, Mc/S sarcoma, B16 melanoma, P8157 mastocytoma, P1798 lymphosarcoma and the Gardner 6C3HED lymphosarcoma. The only tumours to show sensitivity to the enzyme were the Mc/S sarcoma against which a 65% increase in life span was observed at the highest enzyme dose, 1000 U/kg on alternate days over 14 days and the Ehrlich ascites carcinoma where cures were obtained at 250 U/kg on alternate days over 14 days but only at inocula levels of 10 5 and 10 3 cells/animal respectively.

Immunological relatedness of histidine ammonia-lyases from some species of Pseudomonas: Taxonomic implication

1. 1. Histidine ammonia-lyases (histidase EC 4.3.1.3) from Pseudomonas testosteroni NCIB 10808 and Pseudomonas putida NCIB 10807 were purified and specific antibody was raised to each separately in a rabbit. 2. 2. Immunological cross-reactions of each antibody to histidine ammonia-lyases from various species of Pseudomonas were examined by the enzyme inhibition test. 3. 3. The immunological data obtained suggest that these Pseudomonas species can be classified into three groups. These cross-reactions tend to indicate a certain degree of homology within species in a group but not between groups.