Structural basis for the entrance into the phenylpropanoid metabolism catalyzed by phenylalanine ammonia-lyase

Because of its key role in secondary phenylpropanoid metabolism, Phe ammonia-lyase is one of the most extensively studied plant enzymes. To provide a basis for detailed structure-function studies, the enzyme from parsley (Petroselinum crispum) was crystallized, and the structure was elucidated at 1.7-A resolution. It contains the unusual electrophilic 4-methylidene-imidazole-5-one group, which is derived from a tripeptide segment in two autocatalytic dehydration reactions. The enzyme resembles His ammonia-lyase from the general His degradation pathway but contains 207 additional residues, mainly in an N-terminal extension rigidifying a domain interface and in an inserted alpha-helical domain restricting the access to the active center. Presumably, Phe ammonia-lyase developed from His ammonia-lyase when fungi and plants diverged from the other kingdoms. A pathway of the catalyzed reaction is proposed in agreement with established biochemical data. The inactivation of the enzyme by a nucleophile is described in detail.     

Structures of two histidine ammonia-lyase modifications and implications for the catalytic mechanism.

Histidine ammonia-lyase (EC 4.3.1.3) catalyzes the nonoxidative elimination of the alpha-amino group of histidine using a 4-methylidene-imidazole-5-one (MIO), which is formed autocatalytically from the internal peptide segment 142Ala-Ser-Gly. The structure of the enzyme inhibited by a reaction with l-cysteine was established at the very high resolution of 1.0 A. Five active center mutants were produced and their catalytic activities were measured. Among them, mutant Tyr280-->Phe could be crystallized and its structure could be determined at 1.7 A resolution. It contains a planar sp2-hybridized 144-N atom of MIO, in contrast to the pyramidal sp3-hybridized 144-N of the wild-type. With the planar 144-N atom, MIO assumes the conformation of a putative intermediate aromatic state of the reaction, demonstrating that the conformational barrier between aromatic and wild-type states is very low. The data led to a new proposal for the geometry for the catalyzed reaction, which also applies to the closely related phenylalanine ammonia-lyase (EC 4.3.1.5). Moreover, it suggested an intermediate binding site for the released ammonia.     

Expression in Escherichia coli of catalytically active phenylalanine ammonia-lyase from parsley

In parsley (Petroselinum crispum), phenylalanine ammonia-lyase (PAL) is encoded by 4 structurally similar genes. The nucleotide sequence of a near full-length cDNA and the deduced amino acid sequence of PAL-4 are presented and compared with the corresponding sequences of PAL-1, a previously described representative of the gene family. Transformation of Escherichia coli cells with PAL-1 or PAL-4 cDNA yielded catalytically active PAL, suggesting that the catalytic center of the enzyme is formed spontaneously rather than by a plant-specific mechanism.     

Nucleotide sequences at the sites of action of the deoxyribonucleic acid modification enzyme of bacteriophage P1

Labelled oligonucleotides have been fractionated from DNAase digests of phage λ DNA that had been methylated with the phage P1 modification enzyme and S-[methyl-¹⁴C]adenosyl-l-methionine. The longest sequences established are the tetranucleotides pG-ǎ?-T-C4 and p?-T-C-T, which, together with the other sequences determined, particularly pA-G-?, show that the modification enzyme, M.EcoP1, methylates adenine residues within defined sequences and suggest that the oligonucleotide sequence recognized by this enzyme is the hexanucleotide pA-G-A-T-C-T. The duplex formed by base-pairing this hexanucleotide with its complementary sequence resembles the recognition sequence for several restriction enzymes in being bisected by an axis of 2-fold rotational symmetry.     

Molecular characterization of phenyllactate dehydratase and its initiator from Clostridium sporogenes

The heterotrimeric phenyllactate dehydratase from Clostridium sporogenes, FldABC, catalyses the reversible dehydration of (R)-phenyllactate to (E)-cinnamate in two steps: (i) CoA-transfer from the cofactor cinnamoyl-CoA to phenyllactate to yield phenyllactyl-CoA and the product cinnamate mediated by FldA, a (R)-phenyllactate CoA-transferase; followed by (ii) dehydration of phenyllactyl-CoA to cinnamoyl-CoA mediated by heterodimeric FldBC, a phenyllactyl-CoA dehydratase. Phenyllactate dehydratase requires initiation by ATP, MgCl2 and a reducing agent such as dithionite mediated by an extremely oxygen-sensitive initiator protein (FldI) present in the cell-free extract. All four genes coding for these proteins were cloned and shown to be clustered in the order fldAIBC, which shares over 95% sequence identity of nucleotide and protein levels with a gene cluster detected in the genome of the closely related Clostridium botulinum Hall strain A. FldA shows sequence similarities to a new family of CoA-transferases, which apparently do not form covalent enzyme CoA-ester intermediates. An N-terminal Strep II-Tag containing enzymatically active FldI was overproduced and purified from Escherichia coli. FldI was characterized as a homodimeric protein, which contains one [4Fe-4S]1+/2+ cluster with an electron spin S = 3/2 in the reduced form. The amino acid sequence as well as the chemical and EPR-properties of the pure protein are very similar to those of component A of 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans (HgdC), which was able to replace FldI in the activation of phenyllactate dehydratase. Only in the oxidized state, FldI and component A exhibit significant ATPase activity, which appears to be essential for unidirectional electron transfer. Both subunits of phenyllactyl-CoA dehydratase (FldBC) show significant sequence similarities to both subunits of 2-hydroxyglutaryl-CoA dehydratase (HgdAB). The fldAIBC gene cluster resembles the hadAIBC gene cluster in the genome of Clostridium difficile and the hadABC,I genes in C. botulinum. The four subunits of these deduced 2-hydroxyacid dehydratases (65-81% amino acid sequence identity between the had genes) probably code for a 2-hydroxyisocaproate dehydratase involved in leucine fermentation. This enzyme could be the target for metronidazole in the treatment of pseudomembranous enterocolitis caused by C. difficile.     

The estimation of phenylalanine ammonia-lyase (PAL)-activity in intact cells of higher plant tissue

Summary A procedure is described which permits the estimation of the relative activity of phenylalanine ammonia-lyase (E.C. 4.3.1.5.) in intact plant cells, exemplified by buckwheat hypocotyls. Hypocotyl segments are incubated at pH 5.5 with L-[3-³H]phenylalanine. N³HH₂, which is liberated from phenylalanine by the action of phenylalanine ammonia-lyase, equilibrates with tissue water to yield ³HOH, which is recovered by sublimation. Participation of phenylalanine transaminase in the reactions leading to ³HOH formation is excluded, and it is conclusively shown that ³HOH is formed intracellularly and not by enzymatic activity leaking out of wounded tissue.Abbreviation PAL phenylalanine ammonia-lyase (E.C. 4.3.1.5.)     

Melanoma antigens as modified normal gene sequences

Melanomas are highly aggressive tumors with a well-documented antigenic nature. Several melanoma antigens have been reported, four of which, p97, Ia-like antigen, B700, and A have been implicated as having regions in common with normally occurring proteins. P97 has partial sequence homology with transferrin and lactotransferrin, Ia-like antigen is immunologically cross-reactive with alpha and beta chains of Ia-like proteins, A is a variant of alpha actin, and B700 resembles a normal melanosomal membrane protein. In addition, B700 has partial sequence homology to serum albumins. These observations suggest that melanoma tumors can produce antigenic proteins by modification of normally occurring proteins. The possible mechanisms are discussed.     

An active site homology model of phenylalanine ammonia-lyase from Petroselinum crispum.

The plant enzyme phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) shows homology to histidine ammonia-lyase (HAL) whose structure has been solved by X-ray crystallography. Based on amino-acid sequence alignment of the two enzymes, mutagenesis was performed on amino-acid residues that were identical or similar to the active site residues in HAL to gain insight into the importance of this residues in PAL for substrate binding or catalysis. We mutated the following amino-acid residues: S203, R354, Y110, Y351, N260, Q348, F400, Q488 and L138. Determination of the kinetic constants of the overexpressed and purified enzymes revealed that mutagenesis led in each case to diminished activity. Mutants S203A, R354A and Y351F showed a decrease in kcat by factors of 435, 130 and 235, respectively. Mutants F400A, Q488A and L138H showed a 345-, 615- and 14-fold lower kcat, respectively. The greatest loss of activity occurred in the PAL mutants N260A, Q348A and Y110F, which were 2700, 2370 and 75 000 times less active than wild-type PAL. To elucidate the possible function of the mutated amino-acid residues in PAL we built a homology model of PAL based on structural data of HAL and mutagenesis experiments with PAL. The homology model of PAL showed that the active site of PAL resembles the active site of HAL. This allowed us to propose possible roles for the corresponding residues in PAL catalysis.     

Cloning, sequencing, and expression of the genes encoding the adenosylcobalamin-dependent ethanolamine ammonia-lyase of Salmonella typhimurium

Ethanolamine ammonia-lyase is a bacterial enzyme that catalyzes the adenosylcobalamin-dependent conversion of certain vicinal amino alcohols to oxo compounds and ammonia. Studies of ethanolamine ammonia-lyase from Clostridium sp. and Escherichia coli have suggested that the enzyme is a heterodimer composed of subunits of Mr approximately 55,000 and 35,000. Using a partial Sau3A Salmonella typhimurium library ligated into pBR328 and selecting by complementation of a mutant lacking ethanolamine ammonia-lyase activity, we have cloned the genes for the 2 subunits of the S. typhimurium enzyme. The genes were localized to a 6.5-kilobase fragment of S. typhimurium DNA, from which they could be expressed in E. coli under noninducing conditions. Sequencing of a 2526-base pair portion of this 6.5-kilobase DNA fragment revealed two open reading frames separated by 21 base pairs. The open reading frames encoded proteins of 452 and 286 residues whose derived N-terminal sequences were identical to the N-terminal sequences of the 2 subunits of the E. coli ethanolamine ammonia-lyase, except that residue 16 of the large subunit was asparagine in the E. coli sequence and aspartic acid in the S. typhimurium sequence.     

Induction of Phenylalanine Ammonia-lyase in Xanthium Leaf Disks. Photosynthetic Requirement and Effect of Daylength

A cycloheximide-sensitive increase in the activity of phenylalanine ammonia-lyase (EC 4.3.1.5) occurs in Xanthium leaf disks exposed to light. Radioactive ammonia-lyase has been isolated by means of sucrose density gradient centrifugation and starch gel electrophoresis from disks fed l-isoleucine-U-(14)C or l-arginine-U-(14)C. The incorporation of radioactive amino acids into phenylalanine ammonia-lyase together with the inhibitory effects of cycloheximide indicate that the observed increase in enzyme activity involves the induction of lyase synthesis.The light-dependent synthesis of the ammonia-lyase is completely inhibited by 50 mum 3-(4-chlorophenyl)-1,1-dimethylurea (CMU) indicating that photosynthesis is involved. Only a trace quantity of some photosynthetic product must be needed because half light saturation occurs at very low intensity (ca. 30 ft-c). Exogenous carbohydrate is also required for continuing enzyme synthesis over a 72 hr period. But carbohydrate does not replace the photosynthetic requirement in darkness.Enzyme formed in light disappears rapidly from disks placed in the dark. The decay of ammonia-lyase activity follows first order kinetics. The half-life of the lyase ranged from 6 to 15 hr in leaf material used. Cyoloheximide inhibits the decay of lyase activity. Thus the maintenance of turnover in Xanthium leaf disks requires de novo synthesis of protein. That turnover, i.e., degradation as well as synthesis of lyase protein occurs is suggested by the apparent loss of radioactive ammonia-lyase from leaf disks placed in darkness. Light-induced synthesis coupled with rapid turnover can produce a diurnal fluctuation of ammonia-lyase activity in Xanthium leaf disks. Alternating periods of enzyme synthesis and degradation were observed in disks exposed to 24 hr cycles of light and dark. The average level of enzyme activity maintained in the tissue was directly related to the length of the light period. Induction of lyase synthesis was also observed in excised leaves and to a lesser extent in leaves of whole plants.     

Cloning and expression of rat histidase. Homology to two bacterial histidases and four phenylalanine ammonia-lyases

Histidase (histidine ammonia-lyase, EC 4.3.1.3) catalyzes the deamination of histidine to urocanic acid. Apart from phenylalanine ammonia-lyase, which is not expressed in animals, histidase is the only enzyme known to have a dehydroalanine residue in its active site. The amino site precursor and the mechanism of formation of dehydroalanine are not known. As an initial step to determining the precursor of dehydroalanine in histidase, we have isolated a functional cDNA clone for histidase from a rat liver cDNA library using an affinity-purified antiserum. The 2.2-kilobase cDNA has a 1,971-base pair open reading frame coding for a 657-amino acid polypeptide with a predicted molecular mass of 72,165 Da. The cDNA has a rare polyadenylation signal (AAUACA) that appears to inefficiently direct polyadenylation in transfected COS monkey kidney cells. Conversion of this sequence to the consensus polyadenylation signal (AAUAAA) resulted in increased levels of stable mRNA. COS cells transfected with a histidase expression vector produce active histidase. The formation of active histidase in cells that have no endogenous histidase activity suggests either that the requisite modifying enzyme is present in these cells or that the dehydroalanine residue forms by an autocatalytic mechanism. Rat histidase was found to have 41 and 43% amino acid identity to Pseudomonas putida and Bacillus subtilis histidases, respectively. Phenylalanine ammonia-lyases from parsley, kidney bean, and two yeast strains were also found to have approximately 20% amino acid identity to rat histidase. On the basis of the similarity of function of histidase and phenylalanine ammonia-lyase, dehydroalanine at the active sites, and the sequence conservation over a large evolutionary distance (mammals, bacteria, yeast, and plants), we propose that the genes for histidase and phenylalanine ammonia-lyase have diverged from a common ancestral gene, of which the most conserved regions are likely to be involved in catalysis or dehydroalanine formation.     

Influence of amino acid and peptide counterions on the conformation of DNA

We describe fibre diffraction studies on the interaction of DNA with different amino acids and peptides. The B form of DNA, with ten base-pairs per turn, is always found at high levels of humidity. We suggest that this pitch is observed because the DNA molecules are maintained in a straight position. In solution, the DNA molecules are bent and may have a larger pitch. The A form of DNA is never found upon dehydration. Instead, the B form may be either stabilized by the counterions or altered so that the number of base-pairs per helical turn decreases upon dehydration. Alteration is favoured either by small counterions that have a single charge or by large basic polypeptides and proteins. Stabilization is favoured by small counterions that have several charged groups. A third type of behaviour is found with some amino acids that contain hydrophobic groups, which destabilize the secondary structure of DNA, probably due to a modification of its intramolecular interactions. We have not detected any specific effect of amino acid side-chains, although the amino acid sequence has a clear influence on the interaction. We think that these observations are of interest in the pursuit of more detailed crystallographic studies on protein-DNA interactions.     

Autocatalytic peptide cyclization during chain folding of histidine ammonia-lyase.

Histidine ammonia-lyase requires a 4-methylidene-imidazole-5-one group (MIO) that is produced autocatalytically by a cyclization and dehydration step in a 3-residue loop of the polypeptide. The crystal structures of three mutants have been established. Two mutants were inactive and failed to form MIO, but remained unchanged elsewhere. The third mutant showed very low activity and formed MIO, although it differed from an MIO-less mutant only by an additional 329-C(beta) atom. This atom forms one constraint during MIO formation, the other being the strongly connected Asp145. An exploration of the conformational space of the MIO-forming loop showed that the cyclization is probably enforced by a mechanic compression in a late stage of chain folding and is catalyzed by a well-connected internal water molecule. The cyclization of the respective 3-residue loop of green fluorescent protein is likely to occur in a similar reaction.     

Novel characteristics and regulation of a divergent cinnamate 4-hydroxylase (CYP73A15) from French bean: engineering expression in yeast

cDNAs showing high sequence similarity (>70%) over large stretches to plant CYP73A orthologues from other species were isolated from a cDNA library derived from mRNAs expressed in elicitor-treated suspension-cultured cells. These clones appear to code for a full-length 1554 bp open reading frame with a 78 bp 5-untranslated region and a 140 bp 3-untranslated region. The open reading frame, determined by sequence similarity, codes for a protein with a predicted Mr of 59 229 and a pI of 8.8. It contains the conserved cysteine haem-binding site found in all cytochrome P450s. The protein encoded by this cDNA diverges however from other CYP73As in its N- and C-terminus and in four domains internally, so that overall sequence similarity is in the range 58–66%. Many clones contained an identical intron, which may be associated with a novel regulatory mechanism. Sequence similarity is sufficient for it to be classified as CYP73A15, although it is the least similar member of this family classified so far. The cDNA was expressed in yeast. Successful expression of cinnamate 4-hydroxylase activity required removal of the intron. High-level expression also required modification of the N-terminus to that of CYP73A1. Yeast did not process the intron at all and the leader sequence for A15 was not as compatible as that of A1. The mRNA for CYP73A15 was shown to be rapidly induced by elicitor treatment of suspension-cultured cells of French bean but induction was more transient than that of phenylalanine ammonia-lyase (PAL). In contrast, induction in cells undergoing xylogenesis was much more coordinate with PAL. The cloned cDNA may represent a cinnamate 4-hydroxylase isoform, whose expression is more related to differentiation than the responses to stress in which the majority of CYP73As cloned so far are involved.     

A hotspot for novel amplification joints in a mosaic of Alu-like repeats and palindromic A + T-rich DNA.

We have identified, in the amplified domain of adenylate deaminase (AMPD) overproducing Chinese hamster fibroblasts, a 2.6 kb recombinogenic DNA region which is frequently involved in amplification-associated rearrangements. The nucleotide sequence reveals a mosaic organization of four Alu-equivalent repeats of the B1 and B2 families and eight long A + T-rich DNA segments. Part of this region is enriched with long imperfect palindromes. The center of one palindrome contains a putative topoisomerase I cleavage site and this site defines the position of a novel junction which was formed by illegitimate recombination with anther A + T-rich DNA sequence located far apart on the amplified DNA. These findings and their significance are discussed in the context of related data from other systems and in the light of current models for eukaryotic DNA recombination, replication and organization.     

Comparative model of EutB from coenzyme B12-dependent ethanolamine ammonia-lyase reveals a beta8alpha8, TIM-barrel fold and radical catalytic site structural features

The structure of the EutB protein from Salmonella typhimurium, which contains the active site of the coenzyme B12 (adenosylcobalamin)-dependent enzyme, ethanolamine ammonia-lyase, has been predicted by using structural proteomics techniques of comparative modelling. The 453-residue EutB protein displays no significant sequence identity with proteins of known structure. Therefore, secondary structure prediction and fold recognition algorithms were used to identify templates. Multiple three-dimensional template matching (threading) servers identified predominantly beta8alpha8, TIM-barrel proteins, and in particular, the large subunits of diol dehydratase (PDB: 1eex:A, 1dio:A) and glycerol dehydratase (PDB: 1mmf:A), as templates. Consistent with this identification, the dehydratases are, like ethanolamine ammonia-lyase, Class II coenzyme B12-dependent enzymes. Model building was performed by using MODELLER. Models were evaluated by using different programs, including PROCHECK and VERIFY3D. The results identify a beta8alpha8, TIM-barrel fold for EutB. The beta8alpha8, TIM-barrel fold is consistent with a central role of the alpha/beta-barrel structures in radical catalysis conducted by the coenzyme B12- and S-adenosylmethionine-dependent (radical SAM) enzyme superfamilies. The EutB model and multiple sequence alignment among ethanolamine ammonia-lyase, diol dehydratase, and glycerol dehydratase from different species reveal the following protein structural features: (1) a "cap" loop segment that closes the N-terminal region of the barrel, (2) a common cobalamin cofactor binding topography at the C-terminal region of the barrel, and (3) a beta-barrel-internal guanidinium group from EutB R160 that overlaps the position of the active-site potassium ion found in the dehydratases. R160 is proposed to have a role in substrate binding and radical catalysis.     

Detection of protein similarities using nucleotide sequence databases.

A simple procedure is described for finding similarities between proteins using nucleotide sequence databases. The approach is illustrated by several examples of previously unknown correspondences with important biological implications: Drosophila elongation factor Tu is shown to be encoded by two genes that are differently expressed during development; a cluster of three Drosophila genes likely encode maltases; a flesh-fly fat body protein resembles the hypothesized Drosophila alcohol dehydrogenase ancestral protein; an unknown protein encoded at the multifunctional E. coli hisT locus resembles aspartate beta-semialdehyde dehydrogenase; and the E. coli tyrR protein is related to nitrogen regulatory proteins. These and other matches were discovered using a personal computer of the type available in most laboratories collecting DNA sequence data. As relatively few sequences were sampled to find these matches, it is likely that much of the existing data has not been adequately examined.     

Characterization of nuclear factors for elicitor-mediated activation of the promoter of the pea phenylalanine ammonia-lyase gene 1.

The nuclear factors presumably associated with the activation of the gene encoding phenylalanine ammonia-lyase by a fungal elicitor were characterized in pea (Pisum sativum L.) epicotyls. The TATA-proximal region was dissected and putative cis-regulatory elements in the promoter of pea phenylalanine ammonia-lyase gene 1 were examined by gel-mobility shift and in vitro footprinting analyses. Specific binding of the nuclear factors to the promoter-proximal regions of pea phenylalanine ammonia-lyase gene 1 associated with elicitor-mediated activation was detected at a region containing consensus sequence motifs of boxes 2 and 4 and other AT-rich sequences. The analyses of DNA fragments containing the deleted promoter regions suggested that a residue from -183 to -173 (ATTAGTAAGTGAT) was essential for a maximal activity of forming low-mobility complex (LMC) in the gel-mobility shift assay, and synthetic oligonucleotides confirmed the presence of at least one nuclear component associated with the formation of an active LMC. Competition experiments and treatment with Hoechst 33258 provided direct evidence that the formation of LMC with the promoter fragments from genes encoding phenylalanine ammonia-lyase and chalcone synthase in pea contained one or more of the same proteins that recognize AT-rich sequence motifs for binding. It also suggests that common high-mobility group-like proteins might be involved in the regulation of elicitor-inducible genes in pea.     

Structure and Function of Amino Acid Ammonia-lyases

Histidine ammonia-lyase (HAL) and methylaspartate ammonia-lyase (MAL) belong to the family of carbon-nitrogen lyases (EC 4.3.1). The enzymes catalyze the α,β-elimination of ammonia from (S)-His to yield urocanic acid, and (S)-threo-(2S,3S)-3-methylaspartic acid to mesaconic acid, respectively. Based on structural analyses, the peptide at the active center of HAL from Pseudomonas putida is considered to be post-translationally dehydrated to form an electrophilic 4-methylidene-imidazole-one (MIO) group. A reaction mechanism was proposed with the structure. On the other hand, the structure of MAL from Citrobacter amalonaticus was found to be a typical TIM barrel structure with Mg²⁺ coordinated to the 4-carbonyl of the substrate methylaspartate. Unlike HAL, MIO was not observed in MAL, and the reaction of MAL appears to be completely different from phenylalanine ammonia-lyase (PAL), HAL, and other amino acid ammonia-lyases. A reaction mechanism is proposed in which the hydrogen at the β to the amino group of the substrate is abstracted forming an enolate type intermediate and then ammonia is released.