Biochemical identification of residues that discriminate between 3,4-dihydroxyphenylalanine decarboxylase and 3,4-dihydroxyphenylacetaldehyde synthase-mediated reactions

In available insect genomes, there are several L-3,4-dihydroxyphenylalanine (L-dopa) decarboxylase (DDC)-like or aromatic amino acid decarboxylase (AAAD) sequences. This contrasts to those of mammals whose genomes contain only one DDC. Our previous experiments established that two DDC-like proteins from Drosophila actually mediate a complicated decarboxylation-oxidative deamination process of dopa in the presence of oxygen, leading to the formation of 3,4-dihydroxyphenylacetaldehyde (DHPA), CO₂, NH_3, and H₂O₂. This contrasts to the typical DDC-catalyzed reaction, which produces CO₂ and dopamine. These DDC-like proteins were arbitrarily named DHPA synthases based on their critical role in insect soft cuticle formation. Establishment of reactions catalyzed by these AAAD-like proteins solved a puzzle that perplexed researchers for years, but to tell a true DHPA synthase from a DDC in the insect AAAD family remains problematic due to high sequence similarity. In this study, we performed extensive structural and biochemical comparisons between DHPA synthase and DDC. These comparisons identified several target residues potentially dictating DDC-catalyzed and DHPA synthase-catalyzed reactions, respectively. Comparison of DHPA synthase homology models with crystal structures of typical DDC proteins, particularly residues in the active sites, provided further insights for the roles these identified target residues play. Subsequent site-directed mutagenesis of the tentative target residues and activity evaluations of their corresponding mutants determined that active site His192 and Asn192 are essential signature residues for DDC- and DHPA synthase-catalyzed reactions, respectively. Oxygen is required in DHPA synthase-mediated process and this oxidizing agent is reduced to H₂O₂ in the process. Biochemical assessment established that H₂O₂, formed in DHPA synthase-mediated process, can be reused as oxidizing agent and this active oxygen species is reduced to H₂O; thereby avoiding oxidative stress by H₂O₂. Results of our structural and functional analyses provide a reasonable explanation of mechanisms involved in DHPA synthase-mediated reactions. Based on the key active site residue Asn192, identified in Drosophila DHPA synthase, we were able to distinguish all available insect DHPA synthases from DDC sequences primarily.     

Biochemical Evaluation of the Decarboxylation and Decarboxylation-Deamination Activities of Plant Aromatic Amino Acid Decarboxylases

Plant aromatic amino acid decarboxylase (AAAD) enzymes are capable of catalyzing either decarboxylation or decarboxylation-deamination on various combinations of aromatic amino acid substrates. These two different activities result in the production of arylalkylamines and the formation of aromatic acetaldehydes, respectively. Variations in product formation enable individual enzymes to play different physiological functions. Despite these catalytic variations, arylalkylamine and aldehyde synthesizing AAADs are indistinguishable without protein expression and characterization. In this study, extensive biochemical characterization of plant AAADs was performed to identify residues responsible for differentiating decarboxylation AAADs from aldehyde synthase AAADs. Results demonstrated that a tyrosine residue located on a catalytic loop proximal to the active site of plant AAADs is primarily responsible for dictating typical decarboxylase activity, whereas a phenylalanine at the same position is primarily liable for aldehyde synthase activity. Mutagenesis of the active site phenylalanine to tyrosine in Arabidopsis thaliana and Petroselinum crispum aromatic acetaldehyde synthases primarily converts the enzymes activity from decarboxylation-deamination to decarboxylation. The mutation of the active site tyrosine to phenylalanine in the Catharanthus roseus and Papaver somniferum aromatic amino acid decarboxylases changes the enzymes decarboxylation activity to a primarily decarboxylation-deamination activity. Generation of these mutant enzymes enables the production of unusual AAAD enzyme products including indole-3-acetaldehyde, 4-hydroxyphenylacetaldehyde, and phenylethylamine. Our data indicates that the tyrosine and phenylalanine in the catalytic loop region could serve as a signature residue to reliably distinguish plant arylalkylamine and aldehyde synthesizing AAADs. Additionally, the resulting data enables further insights into the mechanistic roles of active site residues.     

Identification of an l-dopa decarboxylase gene from Sorangium cellulosum So ce90

During a screening program intended to identify genes encoding enzymes typical for secondary metabolism in Sorangium cellulosum So ce90, an aromatic amino acid decarboxylase gene (ddc) was detected. Expression of ddc in Escherichia coli and subsequent enzyme assays with cell-free extracts confirmed the proposed function derived from amino acid sequence comparisons. In contrast to other aromatic amino acid decarboxylases of eukaryotic origin, the S. cellulosum Ddc converted only L-dihydroxy phenylalanine. This is the first report of a gene encoding an L-dihydroxy phenylalanine decarboxylase in bacteria.     

Molecular Mapping of a Gene Cluster Flanking the Drosophila Dopa Decarboxylase Gene

Nine lethal complementation groups flanking the Drosophila Dopa decarboxylase (Ddc) gene, have been localized within 100 kb of cloned chromosomal DNA. Six of these complementation groups are within 23 kb of DNA, and all ten complementation groups, including Ddc, lie within 78-82 kb of DNA. The potential significance of this unusually high gene density is discussed.     

Complex evolution of orthologous and paralogous decarboxylase genes

The decarboxylases are involved in neurotransmitter synthesis in animals, and in pathways of secondary metabolism in plants. Different decarboxylase proteins are characterized for their different substrate specificities, but are encoded by homologous genes. We study, within a maximum-likelihood framework, the evolutionary relationships among dopa decarboxylase (Ddc), histidine decarboxylase (Hdc) and alpha-methyldopa hypersensitive (amd) in animals, and tryptophan decarboxylase (Wdc) and tyrosine decarboxylase (Ydc) in plants. The evolutionary rates are heterogeneous. There are differences between paralogous genes in the same lineages: 4.13 x 10(-10) nucleotide substitutions per site per year in mammalian Ddc vs. 1.95 in Hdc; between orthologous genes in different lineages, 7.62 in dipteran Ddc vs. 4.13 in mammalian Ddc; and very large temporal variations in some lineages, from 3.7 up to 54.9 in the Drosophila Ddc lineage. Our results are inconsistent with the molecular clock hypothesis.     

Isolation and characterization of a cDNA clone encoding human aromatic L-amino acid decarboxylase

The nucleotide sequence of a cDNA clone that includes the entire coding region of human aromatic L-amino acid decarboxylase gene is presented. A human pheochromocytoma cDNA library was screened using an oligonucleotide probe which corresponded to a partial amino acid sequence of the enzyme purified from the human pheochromocytoma. The isolated cDNA clone encoded a protein of 480 amino acids with a calculated molecular mass of 53.9 kDa. The amino acid sequence Asn-Phe-Asn-Pro-His-Lys-Trp around a possible cofactor (pyridoxal phosphate) binding site is identical in human, Drosophila, and pig enzymes.     

A kinetic analysis of Drosophila melanogaster dopa decarboxylase

The kinetic mechanism of dopa decarboxylase (3,4-dihydroxy-L-phenylalanine carboxy-lyase, EC 4.1.1.28) was investigated in Drosophila melanogaster. Based on initial velocity and product inhibition studies, an ordered reaction is proposed for dopa decarboxylase. This kinetic mechanism is interpreted in the context of measured enzyme activities and the catecholamine pools in Drosophila. The 1(2)amd gene is immediately adjacent to the gene coding for dopa decarboxylase (Ddc) and determines hypersensitivity to alpha-methyldopa in Drosophila. Dopa decarboxylase does not decarboxylate alpha-methyldopa and hence does not generate a toxic product capable of inhibiting 1(2)amd gene function. We propose that the 1(2)amd gene is involved with an unknown catecholamine pathway involving dopa but not dopamine.     

Nucleotide sequence of the Escherichia coli cad operon: a system for neutralization of low extracellular pH.

Lysine decarboxylase of Escherichia coli has been the subject of enzymological studies, and the gene encoding lysine decarboxylase (cadA) and a regulatory gene (cadR) have been mapped. This enzyme is induced at low pH in the presence of lysine and achieves maximal level under anaerobic conditions. The induction of lysine decarboxylase increases the pH of the extracellular medium and provides a distinctive marker in tests of clinical strains. We report the sequence of the cad operon encoding lysine decarboxylase, a protein of 715 amino acids, and another protein, CadB, of 444 amino acids. The amino acid sequence of lysine decarboxylase showed high homology to that of the lysine decarboxylase of Hafnia alvei with less homology to the sequence of speC, which encodes the biosynthetic ornithine decarboxylase of E. coli. The cadA and cadB genes were separately cloned and placed under the control of lac and tac promoters, respectively, to facilitate independent study of their physiological effects. The cadB gene product had a mobility characteristic of a smaller protein on protein gels, analogous to that found for some other membrane proteins. The CadB sequence showed homology to that of ArcD of Pseudomonas aeruginosa, encoding an arginine/ornithine antiporter. Excretion studies of various strains, the coinduction of cadB and cadA, and the attractive physiological role for an antiport system led to a model for the coupled action of cadA and cadB in uptake of lysine, the reduction of H+ concentration, and excretion of cadaverine.     

Molecular cloning and analysis of a cDNA coding for chorismate synthase from the higher plant Corydalis sempervirens Pers.

Chorismate synthase catalyzes the last common step in the biosynthesis of the three aromatic amino acids in microorganisms and plants. We have cloned a cDNA for this enzyme from the higher plant Corydalis sempervirens. This is the first chorismate synthase cDNA from a eukaryotic organism. The nucleotide sequence was determined and the identity of the cDNA was confirmed by the amino acid sequence of tryptic peptides obtained from purified chorismate synthase. The homology to the two known bacterial sequences is about 48%. The cDNA contains an open reading frame of 1341 base pairs, encoding a protein of 447 amino acids. This protein with a molecular mass of 48,100 daltons resembles a chorismate synthase precursor targeted for chloroplast import. Multiple sites of polyadenylation were observed in chorismate synthase mRNAs.     

The Drosophila virilis dopa decarboxylase gene is developmentally regulated when integrated into Drosophila melanogaster.

The dopa decarboxylase gene (Ddc) has been isolated from Drosophila virilis and introduced into the germ-line of Drosophila melanogaster by P-element mediated transformation. The integrated gene is induced at the correct stages during development with apparently normal tissue specificity, indicating that cis-acting elements required for regulation are functionally conserved between the two species. A comparison of the DNA sequences from the 5' flanking regions reveals a cluster of small (8-16 bp) conserved sequence elements within 150 bp upstream of the RNA startpoint, a region required for normal expression of the D. melanogaster Ddc gene.     

Ddcde1, a Mutant Differentially Affecting Both Stage and Tissue Specific Expression of Dopa Decarboxylase in Drosophila

The isolation and characterization of a unique Dopa decarboxylase (Ddc) mutant in Drosophila melanogaster is reported. This mutant, DdcDE1, exhibits stage- and tissue-specific altered Ddc expression. Homozygous DdcDE1 embryos, central nervous systems (CNSs) at pupariation and newly eclosed adult epidermis all have approximately 5% as much specific dopa decarboxylase (DDC) activity as the pr control stock in which DdcDE1 was induced. In contrast, the DdcDE1 epidermis at pupariation has roughly 50% as much DDC activity as controls, a 10-fold increase over the relative activity detected in other tissues and stages. Although the adult cuticle lacks proper pigmentation as expected in flies with low DDC activity (less than or equal to 5%), the bristles unexpectedly have wild-type black pigmentation. This implies that the bristle forming cells have more DDC activity than the rest of the adult epidermis. This variegated phenotype, black bristles and pale cuticle, plus the fact that DdcDE1 was originally isolated in a reciprocal translocation between proximal X heterochromatin and the euchromatic left arm of the second chromosome, 42 bands from the Ddc locus, suggested that the mutant might be an example of position-effect variegation. All tests for position-effect variegation, including persistence of the mutant phenotype when DdcDE1 was removed from the translocation, were negative. At pupariation DDC cross-reacting material (CRM) levels are similar in DdcDE1 and wild-type controls, but in newly eclosed adults CRM levels are approximately 35% of wild-type controls. This suggests that DDC produced by DdcDE1 adults has less activity per DDC molecule than the DDC produced at pupariation by DdcDE1. If the DDC enzyme produced by DdcDE1 at adult eclosion had full DDC activity (35% DDC CRM = 35% DDC activity) then no mutant phenotype would be exhibited by DdcDE1 since flies with as little as 10% activity have a wild-type phenotype. DDC thermolability assays clearly demonstrate that DDC from DdcDE1 is more thermolabile than control DDC at both pupariation and adult eclosion. Furthermore, DDC from adults in both DdcDE1 and the pr control is more thermolabile than DDC from white prepupae. Mixing experiments indicate the difference in DDC thermolability between pr white prepupae and pr adults is not due to a difference in the white prepupal and adult supernatants. This suggests that in wild-type different isoforms of DDC are produced either by differences in post-translational modification or as a result of a different primary amino acid sequence.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nucleotide sequence of a cDNA for a member of the human 90-kDa heat-shock protein family

This paper describes the isolation and sequence of a human cDNA homologous to a class of proteins commonly referred to as 90-kDa heat-shock proteins. The complete nucleotide sequence of 2563 bp and the deduced amino acid sequence are presented. A single long open reading frame encodes a protein of 83,303 Da, the amino acid composition of which correlates well with that determined for the human 90-kDa heat-shock or 'stress' protein [Welch, W.J. and Feramisco, J.R., J. Biol. Chem. 257 (1982) 14949-14959]. Moreover, sequence analysis of this gene reveals extensive homology with the Drosophila 83-kDa and yeast 90-kDa heat-shock proteins. A comparison of the translated product of the human cDNA to the published yeast 90-kDa heat-shock protein reveals more than 60% homology at both the nucleotide and amino acid levels. Several regions of 50 aa or more show greater than 90% identity. This cDNA also hybridizes with an RNA species which increases upon heat shock of HeLa cells.     

A cis-acting element and associated binding factor required for CNS expression of the Drosophila melanogaster dopa decarboxylase gene.

A cis-acting sequence from the Drosophila melanogaster dopa decarboxylase (Ddc) gene is selectively required for Ddc expression in the central nervous system. We analyze several parameters influencing the function of the sequence element and describe a factor which interacts with it and mediates CNS expression of Ddc. The element, element I, can function in vivo when included on a synthetic oligonucleotide inserted near its normal location, or closer to the RNA startpoint. It displays partial activity when inverted. Two different 2-bp mutations in element I abolish its ability to stimulate neuronal Ddc expression in the CNS. A factor present in embryonic nuclear extracts specifically protects element I in DNase I footprinting assays. The binding affinity of this factor is reduced by each alteration of element I that inhibits neuronal expression, indicating a role in mediating CNS expression of Ddc. Element I alone has no detectable activity when placed adjacent to a heterologous promoter, although 2.2 kb of 5' Ddc sequences direct correct cell-specific expression of a heterologous promoter.