First evidence of a membrane‐bound,tyramine and β‐phenylethylamine producing,tyrosine decarboxylase in Enterococcus faecalis: A two‐dimensional electrophoresis proteomic study

The soluble and membrane proteome of a tyramine producing Enterococcus faecalis, isolated from an Italian goat cheese, was investigated. A detailed analysis revealed that this strain also produces small amounts of β‐phenylethylamine. Kinetics of tyramine and β‐phenylethylamine accumulation, evaluated in tyrosine plus phenylalanine‐enriched cultures (stimulated condition), suggest that the same enzyme, the tyrosine decarboxylase (TDC), catalyzes both tyrosine and phenylalanine decarboxylation: tyrosine was recognized as the first substrate and completely converted into tyramine (100% yield) while phenylalanine was decarboxylated to β‐phenylethylamine (10% yield) only when tyrosine was completely depleted. The presence of an aspecific aromatic amino acid decarboxylase is a common feature in eukaryotes, but in bacteria only indirect evidences of a phenylalanine decarboxylating TDC have been presented so far. Comparative proteomic investigations, performed by 2‐DE and MALDI‐TOF/TOF MS, on bacteria grown in conditions stimulating tyramine and β‐phenylethylamine biosynthesis and in control conditions revealed 49 differentially expressed proteins. Except for aromatic amino acid biosynthetic enzymes, no significant down‐regulation of the central metabolic pathways was observed in stimulated conditions, suggesting that tyrosine decarboxylation does not compete with the other energy‐supplying routes. The most interesting finding is a membrane‐bound TDC highly over‐expressed during amine production. This is the first evidence of a true membrane‐bound TDC, longly suspected in bacteria on the basis of the gene sequence.     

Role of aromatic aldehyde synthase in wounding/herbivory response and flower scent production in different Arabidopsis ecotypes

Aromatic L-amino acid decarboxylases (AADCs) are key enzymes operating at the interface between primary and secondary metabolism. The Arabidopsis thaliana genome contains two genes, At2g20340 and At4g28680, encoding pyridoxal 5'-phosphate-dependent AADCs with high homology to the recently identified Petunia hybrida phenylacetaldehyde synthase involved in floral scent production. The At4g28680 gene product was recently biochemically characterized as an L-tyrosine decarboxylase (AtTYDC), whereas the function of the other gene product remains unknown. The biochemical and functional characterization of the At2g20340 gene product revealed that it is an aromatic aldehyde synthase (AtAAS), which catalyzes the conversion of phenylalanine and 3,4-dihydroxy-L-phenylalanine to phenylacetaldehyde and dopaldehyde, respectively. AtAAS knock-down and transgenic AtAAS RNA interference (RNAi) lines show significant reduction in phenylacetaldehyde levels and an increase in phenylalanine, indicating that AtAAS is responsible for phenylacetaldehyde formation in planta. In A. thaliana ecotype Columbia (Col-0), AtAAS expression was highest in leaves, and was induced by methyl jasmonate treatment and wounding. Pieris rapae larvae feeding on Col-0 leaves resulted in increased phenylacetaldehyde emission, suggesting that the emitted aldehyde has a defensive activity against attacking herbivores. In the ecotypes Sei-0 and Di-G, which emit phenylacetaldehyde as a predominant flower volatile, the highest expression of AtAAS was found in flowers and RNAi AtAAS silencing led to a reduction of phenylacetaldehyde formation in this organ. In contrast to ecotype Col-0, no phenylacetaldehyde accumulation was observed in Sei-0 upon wounding, suggesting that AtAAS and subsequently phenylacetaldehyde contribute to pollinator attraction in this ecotype.     

New tricks for the glycyl radical enzyme family

Glycyl radical enzymes (GREs) are important biological catalysts in both strict and facultative anaerobes, playing key roles both in the human microbiota and in the environment. GREs contain a backbone glycyl radical that is post-translationally installed, enabling radical-based mechanisms. GREs function in several metabolic pathways including mixed acid fermentation, ribonucleotide reduction and the anaerobic breakdown of the nutrient choline and the pollutant toluene. By generating a substrate-based radical species within the active site, GREs enable C–C, C–O and C–N bond breaking and formation steps that are otherwise challenging for nonradical enzymes. Identification of previously unknown family members from genomic data and the determination of structures of well-characterized GREs have expanded the scope of GRE-catalyzed reactions as well as defined key features that enable radical catalysis. Here, we review the structures and mechanisms of characterized GREs, classifying members into five categories. We consider the open questions about each of the five GRE classes and evaluate the tools available to interrogate uncharacterized GREs.     

Structure,Function, and Evolution of the Pseudomonas aeruginosa Lysine Decarboxylase LdcA

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Crystallization of a mammalian ornithine decarboxylase

Crystals of truncated (Δ425-461) pyridoxal-5′-phosphate (PLP)-dependent mouse ornithine decarboxylase (mOrnDC′) have been obtained that diffract to 2.2 Å resolution (P2₁2₁2, a = 119.5 Å, b = 74.3 Å, c = 46.1 Å). OrnDC produces putrescine, which is the precursor for the synthesis of polyamines in eukaryotes. Regulation of activity and understanding of the mechanism of action of this enzyme may aid in the development of compounds against cancer. mOrnDC is a member of group IV PLP-dependent decarboxylases, for which there are no known representative structures.     

Participation of amino acid decarboxylases in biochemical interactions between triticale (Triticosecale; Poaceae) and bird cherry-oat aphid (Rhopalosiphum padi; Aphididae)

The roles of ornithine decarboxylase, lysine decarboxylase and tyrosine decarboxylase in biochemical interactions of two cultivars of winter triticale (Triticosecale), Tornado and Witon, and bird cherry-oat aphid (Rhopalosiphum padi L.) were determined. Results showed the resistant Witon had higher lysine decarboxylase activity than the susceptible Tornado. There was a significant negative correlation between the density of R. padi populations and lysine decarboxylase activity. Such correlations did not occur for the other decarboxylases. Aphid feeding induced a decrease of lysine decarboxylase activity within both cultivars after one week of infestation and increased its activity after two weeks in the moderately resistant Witon. Ornithine decarboxylase activity was induced in tissues of the susceptible Tornado and inhibited in Witon after two weeks of infestation. Aphid infestations did not change tyrosine decarboxylase activity in Witon, whereas in Tornado it decreased in activity after one day of aphid feeding and then increased after two weeks. It was concluded that of the three enzymes studied, lysine decarboxylase was the most important in the response of winter triticale to infestation by R. padi.     

Bacterial sodium ion-coupled energetics

For many bacteria Na⁺ bioenergetics is important as a link between exergonic and endergonic reactions in the membrane. This article focusses on two primary Na⁺ pumps in bacteria, the Na⁺-translocating oxaloacetate decarboxylase ofKlebsiella pneumoniae and the Na⁺-translocating F₁F₀ ATPase ofPropionigenium modestum. Oxaloacetate decarboxylase is an essential enzyme of the citrate fermentation pathway and has the additional function to conserve the free energy of decarboxylation by conversion into a Na⁺ gradient. Oxaloacetate decarboxylase is composed of three different subunits and the related methylmalonyl-CoA decarboxylase consists of five different subunits. The genes encoding these enzymes have been cloned and sequenced. Remarkable are large areas of complete sequence identity in the integral membrane-bound -subunits including two conserved aspartates that may be important for Na⁺ translocation. The coupling ratio of the decarboxylase Na⁺ pumps depended on and decreased from two to zero Na⁺ uptake per decarboxylation event as increased from zero to the steady state level.InP. modestum, is generated in the course of succinate fermentation to propionate and CO₂. This is used by a unique Na⁺-translocating F₁F₀ ATPase for ATP synthesis. The enzyme is related to H⁺-translocating F₁F₀ ATPases. The F₀ part is entirely responsible for the coupling of ion specificity. A hybrid ATPase formed by in vivo complementation of anEscherichia coli deletion mutant was completely functional as a Na⁺-ATP synthase conferring theE. coli strain the ability of Na⁺-dependent growth on succinate. The hybrid consisted of subunits a, c, b, and part of fromP. modestum and of the remaining subunits fromE. coli. Studies on Na⁺ translocation through the F₀ part of theP. modestum ATPase revealed typical transporter-like properties. Sodium ions specifically protected the ATPase from the modification of glutamate-65 in subunit c by dicyclohexylcarbodiimide in a pH-dependent manner indicating that the Na⁺ binding site is at this highly conserved acidic amino acid residue of subunit c within the middle of the membrane.     

Transport of Phosphatidylserine from the Endoplasmic Reticulum to the Site of Phosphatidylserine Decarboxylase2 in Yeast

Over the past two decades, most of the genes specifying lipid synthesis and metabolism in yeast have been identified and characterized. Several of these biosynthetic genes and their encoded enzymes have provided valuable tools for the genetic and biochemical dissection of interorganelle lipid transport processes in yeast. One such pathway involves the synthesis of phosphatidylserine (PtdSer) in the endoplasmic reticulum (ER), and its non‐vesicular transport to the site of phosphatidylserine decarboxylase2 (Psd2p) in membranes of the Golgi and endosomal sorting system. In this review, we summarize the identification and characterization of the yeast phosphatidylserine decarboxylases, and examine their role in studies of the transport‐dependent pathways of de novo synthesis of phosphatidylethanolamine (PtdEtn). The emerging picture of the Psd2p‐specific transport pathway is one in which the enzyme and its non‐catalytic N‐terminal domains act as a hub to nucleate the assembly of a multiprotein complex, which facilitates PtdSer transport at membrane contact sites between the ER and Golgi/endosome membranes. After transport to the catalytic site of Psd2p, PtdSer is decarboxylated to form PtdEtn, which is disseminated throughout the cell to support the structural and functional needs of multiple membranes.      

Molecular Genetic Analyses of Plant Polyamines

Polyamines are small, positively charged aliphatic amines that play a variety of roles in plant physiology. Putrescine, spermidine, and spermine are usually what are collectively meant by the term polyamines, although plants also have a variety of other related compounds and secondary product conjugates to polyamines. Organisms synthesize putrescine, spermidine, and spermine by pathways leading from ornithine, arginine, and SAM, with three of the important enzymes being amino acid decarboxylases. There has been recent progress in understanding plant polyamines, both their function and the regulation of their synthesis, as a result of molecular genetic investigations. The cDNAs for many of the key enzymes have been cloned and se-quenced, and studies on regulation of the enzymes have begun. Mutational and transgenic approaches are being used to perturb the pathway. Some of the phenotypes observed suggest interactions between polyamines and either ethylene or cytokinin, consistent with some of the correlations observed many years previously by polyamine physiologists. These studies, while still in their early stages, should improve our understanding of polyamine synthesis, but difficult problems remain to be solved before we can answer the question: What are the biological functions and associated mechanisms of action of polyamines?