The review examines functional knowledge gathered over two decades of research on the 8-17 DNAzym... more The review examines functional knowledge gathered over two decades of research on the 8-17 DNAzyme, focusing on three aspects: the structural requirements for catalysis, the role of metal ions and the participation of general acid-base catalysis.
The 8-17 deoxyribozyme (DNAzyme) is a catalytic DNA molecule capable of cleaving specific RNA sub... more The 8-17 deoxyribozyme (DNAzyme) is a catalytic DNA molecule capable of cleaving specific RNA substrates. The deoxyribozyme is activated by a wide variety of divalent metal ions, from Mg2+ to Pb2+, with just a few exceptions. It is not clear if metal ions are directly involved in catalysis, or are required to attain an active conformation, or both. In particular, the connection between metal-induced global structural rearrangements and catalysis is not straightforward. To gain more information on the local structural changes induced by metal ions, we introduced fluorescent 2-aminopurine (2-Ap) residues at different positions of the 8-17 'core'. We found that a construct containing 2-Ap at position 15 was best suited to monitor conformational changes in the presence of Mg2+, Ca2+ or Mn2+. Binding of these activating metal ions caused a local rearrangement at position 15, apparently entailing decreased stacking of the 2-Ap base. The metal dependence for such conformational cha...
Proceedings of the National Academy of Sciences of the United States of America, Apr 18, 2017
The mammalian gene Nit1 (nitrilase-like protein 1) encodes a protein that is highly conserved in ... more The mammalian gene Nit1 (nitrilase-like protein 1) encodes a protein that is highly conserved in eukaryotes and is thought to act as a tumor suppressor. Despite being ∼35% sequence identical to ω-amidase (Nit2), the Nit1 protein does not hydrolyze efficiently α-ketoglutaramate (a known physiological substrate of Nit2), and its actual enzymatic function has so far remained a puzzle. In the present study, we demonstrate that both the mammalian Nit1 and its yeast ortholog are amidases highly active toward deaminated glutathione (dGSH; i.e., a form of glutathione in which the free amino group has been replaced by a carbonyl group). We further show that Nit1-KO mutants of both human and yeast cells accumulate dGSH and the same compound is excreted in large amounts in the urine of Nit1-KO mice. Finally, we show that several mammalian aminotransferases (transaminases), both cytosolic and mitochondrial, can form dGSH via a common (if slow) side-reaction and provide indirect evidence that tr...
Serine racemase is the pyridoxal 5&am... more Serine racemase is the pyridoxal 5'-phosphate-dependent enzyme that catalyzes L-serine racemisation to D-serine, and L- and D-serine beta-elimination in mammalian brain. D-serine is the essential co-agonist of the N-methyl-D-aspartate receptor, that mediates neurotransmission, synaptic plasticity, cell migration and long term potentiation. High and low D-serine levels have been associated with distinct neuropathologies, aging-related deficits and psychiatric disorders due to either hyper- or hypo-activation of the receptor. Serine racemase dual activity is regulated by ATP, divalent cations, cysteine nitrosylation, post-translational modifications, and interactions with proteins that bind either at the N- or C-terminus. A detailed elucidation of the molecular basis of catalysis, regulation and conformational plasticity, as well as enzyme and D-serine localization and neurons and astrocytes cross-talk, opens the way to the development of enzyme inhibitors and effectors for tailored therapeutic treatments.
Monovalent cations affect both conformational and catalytic properties of the tryptophan synthase... more Monovalent cations affect both conformational and catalytic properties of the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium. Their influence on the dynamic properties of the enzyme was probed by monitoring the phosphorescence decay of the unique Trp-177 beta, a residue located near the beta-active site, at the interface between alpha- and beta-subunits. In the presence of either Li+, Na+, Cs+, or NH4+, the phosphorescence decay is biphasic and the average lifetime increases indicating a decrease in the flexibility of the N-terminal domain of the beta-subunit. Since amplitudes but not lifetimes are affected, cations appear to shift the equilibrium between preexisting enzyme conformations. The effect on the reaction between indole and L-serine was studied by steady state kinetic methods at room temperature. We found that cations: (i) bind to the L-serine--enzyme derivatives with an apparent dissociation constant, measured as the concentration of cation corresponding to one-half of the maximal activity, that is in the millimolar range and decreases with ion size; (ii) increase kcat with the order of efficacy Cs+ > K+ > Li+ > Na+; (iii) decrease KM for indole, Na+ being the most effective and causing a 30-fold decrease; and (iv) cause an increase of the kcat/KM ratio by 20-40-fold. The influence on the equilibrium distribution between the external aldimine and the alpha-aminoacrylate, intermediates in the reaction of L-serine with the beta-subunits of the enzyme, was found to be cation-specific.(ABSTRACT TRUNCATED AT 250 WORDS)
Pyridoxal 59-phosphate (PLP)-dependent enzymes represent about 4% of the enzymes classified by th... more Pyridoxal 59-phosphate (PLP)-dependent enzymes represent about 4% of the enzymes classified by the Enzyme Commission. The versatility of PLP in carrying out a large variety of reactions exploiting the electron sink effect of the pyridine ring, the conformational changes accompanying the chemical steps and stabilizing distinct catalytic intermediates, and the spectral properties of the different coenzyme–substrate derivatives signaling the reaction progress are some of the key features of PLP and PLP-dependent catalytic action. Progress in PLP-dependent enzymes functional genomics and development of inhibitors with therapeutic activity highlight the continuous novelty of an ‘old’ class of enzymes.
The 8-17 deoxyribozyme is a small DNA catalyst of significant applicative interest. We have analy... more The 8-17 deoxyribozyme is a small DNA catalyst of significant applicative interest. We have analyzed the kinetic features of a well behaved 8-17 construct and determined the influence of several reaction conditions on such features, providing a basis for further exploration of the deoxyribozyme mechan- ism. The 8-17 bound its substrate with a rate con- stant ~10-fold lower than those
Biochemistry and Molecular Biology of Vitamin B6 and PQQ-dependent Proteins, 2000
ABSTRACT Functional properties of the pyridoxal 5'-phosphate-dependent enzymes tryptophan... more ABSTRACT Functional properties of the pyridoxal 5'-phosphate-dependent enzymes tryptophan synthase and O-acetylserine sulfhydrylase immobilized by crystallization or encapsulation in wet porous silica gels were investigated by absorption microspectrophotometry. The enzyme behavior exhibits a few striking differences in the crystals and silica gels compared to solution, likely due the constraints of either lattice forces or silica matrix on the conformational equilibria that accompany catalytic events. The results define the experimental conditions to accumulate metastable catalytic species in the crystalline state, to develop bioreactors with immobilized enzymes and to slow down protein folding processes.
The present review focuses on a subfamily of pyridoxal phosphate (PLP)-dependent enzymes, belongi... more The present review focuses on a subfamily of pyridoxal phosphate (PLP)-dependent enzymes, belonging to the broader fold-type I structural group and whose archetypes can be considered ornithine δ-transaminase and γ-aminobutyrate transaminase. These proteins were originally christened "subgroup-II aminotransferases" (AT-II) but are very often referred to as "class-III aminotransferases". As names suggest, the subgroup includes mainly transaminases, with just a few interesting exceptions. However, at variance with most other PLP-dependent enzymes, catalysts in this subfamily seem specialized at utilizing substrates whose amino function is not adjacent to a carboxylate group. AT-II enzymes are widespread in biology and play mostly catabolic roles. Furthermore, today several transaminases in this group are being used as bioorganic tools for the asymmetric synthesis of chiral amines. We present an overview of the biochemical and structural features of these enzymes, il...
Human O-phosphoethanolamine (PEA) phospho-lyase is a pyridoxal 5'-phosphate (PLP) dependent e... more Human O-phosphoethanolamine (PEA) phospho-lyase is a pyridoxal 5'-phosphate (PLP) dependent enzyme that catalyzes the degradation of PEA to acetaldehyde, phosphate and ammonia. Physiologically, the enzyme is involved in phospholipid metabolism and is expressed mainly in the brain, where its expression becomes dysregulated in the course of neuropsychiatric diseases. Mechanistically, PEA phospho-lyase shows a remarkable substrate selectivity, strongly discriminating against other amino compounds structurally similar to PEA. Herein, we studied the enzyme under steady-state and pre-steady-state conditions, analyzing its kinetic features and getting insights into the factors that contribute to its specificity. The pH dependence of the catalytic parameters and the pattern of inhibition by the product phosphate and by other anionic compounds suggest that the active site of PEA phospho-lyase is optimized to bind dianionic groups and that this is a prime determinant of the enzyme specifi...
Enzymatic catalysis relies on the action of the amino acid side chains arrayed in the enzyme acti... more Enzymatic catalysis relies on the action of the amino acid side chains arrayed in the enzyme active sites. Usually, only two or three 'essential' residues are directly involved in the bond making and breaking steps leading to product formation. For the past 20 years, enzymologists have been addressing the role of such residues by changing them into chemically inert side chains. Removal of an 'essential' group often does not abolish activity, but can significantly alter the catalytic mechanism. Such results underscore the sophistication of enzyme catalysis and the functional plasticity of enzyme active sites.
The review examines functional knowledge gathered over two decades of research on the 8-17 DNAzym... more The review examines functional knowledge gathered over two decades of research on the 8-17 DNAzyme, focusing on three aspects: the structural requirements for catalysis, the role of metal ions and the participation of general acid-base catalysis.
The 8-17 deoxyribozyme (DNAzyme) is a catalytic DNA molecule capable of cleaving specific RNA sub... more The 8-17 deoxyribozyme (DNAzyme) is a catalytic DNA molecule capable of cleaving specific RNA substrates. The deoxyribozyme is activated by a wide variety of divalent metal ions, from Mg2+ to Pb2+, with just a few exceptions. It is not clear if metal ions are directly involved in catalysis, or are required to attain an active conformation, or both. In particular, the connection between metal-induced global structural rearrangements and catalysis is not straightforward. To gain more information on the local structural changes induced by metal ions, we introduced fluorescent 2-aminopurine (2-Ap) residues at different positions of the 8-17 'core'. We found that a construct containing 2-Ap at position 15 was best suited to monitor conformational changes in the presence of Mg2+, Ca2+ or Mn2+. Binding of these activating metal ions caused a local rearrangement at position 15, apparently entailing decreased stacking of the 2-Ap base. The metal dependence for such conformational cha...
Proceedings of the National Academy of Sciences of the United States of America, Apr 18, 2017
The mammalian gene Nit1 (nitrilase-like protein 1) encodes a protein that is highly conserved in ... more The mammalian gene Nit1 (nitrilase-like protein 1) encodes a protein that is highly conserved in eukaryotes and is thought to act as a tumor suppressor. Despite being ∼35% sequence identical to ω-amidase (Nit2), the Nit1 protein does not hydrolyze efficiently α-ketoglutaramate (a known physiological substrate of Nit2), and its actual enzymatic function has so far remained a puzzle. In the present study, we demonstrate that both the mammalian Nit1 and its yeast ortholog are amidases highly active toward deaminated glutathione (dGSH; i.e., a form of glutathione in which the free amino group has been replaced by a carbonyl group). We further show that Nit1-KO mutants of both human and yeast cells accumulate dGSH and the same compound is excreted in large amounts in the urine of Nit1-KO mice. Finally, we show that several mammalian aminotransferases (transaminases), both cytosolic and mitochondrial, can form dGSH via a common (if slow) side-reaction and provide indirect evidence that tr...
Serine racemase is the pyridoxal 5&am... more Serine racemase is the pyridoxal 5'-phosphate-dependent enzyme that catalyzes L-serine racemisation to D-serine, and L- and D-serine beta-elimination in mammalian brain. D-serine is the essential co-agonist of the N-methyl-D-aspartate receptor, that mediates neurotransmission, synaptic plasticity, cell migration and long term potentiation. High and low D-serine levels have been associated with distinct neuropathologies, aging-related deficits and psychiatric disorders due to either hyper- or hypo-activation of the receptor. Serine racemase dual activity is regulated by ATP, divalent cations, cysteine nitrosylation, post-translational modifications, and interactions with proteins that bind either at the N- or C-terminus. A detailed elucidation of the molecular basis of catalysis, regulation and conformational plasticity, as well as enzyme and D-serine localization and neurons and astrocytes cross-talk, opens the way to the development of enzyme inhibitors and effectors for tailored therapeutic treatments.
Monovalent cations affect both conformational and catalytic properties of the tryptophan synthase... more Monovalent cations affect both conformational and catalytic properties of the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium. Their influence on the dynamic properties of the enzyme was probed by monitoring the phosphorescence decay of the unique Trp-177 beta, a residue located near the beta-active site, at the interface between alpha- and beta-subunits. In the presence of either Li+, Na+, Cs+, or NH4+, the phosphorescence decay is biphasic and the average lifetime increases indicating a decrease in the flexibility of the N-terminal domain of the beta-subunit. Since amplitudes but not lifetimes are affected, cations appear to shift the equilibrium between preexisting enzyme conformations. The effect on the reaction between indole and L-serine was studied by steady state kinetic methods at room temperature. We found that cations: (i) bind to the L-serine--enzyme derivatives with an apparent dissociation constant, measured as the concentration of cation corresponding to one-half of the maximal activity, that is in the millimolar range and decreases with ion size; (ii) increase kcat with the order of efficacy Cs+ > K+ > Li+ > Na+; (iii) decrease KM for indole, Na+ being the most effective and causing a 30-fold decrease; and (iv) cause an increase of the kcat/KM ratio by 20-40-fold. The influence on the equilibrium distribution between the external aldimine and the alpha-aminoacrylate, intermediates in the reaction of L-serine with the beta-subunits of the enzyme, was found to be cation-specific.(ABSTRACT TRUNCATED AT 250 WORDS)
Pyridoxal 59-phosphate (PLP)-dependent enzymes represent about 4% of the enzymes classified by th... more Pyridoxal 59-phosphate (PLP)-dependent enzymes represent about 4% of the enzymes classified by the Enzyme Commission. The versatility of PLP in carrying out a large variety of reactions exploiting the electron sink effect of the pyridine ring, the conformational changes accompanying the chemical steps and stabilizing distinct catalytic intermediates, and the spectral properties of the different coenzyme–substrate derivatives signaling the reaction progress are some of the key features of PLP and PLP-dependent catalytic action. Progress in PLP-dependent enzymes functional genomics and development of inhibitors with therapeutic activity highlight the continuous novelty of an ‘old’ class of enzymes.
The 8-17 deoxyribozyme is a small DNA catalyst of significant applicative interest. We have analy... more The 8-17 deoxyribozyme is a small DNA catalyst of significant applicative interest. We have analyzed the kinetic features of a well behaved 8-17 construct and determined the influence of several reaction conditions on such features, providing a basis for further exploration of the deoxyribozyme mechan- ism. The 8-17 bound its substrate with a rate con- stant ~10-fold lower than those
Biochemistry and Molecular Biology of Vitamin B6 and PQQ-dependent Proteins, 2000
ABSTRACT Functional properties of the pyridoxal 5'-phosphate-dependent enzymes tryptophan... more ABSTRACT Functional properties of the pyridoxal 5'-phosphate-dependent enzymes tryptophan synthase and O-acetylserine sulfhydrylase immobilized by crystallization or encapsulation in wet porous silica gels were investigated by absorption microspectrophotometry. The enzyme behavior exhibits a few striking differences in the crystals and silica gels compared to solution, likely due the constraints of either lattice forces or silica matrix on the conformational equilibria that accompany catalytic events. The results define the experimental conditions to accumulate metastable catalytic species in the crystalline state, to develop bioreactors with immobilized enzymes and to slow down protein folding processes.
The present review focuses on a subfamily of pyridoxal phosphate (PLP)-dependent enzymes, belongi... more The present review focuses on a subfamily of pyridoxal phosphate (PLP)-dependent enzymes, belonging to the broader fold-type I structural group and whose archetypes can be considered ornithine δ-transaminase and γ-aminobutyrate transaminase. These proteins were originally christened "subgroup-II aminotransferases" (AT-II) but are very often referred to as "class-III aminotransferases". As names suggest, the subgroup includes mainly transaminases, with just a few interesting exceptions. However, at variance with most other PLP-dependent enzymes, catalysts in this subfamily seem specialized at utilizing substrates whose amino function is not adjacent to a carboxylate group. AT-II enzymes are widespread in biology and play mostly catabolic roles. Furthermore, today several transaminases in this group are being used as bioorganic tools for the asymmetric synthesis of chiral amines. We present an overview of the biochemical and structural features of these enzymes, il...
Human O-phosphoethanolamine (PEA) phospho-lyase is a pyridoxal 5'-phosphate (PLP) dependent e... more Human O-phosphoethanolamine (PEA) phospho-lyase is a pyridoxal 5'-phosphate (PLP) dependent enzyme that catalyzes the degradation of PEA to acetaldehyde, phosphate and ammonia. Physiologically, the enzyme is involved in phospholipid metabolism and is expressed mainly in the brain, where its expression becomes dysregulated in the course of neuropsychiatric diseases. Mechanistically, PEA phospho-lyase shows a remarkable substrate selectivity, strongly discriminating against other amino compounds structurally similar to PEA. Herein, we studied the enzyme under steady-state and pre-steady-state conditions, analyzing its kinetic features and getting insights into the factors that contribute to its specificity. The pH dependence of the catalytic parameters and the pattern of inhibition by the product phosphate and by other anionic compounds suggest that the active site of PEA phospho-lyase is optimized to bind dianionic groups and that this is a prime determinant of the enzyme specifi...
Enzymatic catalysis relies on the action of the amino acid side chains arrayed in the enzyme acti... more Enzymatic catalysis relies on the action of the amino acid side chains arrayed in the enzyme active sites. Usually, only two or three 'essential' residues are directly involved in the bond making and breaking steps leading to product formation. For the past 20 years, enzymologists have been addressing the role of such residues by changing them into chemically inert side chains. Removal of an 'essential' group often does not abolish activity, but can significantly alter the catalytic mechanism. Such results underscore the sophistication of enzyme catalysis and the functional plasticity of enzyme active sites.
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Papers by Alessio Peracchi