David Merkler - Academia.edu (original) (raw)

Papers by David Merkler

Peptides play a key role in controlling many physiological and neurobiological pathways. Many bio... more Peptides play a key role in controlling many physiological and neurobiological pathways. Many bioactive peptides require a C-terminal α-amide for full activity. The bifunctional enzyme catalyzing α-amidation, peptidylglycine α-amidating monooxygenase (PAM), is the sole enzyme responsible for amidated peptide biosynthesis, from Chlamydomonas reinhardtii to Homo sapiens. Many neuronal and endocrine functions are dependent upon amidated peptides; additional amidated peptides are growth promoters in tumors. The amidation reaction occurs in two steps, glycine α-hydroxylation followed by dealkylation to generate the α-amide product. Currently, most potentially useful inhibitors target the first reaction, which is rate-limiting. PAM is a membrane-bound enzyme that visits the cell surface during peptide secretion. PAM is then used again in the biosynthetic pathway, meaning that cell-impermeable inhibitors or inactivators could have therapeutic value for the treatment of cancer or psychiatri...

Journal of biology and nature, 2017

Long-chain -fatty acylglycines, R-CO-NH-CH-COOH (where "R" refers to an unsaturated or ... more Long-chain -fatty acylglycines, R-CO-NH-CH-COOH (where "R" refers to an unsaturated or saturated alkyl chain of at least 14 carbons) are found in mammals and insects and are structurally related to the cell-signaling, lipid-like, -fatty acylethanolamines, R-CO-NH-CH-CH-OH (where "R" refers to an alkyl chain of at least 14 carbons). Accumulating evidence demonstrates that the -fatty acylglycines have important cellular functions, but much work remains in order to fully appreciate and understand these biomolecules including: () more work on their functions , () measuring their concentrations in the cell, ( defining the pathways for the biosynthesis and degradation, and () understanding the metabolic interconversion(s) between the -fatty acylglycines and other fatty acid amides. The purpose of reviewing the current state-of-knowledge about the -fatty acylglycines is to stimulate future research about this intriguing family of biomolecules.

Research has clearly established a link between omega-3 fatty acids and general health, particula... more Research has clearly established a link between omega-3 fatty acids and general health, particularly cardiovascular health. Omega-3 Fatty Acids in Brain and Neurological Health is the first book to focus exclusively on the role of omega-3 fatty acids on general brain health. The articles in this collection illustrate omega-3 fatty acids' importance in longevity, cognitive impairment, and structure and function of the brain's neurons. Research has established links between omega-3 fatty acids and the developing brain, aging, dementia, Alzheimer's disease and multiple sclerosis. This book encompasses some of the most recent research, including the role of omega-3 fatty acid supplements on hippocampal neurogenesis, substantia nigra modulation, migraine headaches, the developing brain in animals, sleep, and neurodegenerative diseases. This collection helps to push research forward toward a complete understanding of omega-3 fatty acids' relationship to brain and neurologi...

Biochimica et biophysica acta, Jan 26, 1981

Under conditions of low ionic strength, ribonuclease A, which binds more tightly to single- than ... more Under conditions of low ionic strength, ribonuclease A, which binds more tightly to single- than to double-stranded DNA, lowers the melting temperature of DNA helices (Jensen and von Hippel (1976) J. Biol. Chem. 251, 7198-7214). The effects of chemical modification of lysine and arginine residues on the helix-destabilizing properties of this protein have been examined. Removal of the positive charge on the lysine epsilon-amino group, either by maleylation or acetylation, destroys the ability of RNAase A to lower the Tm of poly[d(A-T)]. However, reductive alkylation of these residues, which has not effect on charge, yields derivatives which lower the Tm by only about one-half that seen with unmodified controls. Phenylglyoxalation of arginines can largely remove the Tm-depressing activity of RNAase A. RNAase S, which is produced by cleavage of RNAase A between amino acids 20 and 21, possesses DNA helix-destabilizing activity comparable to that of the parent protein, whereas S-protein ...

Biochemistry, 2014

Arylalkylamine N-acetyltransferase (AANAT) catalyzes the penultimate step in the biosynthesis of ... more Arylalkylamine N-acetyltransferase (AANAT) catalyzes the penultimate step in the biosynthesis of melatonin and other N-acetylarylalkylamides from the corresponding arylalkylamine and acetyl-CoA. The N-acetylation of arylalkylamines is a critical step in Drosophila melanogaster for the inactivation of the bioactive amines and the sclerotization of the cuticle. Two AANAT variants (AANATA and AANATB) have been identified in D. melanogaster, in which AANATA differs from AANATB by the truncation of 35 amino acids from the N-terminus. We have expressed and purified both D. melanogaster AANAT variants (AANATA and AANATB) in Escherichia coli and used the purified enzymes to demonstrate that this N-terminal truncation does not affect the activity of the enzyme. Subsequent characterization of the kinetic and chemical mechanism of AANATA identified an ordered sequential mechanism, with acetyl-CoA binding first, followed by tyramine. We used a combination of pH−activity profiling and site-directed mutagenesis to study prospective residues believed to function in AANATA catalysis. These data led to an assignment of Glu-47 as the general base in catalysis with an apparent pK a of 7.0. Using the data generated for the kinetic mechanism, structure−function relationships, pH−rate profiles, and site-directed mutagenesis, we propose a chemical mechanism for AANATA.

Natural Product Reports, 1999

Biochemistry, 1993

The transition state of the allosteric AMP deaminase from Saccharomyces cerevisiae has been chara... more The transition state of the allosteric AMP deaminase from Saccharomyces cerevisiae has been characterized by 14C and 15N VmaJKm heavy-atom kinetic isotope effects. The primary 6J4C isotope effect was measured with [6-I4C]AMP, and the 6J5N primary isotope effect was measured by isotope ratio mass spectrometry using the natural abundance of 15N in AMP and by using I5N release from ATP as a slow substrate. Isotope effects for AMP as the substrate were measured in the presence and absence of ATP as an allosteric activator and GTP as an allosteric inhibitor. Kinetic isotope effects with [6-14C]AMP were 1.030 f 0.003, 1.038 f 0.004, and 1.042 f 0.003 in the absence of effectors and in the presence of ATP and GTP, respectively. Isotope effects for [6-15N]AMP averaged 1.010 f 0.002. Allosteric activation increased the 15N isotope effect to 1.016 f 0.003. A primary I5N kinetic isotope effect with ATP, which has a Vmax/Km 10" that for AMP, was 1 .O 13 i 0,001. The presence of D2O as solvent caused a marginally significant decrease in the [6-l5N]AMP kinetic isotope effect from 1.01 1 f 0.001 to 1.007 * 0.002. Previous studies have established that the solvent D2O effect is inverse (0.34) for slow substrates with two or more protons transferred prior to transition state formation and remains inverse (0.79) with AMP as substrate [Merkler, D. J., & Schramm, V. L. (1 993) Biochemistry 32, 579247991. Bond vibrational analysis was used to identify transition states for AMP deaminase that are consistent with all kinetic isotope effects. Fully concerted reaction mechanisms can be eliminated, since these would result in normal D2O solvent isotope effects and are inconsistent with 14C and 15N kinetic isotope effects. The transition state most consistent with the data is characterized by an attacking hydroxyl with a bond order near 0.8, a fully bonded NH2, nearly complete conversion to sp3 at C6, and highly asymmetric, nearly complete protonation of N1. This transition state leads to the formation of a short-lived tetrahedral intermediate. Formation of the tetrahedral intermediate is slow, while protonation of NH2 in the intermediate and departure of NH3 occur in rapid steps.

Frontiers in molecular biosciences, 2018

Arylalkylamine -acyltransferases (AANATs) catalyze the formation of an -acylamide from an acyl-Co... more Arylalkylamine -acyltransferases (AANATs) catalyze the formation of an -acylamide from an acyl-CoA thioester and an amine. One well known example is the production of -acetylserotonin from acetyl-CoA and serotonin, a reaction in the melatonin biosynthetic pathway from tryptophan. AANATs have been identified from a variety of vertebrates and invertebrates. Considerable efforts have been devoted to the mammalian AANAT because a cell-permeable inhibitor specifically targeted against this enzyme could prove useful to treat diseases related to dysfunction in melatonin production. Insects are an interesting model for the study of AANATs because more than one isoform is typically expressed by a specific insect and the different insect AANATs (iAANATs) serve different roles in the insect cell. In contrast, mammals express only one AANAT. The major role of iAANATs seem to be in the production of -acetyldopamine, a reaction important in the tanning and sclerotization of the cuticle. Metabolit...

Biochemistry & Molecular Biology Journal

Crop protection against destructive pests has been at the forefront of recent agricultural advanc... more Crop protection against destructive pests has been at the forefront of recent agricultural advancements. Rapid adaptive evolution has led to insects becoming immune to the chemicals employed to quell their damage. Insecticide resistance is a serious problem that negatively impacts food production, food storage, human health, and the environment. To make matters more complicated are the strict regulations in place on insecticide development, driven by rising public concern relating to the harmful effects these chemicals have on the environment and on society. A key component to solving the problem of insecticide resistance, while keeping public welfare in mind, is the identification of novel insect-specific protein targets. One unexplored target for the development of new targeted insecticides are the insect arylalkylamine -acetyltransferases (iAANATs). This group of enzymes, shown to be intrinsic in the development of the insect cuticle, is an untapped well of potential for target-specific inhibition, while offering enough variety to ensure protection for non-target enzymes. In this review, we highlight kinetic, genetic and bioinformatic data showing that the iAANATs are intriguing insecticide targets that should be specific only for particular insect pests. Such a pest-specific insecticide would minimize environmental harm by eliminating such non-discriminate attacks which have made insecticides such a highly regulated industry, and would have negligible toxicity to humans and other mammals.

Prostaglandins, leukotrienes, and essential fatty acids, 2018

The purpose of this research is to unravel the substrate specificity and kinetic properties of an... more The purpose of this research is to unravel the substrate specificity and kinetic properties of an insect arylalkylamine N-acyltransferase from Bombyx mori (Bm-iAANAT) and to determine if this enzyme will catalyze the formation of long chain N-acylarylalkylamides in vitro. However, the determination of substrates and products for Bm-iAANAT in vitro is no guarantee that these same molecules are substrates and products for the enzyme in the organism. Therefore, RT-PCR was performed to detect the Bm-iAANAT transcripts and liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QToF-MS) analysis was performed on purified lipid extracts from B. mori larvae (fourth instar, Bmi4) to determine if long chain fatty acid amides are produced in B. mori. Ultimately, we found that recombinant Bm-iAANAT will utilize long-chain acyl-CoA thioesters as substrates and identified Bm-iAANAT transcripts and long-chain fatty acid amides in Bmi4. Together, these data show Bm-iAANAT will cataly...

Journal of biology and nature, 2017

Long-chain -fatty acylglycines, R-CO-NH-CH-COOH (where "R" refers to an unsaturated or ... more Long-chain -fatty acylglycines, R-CO-NH-CH-COOH (where "R" refers to an unsaturated or saturated alkyl chain of at least 14 carbons) are found in mammals and insects and are structurally related to the cell-signaling, lipid-like, -fatty acylethanolamines, R-CO-NH-CH-CH-OH (where "R" refers to an alkyl chain of at least 14 carbons). Accumulating evidence demonstrates that the -fatty acylglycines have important cellular functions, but much work remains in order to fully appreciate and understand these biomolecules including: () more work on their functions , () measuring their concentrations in the cell, ( defining the pathways for the biosynthesis and degradation, and () understanding the metabolic interconversion(s) between the -fatty acylglycines and other fatty acid amides. The purpose of reviewing the current state-of-knowledge about the -fatty acylglycines is to stimulate future research about this intriguing family of biomolecules.

Biochemistry & molecular biology journal, 2018

The non-mevalonate dependent (NMVA) pathway for the biosynthesis of isopentenyl pyrophosphate and... more The non-mevalonate dependent (NMVA) pathway for the biosynthesis of isopentenyl pyrophosphate and dimethylallyl pyrophosphate is the sole source of these terpenoids for the production of isoprenoids in the apicomplexan parasites, in many eubacteria, and in plants. The absence of this pathway in higher organisms has opened a new platform for the development of novel antibiotics and anti-malarials. The enzyme catalyzing the first step of the NMVA pathway is 1-deoxy-D-xylulose-5-phosphate synthase (DXPS). DXPS catalyzes the thiamine pyrophosphate- and Mg (II)-dependent conjugation of pyruvate and D-glyceraldehyde-3-phosphate to form 1-deoxy-D-xylulose-5-phosphate and CO. The kinetic mechanism of DXPS from most consistent with our data is random sequential as shown using a combination of kinetic analysis and product and dead-end inhibition studies. The role of active site amino acids, identified by sequence alignment to other DXPS proteins, was probed by constructing and analyzing the c...

PLOS ONE

The transfer of an acetyl group from acetyl-CoA to an acceptor amine is a ubiquitous biochemical ... more The transfer of an acetyl group from acetyl-CoA to an acceptor amine is a ubiquitous biochemical transformation catalyzed by Gcn5-related N-acetyltransferases (GNATs). Although it is established that the reaction proceeds through a sequential ordered mechanism, the role of the acetyl group in driving the ordered formation of binary and ternary complexes remains elusive. Herein, we show that CoA and acetyl-CoA alter the conformation of the substrate binding site of an arylalkylamine N-acetyltransferase (AANAT) to facilitate interaction with acceptor substrates. However, it is the presence of the acetyl group within the catalytic funnel that triggers high affinity binding. Acetyl group occupancy is relayed through a conserved salt bridge between the P-loop and the acceptor binding site, and is manifested as differential dynamics in the CoA and acetyl-CoA-bound states. The capacity of the acetyl group carried by an acceptor to promote its tight binding even in the absence of CoA, but also its mutually exclusive position to the acetyl group of acetyl-CoA underscore its importance in coordinating the progression of the catalytic cycle.

The journal of physical chemistry. B, Sep 22, 2016

1-Deoxy-d-xylulose 5-phosphate synthase (DXS) is a thiamin diphosphate (TDP) dependent enzyme tha... more 1-Deoxy-d-xylulose 5-phosphate synthase (DXS) is a thiamin diphosphate (TDP) dependent enzyme that marks the beginning of the methylerythritol 4-phosphate isoprenoid biosynthesis pathway. The mechanism of action for DXS is still poorly understood and begins with the formation of a thiazolium ylide. This TDP activation step is thought to proceed through an intramolecular deprotonation by the 4'-aminopyrimidine ring of TDP; however, this step would occur only after an initial deprotonation of its own 4'-amino group. The mechanism of the initial deprotonation has been hypothesized, by analogy to transketolases, to occur via a histidine or an active site water molecule. Results from hybrid quantum mechanical/molecular mechanical (QM/MM) reaction path calculations reveal an ∼10 kcal/mol difference in transition state energies, favoring a water mediated mechanism over direct deprotonation by histidine. This difference was determined to be largely governed by electrostatic changes ...

Biochemistry, 1992

The biosynthesis of C-terminal alpha-amidated peptides from their corresponding C-terminal glycin... more The biosynthesis of C-terminal alpha-amidated peptides from their corresponding C-terminal glycine-extended precursors is catalyzed by peptidylglycine alpha-amidating enzyme (alpha-AE) in a reaction that requires copper, ascorbate, and molecular oxygen. Using bifunctional type A rat alpha-AE, we have shown that O2 is the source of the alpha-carbonyl oxygen of pyruvate produced during the amidation of dansyl-Tyr-Val-[alpha-13C]-D-Ala, as demonstrated by the 18O isotopic shift in the 13C NMR spectrum of [alpha-13C]lactate generated from [alpha-13C]pyruvate in the presence of lactate dehydrogenase and NADH. In addition, one-to-one stoichiometries have been determined for glyoxylate formed/dansyl-Tyr-Val-Gly consumed, pyruvate formed/dansyl-Tyr-Val-D-Ala consumed, dansyl-Tyr-Val-NH2 formed/ascorbate oxidized, and dansyl-Tyr-Val-NH2 formed/O2 consumed. Quantitative coupling of NADH oxidation to dansyl-Tyr-Val-NH2 production using Neurospora crassa semidehydroascorbate reductase showed that two one-electron reductions by ascorbate occurred per alpha-AE turnover. The stoichiometry of approximately 1.0 dansyl-Tyr-Val-NH2 produced/ascorbate oxidized observed in the absence of a semidehydroascorbate trap resulted from the disproportionation of two semidehydroascorbate molecules to ascorbate and dehydroascorbate.

Arch Biochem Biophys, 1991

International Journal of Peptide and Protein Research, 2009

The amino acid composition of more than 20 enzymes and protein from various closely related mesop... more The amino acid composition of more than 20 enzymes and protein from various closely related mesophilic and thermophilic micro-organisms (esp. Bacillus) have been used to calculate a variety of macroscopic parameters. These included the hydrophobic index (H phi ), the ratio of polar to non-polar volumes (rho), the ratios of Arg/(Arg + Lys), and (Arg + Lys) or (Glx + Asx) to total amino acids, % H-bonding amino acids, % alpha-helix- or beta-sheet-forming amino acids, the theoretical melting temperature (TCalcm), the total volume to total amino acid ratio (VR), and the % non-polar residues (NPS). In contrast to previous similar comparisons with proteins from divergent sources, it was found that thermophilic vs mesophilic proteins from the same genus show correlations between thermostability and increased H phi, decreased rho, and increased Arg/(Arg + Lys), as well as increased alpha-index and beta-index. Weaker correlations were seen for VR, TCalcm, aliphatic index, and NPS, all derived from, or related to, H phi. No correlations existed for the other calculated parameters. These results are consistent with recent results of Argos et al. (1979) [Biochemistry 18, 5698-5703] on sequence analyses of glyceraldehyde-3-P dehydrogenases, where thermophilic proteins showed multiple amino acid replacements that caused increased internal hydrophobicity and increased external polarity. No trends were observed in any of the parameters calculated from amino acid compositions for crude cytoplasmic protein extracts from mesophilic vs thermophilic Bacilli.

Peptides play a key role in controlling many physiological and neurobiological pathways. Many bio... more Peptides play a key role in controlling many physiological and neurobiological pathways. Many bioactive peptides require a C-terminal α-amide for full activity. The bifunctional enzyme catalyzing α-amidation, peptidylglycine α-amidating monooxygenase (PAM), is the sole enzyme responsible for amidated peptide biosynthesis, from Chlamydomonas reinhardtii to Homo sapiens. Many neuronal and endocrine functions are dependent upon amidated peptides; additional amidated peptides are growth promoters in tumors. The amidation reaction occurs in two steps, glycine α-hydroxylation followed by dealkylation to generate the α-amide product. Currently, most potentially useful inhibitors target the first reaction, which is rate-limiting. PAM is a membrane-bound enzyme that visits the cell surface during peptide secretion. PAM is then used again in the biosynthetic pathway, meaning that cell-impermeable inhibitors or inactivators could have therapeutic value for the treatment of cancer or psychiatri...

Journal of biology and nature, 2017

Long-chain -fatty acylglycines, R-CO-NH-CH-COOH (where "R" refers to an unsaturated or ... more Long-chain -fatty acylglycines, R-CO-NH-CH-COOH (where "R" refers to an unsaturated or saturated alkyl chain of at least 14 carbons) are found in mammals and insects and are structurally related to the cell-signaling, lipid-like, -fatty acylethanolamines, R-CO-NH-CH-CH-OH (where "R" refers to an alkyl chain of at least 14 carbons). Accumulating evidence demonstrates that the -fatty acylglycines have important cellular functions, but much work remains in order to fully appreciate and understand these biomolecules including: () more work on their functions , () measuring their concentrations in the cell, ( defining the pathways for the biosynthesis and degradation, and () understanding the metabolic interconversion(s) between the -fatty acylglycines and other fatty acid amides. The purpose of reviewing the current state-of-knowledge about the -fatty acylglycines is to stimulate future research about this intriguing family of biomolecules.

Research has clearly established a link between omega-3 fatty acids and general health, particula... more Research has clearly established a link between omega-3 fatty acids and general health, particularly cardiovascular health. Omega-3 Fatty Acids in Brain and Neurological Health is the first book to focus exclusively on the role of omega-3 fatty acids on general brain health. The articles in this collection illustrate omega-3 fatty acids' importance in longevity, cognitive impairment, and structure and function of the brain's neurons. Research has established links between omega-3 fatty acids and the developing brain, aging, dementia, Alzheimer's disease and multiple sclerosis. This book encompasses some of the most recent research, including the role of omega-3 fatty acid supplements on hippocampal neurogenesis, substantia nigra modulation, migraine headaches, the developing brain in animals, sleep, and neurodegenerative diseases. This collection helps to push research forward toward a complete understanding of omega-3 fatty acids' relationship to brain and neurologi...

Biochimica et biophysica acta, Jan 26, 1981

Under conditions of low ionic strength, ribonuclease A, which binds more tightly to single- than ... more Under conditions of low ionic strength, ribonuclease A, which binds more tightly to single- than to double-stranded DNA, lowers the melting temperature of DNA helices (Jensen and von Hippel (1976) J. Biol. Chem. 251, 7198-7214). The effects of chemical modification of lysine and arginine residues on the helix-destabilizing properties of this protein have been examined. Removal of the positive charge on the lysine epsilon-amino group, either by maleylation or acetylation, destroys the ability of RNAase A to lower the Tm of poly[d(A-T)]. However, reductive alkylation of these residues, which has not effect on charge, yields derivatives which lower the Tm by only about one-half that seen with unmodified controls. Phenylglyoxalation of arginines can largely remove the Tm-depressing activity of RNAase A. RNAase S, which is produced by cleavage of RNAase A between amino acids 20 and 21, possesses DNA helix-destabilizing activity comparable to that of the parent protein, whereas S-protein ...

Biochemistry, 2014

Arylalkylamine N-acetyltransferase (AANAT) catalyzes the penultimate step in the biosynthesis of ... more Arylalkylamine N-acetyltransferase (AANAT) catalyzes the penultimate step in the biosynthesis of melatonin and other N-acetylarylalkylamides from the corresponding arylalkylamine and acetyl-CoA. The N-acetylation of arylalkylamines is a critical step in Drosophila melanogaster for the inactivation of the bioactive amines and the sclerotization of the cuticle. Two AANAT variants (AANATA and AANATB) have been identified in D. melanogaster, in which AANATA differs from AANATB by the truncation of 35 amino acids from the N-terminus. We have expressed and purified both D. melanogaster AANAT variants (AANATA and AANATB) in Escherichia coli and used the purified enzymes to demonstrate that this N-terminal truncation does not affect the activity of the enzyme. Subsequent characterization of the kinetic and chemical mechanism of AANATA identified an ordered sequential mechanism, with acetyl-CoA binding first, followed by tyramine. We used a combination of pH−activity profiling and site-directed mutagenesis to study prospective residues believed to function in AANATA catalysis. These data led to an assignment of Glu-47 as the general base in catalysis with an apparent pK a of 7.0. Using the data generated for the kinetic mechanism, structure−function relationships, pH−rate profiles, and site-directed mutagenesis, we propose a chemical mechanism for AANATA.

Natural Product Reports, 1999

Biochemistry, 1993

The transition state of the allosteric AMP deaminase from Saccharomyces cerevisiae has been chara... more The transition state of the allosteric AMP deaminase from Saccharomyces cerevisiae has been characterized by 14C and 15N VmaJKm heavy-atom kinetic isotope effects. The primary 6J4C isotope effect was measured with [6-I4C]AMP, and the 6J5N primary isotope effect was measured by isotope ratio mass spectrometry using the natural abundance of 15N in AMP and by using I5N release from ATP as a slow substrate. Isotope effects for AMP as the substrate were measured in the presence and absence of ATP as an allosteric activator and GTP as an allosteric inhibitor. Kinetic isotope effects with [6-14C]AMP were 1.030 f 0.003, 1.038 f 0.004, and 1.042 f 0.003 in the absence of effectors and in the presence of ATP and GTP, respectively. Isotope effects for [6-15N]AMP averaged 1.010 f 0.002. Allosteric activation increased the 15N isotope effect to 1.016 f 0.003. A primary I5N kinetic isotope effect with ATP, which has a Vmax/Km 10" that for AMP, was 1 .O 13 i 0,001. The presence of D2O as solvent caused a marginally significant decrease in the [6-l5N]AMP kinetic isotope effect from 1.01 1 f 0.001 to 1.007 * 0.002. Previous studies have established that the solvent D2O effect is inverse (0.34) for slow substrates with two or more protons transferred prior to transition state formation and remains inverse (0.79) with AMP as substrate [Merkler, D. J., & Schramm, V. L. (1 993) Biochemistry 32, 579247991. Bond vibrational analysis was used to identify transition states for AMP deaminase that are consistent with all kinetic isotope effects. Fully concerted reaction mechanisms can be eliminated, since these would result in normal D2O solvent isotope effects and are inconsistent with 14C and 15N kinetic isotope effects. The transition state most consistent with the data is characterized by an attacking hydroxyl with a bond order near 0.8, a fully bonded NH2, nearly complete conversion to sp3 at C6, and highly asymmetric, nearly complete protonation of N1. This transition state leads to the formation of a short-lived tetrahedral intermediate. Formation of the tetrahedral intermediate is slow, while protonation of NH2 in the intermediate and departure of NH3 occur in rapid steps.

Frontiers in molecular biosciences, 2018

Arylalkylamine -acyltransferases (AANATs) catalyze the formation of an -acylamide from an acyl-Co... more Arylalkylamine -acyltransferases (AANATs) catalyze the formation of an -acylamide from an acyl-CoA thioester and an amine. One well known example is the production of -acetylserotonin from acetyl-CoA and serotonin, a reaction in the melatonin biosynthetic pathway from tryptophan. AANATs have been identified from a variety of vertebrates and invertebrates. Considerable efforts have been devoted to the mammalian AANAT because a cell-permeable inhibitor specifically targeted against this enzyme could prove useful to treat diseases related to dysfunction in melatonin production. Insects are an interesting model for the study of AANATs because more than one isoform is typically expressed by a specific insect and the different insect AANATs (iAANATs) serve different roles in the insect cell. In contrast, mammals express only one AANAT. The major role of iAANATs seem to be in the production of -acetyldopamine, a reaction important in the tanning and sclerotization of the cuticle. Metabolit...

Biochemistry & Molecular Biology Journal

Crop protection against destructive pests has been at the forefront of recent agricultural advanc... more Crop protection against destructive pests has been at the forefront of recent agricultural advancements. Rapid adaptive evolution has led to insects becoming immune to the chemicals employed to quell their damage. Insecticide resistance is a serious problem that negatively impacts food production, food storage, human health, and the environment. To make matters more complicated are the strict regulations in place on insecticide development, driven by rising public concern relating to the harmful effects these chemicals have on the environment and on society. A key component to solving the problem of insecticide resistance, while keeping public welfare in mind, is the identification of novel insect-specific protein targets. One unexplored target for the development of new targeted insecticides are the insect arylalkylamine -acetyltransferases (iAANATs). This group of enzymes, shown to be intrinsic in the development of the insect cuticle, is an untapped well of potential for target-specific inhibition, while offering enough variety to ensure protection for non-target enzymes. In this review, we highlight kinetic, genetic and bioinformatic data showing that the iAANATs are intriguing insecticide targets that should be specific only for particular insect pests. Such a pest-specific insecticide would minimize environmental harm by eliminating such non-discriminate attacks which have made insecticides such a highly regulated industry, and would have negligible toxicity to humans and other mammals.

Prostaglandins, leukotrienes, and essential fatty acids, 2018

The purpose of this research is to unravel the substrate specificity and kinetic properties of an... more The purpose of this research is to unravel the substrate specificity and kinetic properties of an insect arylalkylamine N-acyltransferase from Bombyx mori (Bm-iAANAT) and to determine if this enzyme will catalyze the formation of long chain N-acylarylalkylamides in vitro. However, the determination of substrates and products for Bm-iAANAT in vitro is no guarantee that these same molecules are substrates and products for the enzyme in the organism. Therefore, RT-PCR was performed to detect the Bm-iAANAT transcripts and liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QToF-MS) analysis was performed on purified lipid extracts from B. mori larvae (fourth instar, Bmi4) to determine if long chain fatty acid amides are produced in B. mori. Ultimately, we found that recombinant Bm-iAANAT will utilize long-chain acyl-CoA thioesters as substrates and identified Bm-iAANAT transcripts and long-chain fatty acid amides in Bmi4. Together, these data show Bm-iAANAT will cataly...

Journal of biology and nature, 2017

Long-chain -fatty acylglycines, R-CO-NH-CH-COOH (where "R" refers to an unsaturated or ... more Long-chain -fatty acylglycines, R-CO-NH-CH-COOH (where "R" refers to an unsaturated or saturated alkyl chain of at least 14 carbons) are found in mammals and insects and are structurally related to the cell-signaling, lipid-like, -fatty acylethanolamines, R-CO-NH-CH-CH-OH (where "R" refers to an alkyl chain of at least 14 carbons). Accumulating evidence demonstrates that the -fatty acylglycines have important cellular functions, but much work remains in order to fully appreciate and understand these biomolecules including: () more work on their functions , () measuring their concentrations in the cell, ( defining the pathways for the biosynthesis and degradation, and () understanding the metabolic interconversion(s) between the -fatty acylglycines and other fatty acid amides. The purpose of reviewing the current state-of-knowledge about the -fatty acylglycines is to stimulate future research about this intriguing family of biomolecules.

Biochemistry & molecular biology journal, 2018

The non-mevalonate dependent (NMVA) pathway for the biosynthesis of isopentenyl pyrophosphate and... more The non-mevalonate dependent (NMVA) pathway for the biosynthesis of isopentenyl pyrophosphate and dimethylallyl pyrophosphate is the sole source of these terpenoids for the production of isoprenoids in the apicomplexan parasites, in many eubacteria, and in plants. The absence of this pathway in higher organisms has opened a new platform for the development of novel antibiotics and anti-malarials. The enzyme catalyzing the first step of the NMVA pathway is 1-deoxy-D-xylulose-5-phosphate synthase (DXPS). DXPS catalyzes the thiamine pyrophosphate- and Mg (II)-dependent conjugation of pyruvate and D-glyceraldehyde-3-phosphate to form 1-deoxy-D-xylulose-5-phosphate and CO. The kinetic mechanism of DXPS from most consistent with our data is random sequential as shown using a combination of kinetic analysis and product and dead-end inhibition studies. The role of active site amino acids, identified by sequence alignment to other DXPS proteins, was probed by constructing and analyzing the c...

PLOS ONE

The transfer of an acetyl group from acetyl-CoA to an acceptor amine is a ubiquitous biochemical ... more The transfer of an acetyl group from acetyl-CoA to an acceptor amine is a ubiquitous biochemical transformation catalyzed by Gcn5-related N-acetyltransferases (GNATs). Although it is established that the reaction proceeds through a sequential ordered mechanism, the role of the acetyl group in driving the ordered formation of binary and ternary complexes remains elusive. Herein, we show that CoA and acetyl-CoA alter the conformation of the substrate binding site of an arylalkylamine N-acetyltransferase (AANAT) to facilitate interaction with acceptor substrates. However, it is the presence of the acetyl group within the catalytic funnel that triggers high affinity binding. Acetyl group occupancy is relayed through a conserved salt bridge between the P-loop and the acceptor binding site, and is manifested as differential dynamics in the CoA and acetyl-CoA-bound states. The capacity of the acetyl group carried by an acceptor to promote its tight binding even in the absence of CoA, but also its mutually exclusive position to the acetyl group of acetyl-CoA underscore its importance in coordinating the progression of the catalytic cycle.

The journal of physical chemistry. B, Sep 22, 2016

1-Deoxy-d-xylulose 5-phosphate synthase (DXS) is a thiamin diphosphate (TDP) dependent enzyme tha... more 1-Deoxy-d-xylulose 5-phosphate synthase (DXS) is a thiamin diphosphate (TDP) dependent enzyme that marks the beginning of the methylerythritol 4-phosphate isoprenoid biosynthesis pathway. The mechanism of action for DXS is still poorly understood and begins with the formation of a thiazolium ylide. This TDP activation step is thought to proceed through an intramolecular deprotonation by the 4'-aminopyrimidine ring of TDP; however, this step would occur only after an initial deprotonation of its own 4'-amino group. The mechanism of the initial deprotonation has been hypothesized, by analogy to transketolases, to occur via a histidine or an active site water molecule. Results from hybrid quantum mechanical/molecular mechanical (QM/MM) reaction path calculations reveal an ∼10 kcal/mol difference in transition state energies, favoring a water mediated mechanism over direct deprotonation by histidine. This difference was determined to be largely governed by electrostatic changes ...

Biochemistry, 1992

The biosynthesis of C-terminal alpha-amidated peptides from their corresponding C-terminal glycin... more The biosynthesis of C-terminal alpha-amidated peptides from their corresponding C-terminal glycine-extended precursors is catalyzed by peptidylglycine alpha-amidating enzyme (alpha-AE) in a reaction that requires copper, ascorbate, and molecular oxygen. Using bifunctional type A rat alpha-AE, we have shown that O2 is the source of the alpha-carbonyl oxygen of pyruvate produced during the amidation of dansyl-Tyr-Val-[alpha-13C]-D-Ala, as demonstrated by the 18O isotopic shift in the 13C NMR spectrum of [alpha-13C]lactate generated from [alpha-13C]pyruvate in the presence of lactate dehydrogenase and NADH. In addition, one-to-one stoichiometries have been determined for glyoxylate formed/dansyl-Tyr-Val-Gly consumed, pyruvate formed/dansyl-Tyr-Val-D-Ala consumed, dansyl-Tyr-Val-NH2 formed/ascorbate oxidized, and dansyl-Tyr-Val-NH2 formed/O2 consumed. Quantitative coupling of NADH oxidation to dansyl-Tyr-Val-NH2 production using Neurospora crassa semidehydroascorbate reductase showed that two one-electron reductions by ascorbate occurred per alpha-AE turnover. The stoichiometry of approximately 1.0 dansyl-Tyr-Val-NH2 produced/ascorbate oxidized observed in the absence of a semidehydroascorbate trap resulted from the disproportionation of two semidehydroascorbate molecules to ascorbate and dehydroascorbate.

Arch Biochem Biophys, 1991

International Journal of Peptide and Protein Research, 2009

The amino acid composition of more than 20 enzymes and protein from various closely related mesop... more The amino acid composition of more than 20 enzymes and protein from various closely related mesophilic and thermophilic micro-organisms (esp. Bacillus) have been used to calculate a variety of macroscopic parameters. These included the hydrophobic index (H phi ), the ratio of polar to non-polar volumes (rho), the ratios of Arg/(Arg + Lys), and (Arg + Lys) or (Glx + Asx) to total amino acids, % H-bonding amino acids, % alpha-helix- or beta-sheet-forming amino acids, the theoretical melting temperature (TCalcm), the total volume to total amino acid ratio (VR), and the % non-polar residues (NPS). In contrast to previous similar comparisons with proteins from divergent sources, it was found that thermophilic vs mesophilic proteins from the same genus show correlations between thermostability and increased H phi, decreased rho, and increased Arg/(Arg + Lys), as well as increased alpha-index and beta-index. Weaker correlations were seen for VR, TCalcm, aliphatic index, and NPS, all derived from, or related to, H phi. No correlations existed for the other calculated parameters. These results are consistent with recent results of Argos et al. (1979) [Biochemistry 18, 5698-5703] on sequence analyses of glyceraldehyde-3-P dehydrogenases, where thermophilic proteins showed multiple amino acid replacements that caused increased internal hydrophobicity and increased external polarity. No trends were observed in any of the parameters calculated from amino acid compositions for crude cytoplasmic protein extracts from mesophilic vs thermophilic Bacilli.