Structure-Function Relationships in Flavoenzyme-dependent Amine Oxidations

Binda, Claudia; Mattevi, Andrea; Edmondson, Dale E.

doi:10.1074/jbc.r200005200

Cited by 160 publications

(140 citation statements)

References 28 publications

Supporting

Mentioning

134

Contrasting

Unclassified

Order By: Relevance

“…They also support the identification of TMO as a member of the LAAO family and of the larger flavin amine oxidase superfamily (17). The combination of steady state and rapid reaction kinetic data for the mutant enzymes allows determination of the effects of the mutations on each of the kinetic constants in the mechanism of Scheme 2 ( Table 4).…”

Section: Discussionsupporting

confidence: 53%

Analysis of the Role of the Active Site Residue Arg98 in the Flavoprotein Tryptophan 2-Monooxygenase, a Member of the l-Amino Oxidase Family

Sobrado

Fitzpatrick

2003

Biochemistry

View full text Add to dashboard Cite

The flavoprotein tryptophan 2-monooxygenase catalyzes the oxidative decarboxylation of tryptophan to indoleacetamide. We have previously identified tryptophan 2-monooxygenase as a homologue of L-amino acid oxidase [Sobrado, P., and Fitzpatrick, P. F. (2002) Arch. Biochem. Biophys. 402,[24][25][26][27][28][29][30]. On the basis of the sequence comparisons of the different LAAO family members, Arg98 of tryptophan 2-monooxygenase can be identified as an active site residue which interacts with the carboxylate of the amino acid substrate. The catalytic properties of R98K and R98A tryptophan 2-monooxygenase have been characterized to evaluate the role of this residue. Mutation of Arg98 to lysine decreases the first-order rate constant for flavin reduction by 180-fold and the second-order rate constant for flavin oxidation by 26-fold, has no significant effect on the K d value for tryptophan or the K i value for the competitive inhibitor indoleacetamide, and increases the K i value for indolepyruvate less than 2-fold. Mutation of this residue to alanine decreases the rate constants for reduction and oxidation an additional 5-and 2-fold, respectively, and increases the K d value for tryptophan and the K i value for indolepyruvate by 31-and 17-fold, respectively, while having an only 2-fold effect on the K i value for indoleacetamide. Both mutations increase the value of the primary deuterium isotope effect with tryptophan as a substrate, consistent with a later transition state. Both mutant enzymes catalyze a simple oxidase reaction, producing indolepyruvate and hydrogen peroxide. The pH dependences of the V/K trp values for the mutant enzymes show that the anionic form of the substrate is preferred but that the zwitterionic form is a substrate. The results are consistent with the interaction between Arg98 and the carboxylate of the amino acid substrate being critical for correct positioning of the substrate in the active site for efficient catalysis.Tryptophan 2-monooxygenase (TMO) 1 is an FAD-containing enzyme that catalyzes the oxidative decarboxylation of tryptophan to indoleacetamide (Scheme 1). This reaction is the first step in the biosynthesis of the plant hormone indoleacetic acid by plant pathogens such as Agrobacterium tumefaciens and Pseudomonas savastanoi; the resulting production of indoleacetic acid induces the formation of galls at the site of infection (1,2). The kinetic mechanism of TMO has been determined with tryptophan as a substrate and can be divided † This work was supported in part by grants to P.F.F. from the NIH (GM 58698) and from the Robert A. Welch Foundation (A-1245) and by a fellowship from CONICIT-Costa Rica (02-2002) to P.S. * To whom correspondence should be addressed: Department of Biochemistry and Biophysics, 2128 TAMU, College Station, TX 77843-2128. Phone: (979) into two half-reactions (Scheme 2). In the reductive half-reaction, the α-C-H bond of the amino acid is broken and a hydride equivalent is transferred to the FAD, forming the reduced enzyme with the imin...

show abstract

Section: Discussionsupporting

confidence: 53%

Analysis of the Role of the Active Site Residue Arg98 in the Flavoprotein Tryptophan 2-Monooxygenase, a Member of the l-Amino Oxidase Family

Sobrado

Fitzpatrick

2003

Biochemistry

View full text Add to dashboard Cite

show abstract

“…Polyamine oxidase, the other known FAD-containing oxidase, is an intracellular oxidase that metabolizes spermine and spermidine and regulates cell growth (13). Unlike MAO-A and MAO-B, which are anchored through the carboxyl ter- minus to the outer mitochondrial membrane (14) and confined to intracellular compartments, renalase is secreted into the blood, where it is detectable by Western blotting.…”

Section: Discussionmentioning

confidence: 99%

Renalase is a novel, soluble monoamine oxidase that regulates cardiac function and blood pressure

Xu¹,

Li²,

Wang³

et al. 2005

J. Clin. Invest.

138

307

View full text Add to dashboard Cite

The kidney not only regulates fluid and electrolyte balance but also functions as an endocrine organ. For instance, it is the major source of circulating erythropoietin and renin. Despite currently available therapies, there is a marked increase in cardiovascular morbidity and mortality among patients suffering from end-stage renal disease. We hypothesized that the current understanding of the endocrine function of the kidney was incomplete and that the organ might secrete additional proteins with important biological roles. Here we report the identification of a novel flavin adenine dinucleotide-dependent amine oxidase (renalase) that is secreted into the blood by the kidney and metabolizes catecholamines in vitro (renalase metabolizes dopamine most efficiently, followed by epinephrine, and then norepinephrine). In humans, renalase gene expression is highest in the kidney but is also detectable in the heart, skeletal muscle, and the small intestine. The plasma concentration of renalase is markedly reduced in patients with end-stage renal disease, as compared with healthy subjects. Renalase infusion in rats caused a decrease in cardiac contractility, heart rate, and blood pressure and prevented a compensatory increase in peripheral vascular tone. These results identify renalase as what we believe to be a novel amine oxidase that is secreted by the kidney, circulates in blood, and modulates cardiac function and systemic blood pressure.

show abstract

“…Edmondson and co-workers have proposed a mechanism involving transfer of two electrons from the amine of the substrate to the flavin concerted with abstraction of the α-hydrogen as a proton by N5 of the flavin (17,18). Alternatively, Silverman and co-workers have proposed a mechanism involving a substrate aminium radical cation as a discrete intermediate (19).…”

mentioning

confidence: 99%

Mechanistic Studies of Mouse Polyamine Oxidase with N1,N12-Bisethylspermine as a Substrate

Royo

Fitzpatrick

2005

Biochemistry

View full text Add to dashboard Cite

In mammalian cells, the flavoprotein polyamine oxidase catalyzes a key step in the catabolism of polyamines, the oxidation of N1-acetylspermine and N1-acetylspermidine to spermidine and putrescine, respectively. The mechanism of the mouse enzyme has been studied with N1,N12-bisethylspermine (BESPM) as a substrate. At pH 10, the pH optimum, the limiting rate of reduction of the flavin in the absence of oxygen is comparable to the k cat value for turnover, establishing reduction as rate-limiting. Oxidation of the reduced enzyme is a simple second-order reaction. No intermediates are seen in the reductive or oxidative half-reactions. The k cat value decreases below a pK a of 9.0. The k cat /K m value for BESPM exhibits a bell-shaped pH profile, with pK a values of 9.8 and 10.8. These pK a values are assigned to the substrate nitrogens. The rate constant for the reaction of the reduced enzyme with oxygen is not affected by a pH between 7.5 and 10. Active site residue Tyr430 is conserved in the homologous protein monoamine oxidase. Mutation of this residue to phenylalanine results in a 6-fold decrease in the k cat value and the k cat /K m value for oxygen due to a comparable decrease in the rate constant for flavin reduction. This moderate change is not consistent with this residue forming a tyrosyl radical during catalysis.The polyamines spermine, spermidine, and putrescine are ubiquitous in cells. Higher concentrations are found in rapidly growing cells (1-3), and compounds which deplete polyamines from cells inhibit cell growth (2). These observations have led to the general conclusion that polyamines are essential for cell growth, although their specific role in the cell is still a matter of discussion. Consequently, a variety of polyamine analogues have been examined as anticancer drugs (4-7); a number of clinical trials are underway, and analogues with cytotoxic potential have been developed (8,9). The metabolic pathways for synthesis and degradation of polyamines are generally conserved (1). In mammals, the biosynthetic pathway involves decarboxylation of ornithine to putrescine by ornithine decarboxylase, extension of putrescine to spermidine by spermidine synthase using decarboxylated S-adenosylmethionine as the propylamine donor, and a subsequent extension of spermidine with another propylamine moiety to form spermine catalyzed by the enzyme spermine synthase. Depletion of spermine and spermidine from the cell involves the action of two enzymes: spermidine/spermine N1-acetyltransferase converts spermine and spermidine to the respective N1-acetylated compound, and polyamine oxidase oxidizes N1-acetylspermidine and N1-acetylspermine to putrescine and spermidine, respectively, and 3-acetamidopropanal (Scheme 1). While polyamine oxidase can also oxidize spermine, the enzyme strongly prefers N1-acetylated polyamines as substrates (10). Instead, the related enzyme spermine oxidase is responsible for direct oxidation of † This work was supported in part by grants from the NIH (R01 GM58698) and The Welch Fo...

show abstract

Structure-Function Relationships in Flavoenzyme-dependent Amine Oxidations

Cited by 160 publications

References 28 publications

Analysis of the Role of the Active Site Residue Arg98 in the Flavoprotein Tryptophan 2-Monooxygenase, a Member of the l-Amino Oxidase Family

Analysis of the Role of the Active Site Residue Arg98 in the Flavoprotein Tryptophan 2-Monooxygenase, a Member of the l-Amino Oxidase Family

Renalase is a novel, soluble monoamine oxidase that regulates cardiac function and blood pressure

Mechanistic Studies of Mouse Polyamine Oxidase with N1,N12-Bisethylspermine as a Substrate

Contact Info

Product

Resources

About