Anaerobic degradation of malonatevia malonyl-CoA bySporomusa malonica,Klebsiella oxytoca,andRhodobacter capsulatus

Anaerobic decarboxylation of malonate to acetate was studied withSporomusa malonica, Klebsiella oxytoca, andRhodobacter capsulatus. WhereasS. malonica could grow with malonate as sole substrate (Y=2.0 g·mol^–1), malonate decarboxylation byK. oxytoca was coupled with anaerobic growth only in the presence of a cosubstrate, e.g. sucrose or yeast extract (Y _s =1.1–1.8 g·mol malonate^–1).R. capsulatus used malonate anaerobically only in the light, and growth yields with acetate and malonate were identical. Malonate decarboxylation in cell-free extracts of all three bacteria was stimulated by catalytic amounts of malonyl-CoA, acetyl-CoA, or Coenzyme A plus ATP, indicating that actually malonyl-CoA was the substrate of decarboxylation. Less than 5% of malonyl-CoA decarboxylase activity was found associated with the cytoplasmic membrane. Avidin (except forK. oxytoca) and hydroxylamine inhibited the enzyme completely, EDTA inhibited partially. InS. malonica andK. oxytoca, malonyl-CoA decarboxylase was active only after growth with malonate; malonyl-CoA: acetate CoA transferase was found as well. These results indicate that malonate fermentation by these bacteria proceedsvia malonyl-CoA mediated by a CoA transferase and that subsequent decarboxylation to acetyl-CoA is catalyzed, at least withS. malonica andR. capsulatus, by a biotin enzyme.Abbreviations CoASH Coenzyme A - EDTA ethylenediamine tetraacetate     

Enzymic and genetic basis for bacterial growth on malonate

Various bacteria are able to grow aerobically or anaerobically on malonate as sole source of carbon and energy. Independent of the mechanism for energy conservation, the decarboxylation of malonate is the key reaction in the decomposition of this compound. To achieve malonate decarboxylation under physiological conditions, the substrate must be converted into an activated (thioester) derivative. We report here on the malonate decarboxylases of Malonomonas rubra and Klebsiella pneumoniae. These enzymes perform an interesting substrate activation mechanism by generating a malonyl thioester with the enzyme. Formation of the malonyl-S-enzyme involves an 'activation module' that comprises the acetylation of a specific thiol group of an acyl carrier protein (ACP) and the transfer of the ACP moiety to malonate, yielding malonyl-S-ACP and acetate. The malonyl-S-ACP is subsequently decarboxylated with regeneration of the acetyl-ACP. The malonate activation mechanism is related to the activation of citrate by citrate lyase. The relationship extends to the identical 2'-(5'-phosphoribosyl)-3'-dephospho-CoA thiol cofactor that is bound covalently to the corresponding ACP subunit. In Klebsiella pneumoniae, malonate is decarboxylated by a water-soluble enzyme complex. In the anaerobic bacterium Malonomonas rubra, malonate decarboxylation is catalysed by a set of water-soluble as well as membrane-bound enzymes that function together in converting the free energy of the decarboxylation reaction into delta muNa+. Therefore, this malonate decarboxylase includes a biotin carrier protein that accepts the CO2 moiety from malonyl-S-ACP and delivers it to a membrane-bound decarboxylase acting as a Na+ pump. Genes encoding the individual protein components that perform the decarboxylation of malonate in K. pneumoniae or M. rubra have been identified within the mdc and mad gene clusters respectively. The function of most of the derived proteins could be envisaged from sequence similarities with proteins of known functions. The genetic evidence firmly supports the idea that malonate decarboxylation is carried out by the two different decarboxylases, as deduced from the biochemical studies of the enzymes.     

Histochemical modification of the active site of succinate dehydrogenase with N-acetylimidazole

The kinetics of acetylation of mitochondrial succinate dehydrogenase [EC 1.3.99.1] in the two fibre types (A and C) of rat gastrocnemius with N-acetylimidazole was studied by a newly modified histochemical technique. Acetylimidazole partially inactivated the enzyme, but subsequent deacetylation with hydroxylamine restored the enzyme activity completely. Inactivation of the enzyme by acetylimidazole was prevented by malonate, which is a competitive inhibitor of the enzyme. The value of the inhibition constant (Ki = 34 microM) for malonate, obtained from the dependence of the pseudo-first order rate constant of acetylation of the enzyme with acetylimidazole on the malonate concentration, was in good agreement with the Ki value (33 microM) obtained by a different method, the dependence of the initial velocity of succinate oxidation by the dehydrogenase on the substrate concentration in the presence of malonate. These findings suggest that a tyrosyl residue is located in the malonate binding site (the active site) of succinate dehydrogenase in the gastrocnemius and plays a role in substrate binding, but is not a catalytic group.     

Mechanistic characterization of a bacterial malonate semialdehyde decarboxylase: identification of a new activity on the tautomerase superfamily

Malonate semialdehyde decarboxylase (MSAD) has been identified as the protein encoded by the orf130 gene from Pseudomonas pavonaceae 170 on the basis of the genomic context of the gene as well as its ability to catalyze the decarboxylation of malonate semialdehyde to generate acetaldehyde. The enzyme is found in a degradative pathway for the xenobiotic nematocide trans-1,3-dichloropropene. MSAD has no sequence homology to previously characterized decarboxylases, but the presence of a conserved motif (Pro1-(X)8 -Gly-Arg11-X-Asp-X-Gln) in its N-terminal region suggested a relationship to the tautomerase superfamily. Sequence analysis identified Pro1 and Arg75 as potential active site residues that might be involved in the MSAD activity. The results of site-directed mutagenesis experiments confirmed the importance of these residues to activity and provided further evidence to implicate MSAD as a new member of the tautomerase superfamily. MSAD is the first identified decarboxylase in the superfamily and is possibly the first characterized member of a new and distinct family within this superfamily. Malonate semialdehyde is analogous to a beta-keto acid, and enzymes that catalyze the decarboxylation of these acids generally utilize metal ion catalysis, a Schiff base intermediate, or polarization of the carbonyl group by hydrogen bonding and/or electrostatic interactions. A mechanistic analysis shows that the rate of the reaction is not affected by the presence of a metal ion or EDTA while the incubation of MSAD with the substrate in the presence of sodium cyanoborohydride results in the irreversible inactivation of the enzyme. The site of modification is Pro1. These observations are consistent with the latter two mechanisms, but do not exclude the first mechanism. Based on the sequence analysis, the outcome of the mutagenesis and mechanistic experiments, and the roles determined for Pro1 and the conserved arginine in all tautomerase superfamily members characterized thus far, two mechanistic scenarios are proposed for the MSAD-catalyzed reaction in which Pro1 and Arg75 play prominent roles.     

Purification and characterization of a cytoplasmic enzyme component of the Na+-activated malonate decarboxylase system of Malonomonas rubra: acetyl-S-acyl carrier protein: malonate acyl carrier protein-SH transferase

Malonate decarboxylation by crude extracts of Malonomonas rubra was specifically activated by Na⁺ and less efficiently by Li⁺ ions. The extracts contained an enzyme catalyzing CoA transfer from malonyl-CoA to acetate, yielding acetyl-CoA and malonate. After about a 26-fold purification of the malonyl-CoA:acetate CoA transferase, an almost pure enzyme was obtained, indicating that about 4% of the cellular protein consisted of the CoA transferase. This abundance of the transferase is in accord with its proposed role as an enzyme component of the malonate decarboxylase system, the key enzyme of energy metabolism in this organism. The apparent molecular weight of the polypeptide was 67,000 as revealed from SDS-polyacrylamide gel electrophoresis. A similar molecular weight was estimated for the native transferase by gel chromatography, indicating that the enzyme exists as a monomer. Kinetic analyses of the CoA transferase yielded the following: pH-optimum at pH 5.5, an apparent K_m for malonyl-CoA of 1.9mM, for acetate of 54mM, for acetyl-CoA of 6.9mM, and for malonate of 0.5mM. Malonate or citrate inhibited the enzyme with an apparent K_i of 0.4mM and 3.0mM, respectively. The isolated CoA transferase increased the activity of malonate decarboxylase of a crude enzyme system, in which part of the endogenous CoA transferase was inactivated by borohydride, about three-fold. These results indicate that the CoA transferase functions physiologically as a component of the malonate decarboxylase system, in which it catalyzes the transfer of acyl carrier protein from acetyl acyl carrier protein and malonate to yield malonyl acyl carrier protein and acetate. Malonate is thus activated on the enzyme by exchange for the catalytically important enzymebound acetyl thioester residues noted previously. This type of substrate activation resembles the catalytic mechanism of citrate lyase and citramalate lyase.Abbreviations DTNB 5,5 Dithiobis (2-nitrobenzoate) - MES 2-(N-Morpholino)ethanesulfonic acid - TAPS N-[Tris(hydroxymethyl)-methyl]-3-aminopropanesulfonic acid - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Decarboxylation of malonyl-CoA and biosynthesis of mevalonic acid in rat liver]

Biosynthesis of mevalonic acid (MVA), total formation of 14CO2 from [1,3-14C]malonyl-CoA and the activity of malonyl-CoA decarboxylase in subcellular fractions of rat liver were studied. The dependence of the rate of MVA biosynthesis on malonyl-CoA concentration was found to be linear both in 140,000 g supernatant and solubilized microsomal fractions. It was shown that in a composite system (140,000 g supernatant fraction added to washed microsomes, 10 : 1) the optimal concentration ratio for the substrates of MVA biosynthesis (malonyl-CoA and acetyl-CoA) is 1 to 2. In the absence of acetyl-CoA decarboxylation of [1,3-14C]malonyl-CoA was prevalent. In all subcellular fractions studied decarboxylation of [1,3-14C]malonyl-CoA prevailed over its incorporation into MVA, total non-saponified lipid fraction and fatty acids. The degree of malonyl-CoA, decarboxylation was not correlated with the rate of its incorporation into MVA, i. e. the increase in the 14CO2 formation was not accompanied by stimulation of [1,3-14C]malonyl-CoA incorporation either into MVA or into total non-saponified lipid fractions. The incorporation of [1-14C]acetyl-CoA into MVA under the same conditions was considerably lower than that of [1,3-14C]malonyl-CoA. In all subcellular fractions under study the activity of malonyl-CoA decarboxylase was found. The experimental data suggest that a remarkable part of malonyl-CoA is incorporated into MVA without preliminary decarboxylation. A possible role of malonyl-CoA decarboxylase as an enzyme which protects the cell against accumulation of malonyl-CoA and its immediate metabolites -- malonate and methylmalonyl-CoA is disucssed.     

The malonate decarboxylase enzyme system of Malonomonas rubra: evidence for the cytoplasmic location of the biotin-containing component

Malonate decarboxylase of Malonomonas rubra is a complex enzyme system involving cytoplasmic and membrane-bound components. One of these is a biotin-containing protein of M_r 120'000, the location of which in the cytoplasm was deduced from the following criteria: (i) If the cytoplasm was incubated with avidin and the malonate decarboxylase subsequently completed with the membrane fraction the decarboxylase activity was abolished. The corresponding incubation of the membrane with avidin, however, was without effect. (ii) Western blot analysis identified the single biotin-containing polypeptide of M_r 120'000 within the cytoplasm. (iii) Transmission electron micrographs of immuno-gold labeled M. rubra cells clearly showed the location of the biotinyl protein within the cytoplasm, whereas the same procedure with Propionigenium modestum cells indicated the location of the biotin enzyme methylmalonyl-CoA decarboxylase in the cell membrane. The biotin-containing protein of the M. rubra malonate decarboxylase enzyme system was not retained by monomeric avidin-Sepharose columns but could be isolated with this column in a catalytically inactive form in the presence of detergents. If the high binding affinity of tetrameric avidin towards biotin was reduced by destructing part of the tryptophan residues by irradiation or oxidation with periodate, the inhibition of malonate decarboxylase by the modified avidin was partially reversed with an excess of biotin. Attempts to purify the biotin protein in its catalytically active state using modified avidin columns were without success.     

Reversible and nonoxidative gamma-resorcylic acid decarboxylase: characterization and gene cloning of a novel enzyme catalyzing carboxylation of resorcinol, 1,3-dihydroxybenzene, from Rhizobium radiobacter

We found a gamma-resorcylic acid (gamma-RA, 2,6-dihydroxybenzoic acid) decarboxylase, as a novel enzyme applicable to carboxylation of resorcinol (RE, 1,3-dihydroxybenzene) to form gamma-RA, in a bacterial strain Rhizobium radiobacter WU-0108 isolated through the screening of gamma-RA degrading microorganisms. The activities for carboxylation of RE and decarboxylation of gamma-RA were detected in the cell-free extracts of R. radiobacter WU-0108 grown aerobically with gamma-RA. The enzyme, gamma-RA decarboxylase, was purified to homogeneity on SDS-PAGE through the steps of one ion-exchange chromatography and two kinds of hydrophobic chromatography. The molecular weight of the enzyme was estimated to be 130 kDa by gel-filtration, and that of the subunit was determined to be 34 kDa by SDS-PAGE, suggesting that the enzyme is a homotetrameric structure. The enzyme catalyzed the decarboxylation of gamma-RA, but not alpha-RA or beta-RA. Without addition of any cofactors, the enzyme catalyzed the regio-selective carboxylation of RE to form gamma-RA, without formation of alpha-RA and beta-RA, and of catechol to 2,3-dihydroxybenzoic acid. In the presence of oxygen, this gamma-RA decarboxylase showed no decrease in both of the activities as for decarboxylation of gamma-RA and carboxylation of RE, different from other decarboxylases reported so far. The gene, rdc, encoding the gamma-RA decarboxylase was cloned into Escherichia coli, sequenced, and subjected to over-expression. The deduced amino acid sequence of the rdc gene consists of 327 amino acid residues corresponding to 34 kDa protein, and shows 42% and 30% identity to those of a 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus niger and a 5- carboxyvanillate decarboxylase from Sphingomonas paucimobilis SYK-6. A site-directed mutagenesis study revealed the two histidine residues at positions of 164 and 218 in Rdc to be essential for the catalytic activities of decarboxylation of gamma-RA and carboxylation of RE.     

The role of biotin and sodium in the decarboxylation of oxaloacetate by the membrane-bound oxaloacetate decarboxylase from Klebsiella aerogenes

The biotin-containing oxaloacetate decarboxylase from Klebsiella aerogenes catalyzed the Na+-dependent decarboxylation of oxaloacetate to pyruvate and bicarbonate (or CO2) but not the reversal of this reaction, not even in the presence of an oxaloacetate trapping system. The enzyme catalyzed an avidin-sensitive isotopic exchange between [1-14C]pyruvate and oxaloacetate, which indicated the intermediate formation of a carboxybiotin enzyme. Sodium ions were not required for this partial reaction, but promoted the second partial reaction, the decarboxylation of the carboxybiotin enzyme, thus accounting for the Na+ requirement of the overall reaction. Therefore, the 14CO2-enzyme which was formed upon incubation of the decarboxylase with [4-15C]oxaloacetate, could only be isolated if Na+ ions were excluded. Preincubation of the decarboxylase with avidin also prevented its labelling with 14CO2. The isolated 14CO2-labelled oxaloacetate decarboxylase revealed the following properties. It was slowly decarboxylated at neutral pH and rapidly upon acidification. The 14CO2 residues of the 14CO2-enzyme could be transferred to pyruvate yielding [4-14C]oxaloacetate. In the presence of Na+ this 14CO2 transfer was repressed by the simultaneous decarboxylation of the 14CO2-enzyme. However, Na+ alone was insufficient as a cofactor for the decarboxylation of the isolated 14CO2-enzyme, since this required pyruvate in addition to Na+. It is therefore concluded that the decarboxylation of oxaloacetate proceeds over a CO2-enzyme--pyruvate complex and that free CO2-enzyme is an abortive reaction intermediate. The activation energy of the enzymic decarboxylation of oxaloacetate changed with temperature and was about 113 kJ below 11 degrees C, 60 kJ between 11 degrees C and 31 degrees C and 36 kJ between 31--45 degrees C.     

Novel sites in the p65 subunit of NF-kappaB interact with TFIIB to facilitate NF-kappaB induced transcription

The citM gene from Lactococcus lactis CRL264 was demonstrated to encode for an oxaloacetate decarboxylase. The enzyme exhibits high levels of similarity to malic enzymes (MEs) from other organisms. CitM was expressed in Escherichia coli, purified and its oxaloacetate decarboxylase activity was demonstrated by biochemical and genetic studies. The highest oxaloacetate decarboxylation activity was found at low pH in the presence of manganese, and the K_m value for oxaloacetate was 0.52 ± 0.03 mM. However, no malic activity was found for this enzyme. Our studies clearly show a new group of oxaloacetate decarboxylases associated with the citrate fermentation pathway in gram-positive bacteria. Furthermore, the essential catalytic residues were found to be conserved in all members of the ME family, suggesting a common mechanism for oxaloacetate decarboxylation.     

Oxalacetate decarboxylase and pyruvate carboxylase activities, and effect of sulfhydryl reagents in malic enzyme from Sulfolobus solfataricus

Malic enzyme (S)-malate: NADP+ oxidoreductase (oxaloacetate-decarboxylating, EC 1.1.1.40) purified from the thermoacidophilic archaebacterium Sulfolobus solfataricus, strain MT-4, catalyzed the metal-dependent decarboxylation of oxaloacetate at optimum pH 7.6 at a rate comparable to the decarboxylation of L-malate. The oxaloacetate decarboxylase activity was stimulated about 50% by NADP but only in the presence of MgCl2, and was strongly inhibited by L-malate and NADPH which abolished the NADP activation. In the presence of MnCl2 and in the absence of NADP, the Michaelis constant and Vm for oxaloacetate were 1.7 mM and 2.3 mumol.min-1.mg-1, respectively. When MgCl2 replaced MnCl2, the kinetic parameters for oxaloacetate remained substantially unvaried, whereas the Km and Vm values for L-malate have been found to vary depending on the metal ion. The enzyme carried out the reverse reaction (malate synthesis) at about 70% of the forward reaction, at pH 7.2 and in the presence of relatively high concentrations of bicarbonate and pyruvate. Sulfhydryl residues (three cysteine residues per subunit) have been shown to be essential for the enzymatic activity of the Sulfolobus solfataricus malic enzyme. 5,5'-Dithiobis(2-nitrobenzoic acid), p-hydroxymercuribenzoate and N-ethylmaleimide caused the inactivation of the oxidative decarboxylase activity, but at different rates. The inactivation of the overall activity by p-hydroxymercuribenzoate was partially prevented by NADP singly or in combination with both L-malate and MnCl2, and strongly enhanced by the carboxylic acid substrates; NADP + malate + MnCl2 afforded total protection. The inactivation of the oxaloacetate decarboxylase activity by p-hydroxymercuribenzoate treatment was found to occur at a slower rate than that of the oxidative decarboxylase activity.     

Inactivation of malonate semialdehyde decarboxylase by 3-halopropiolates: evidence for hydratase activity

Malonate semialdehyde decarboxylase (MSAD) from Pseudomonas pavonaceae 170 catalyzes the metal ion-independent decarboxylation of malonate semialdehyde and represents one of three known enzymatic activities in the tautomerase superfamily. The characterized members of this superfamily are structurally homologous proteins that share a beta-alpha-beta fold and a catalytic amino-terminal proline. Sequence analysis, chemical labeling studies, site-directed mutagenesis, and NMR studies of MSAD identified Pro-1 as a key active site residue in which the amino group has a pKa value of 9.2. The available evidence suggests a mechanism involving polarization of the C-3 carbonyl group of malonate semialdehyde by the cationic Pro-1. A second critical active site residue, Arg-75, could assist in the reaction by placing the substrate's carboxylate group in a favorable conformation for decarboxylation. In addition to the decarboxylase activity, MSAD has a hydratase activity as demonstrated by the MSAD-catalyzed conversion of 2-oxo-3-pentynoate to acetopyruvate. In view of this activity, MSAD was incubated with 3-bromo- and 3-chloropropiolate, and the subsequent reactions were characterized. Both compounds result in the irreversible inactivation of MSAD, making them the first identified inhibitors of MSAD. Inactivation by 3-chloropropiolate occurs in a time- and concentration-dependent manner and is due to the covalent modification of Pro-1. The proposed mechanism for inactivation involves the initial hydration of the 3-halopropiolate followed by a rearrangement to an alkylating agent, either an acyl halide or a ketene. The results provide additional evidence for the hydratase activity of MSAD and further support for the hypothesis that MSAD and trans-3-chloroacrylic acid dehalogenase, the preceding enzyme in the trans-1,3-dichloropropene catabolic pathway, diverged from a common ancestor but conserved the necessary catalytic machinery for the conjugate addition of water.     

Identification of bound pyruvate essential for the activity of phosphatidylserine decarboxylase of Escherichia coli.

Phosphatidylserine decarboxylase, an intrinsic membrane protein of Escherichia coli, catalyzes the decarboxylation of phosphatidylserine, the final step in the biosynthesis of phosphatidylethanolamine, the principal membrane lipid of this organism. The purified enzyme lacks the absorption spectrum characteristic of pyridoxal-containing enzymes, and it has now been found to contain bound pyruvate, the carbonyl function of which is essential for catalytic activity. The decarboxylase is inactivated by treatment with a number of reagents that attack carbonyl groups, including sodium borohydride. Reduction with tritiated borohydride leads to the introduction of stably bound radioactivity, which, after acid hydrolysis, has been identified as tritiated lactate by several chromatographic procedures and by treatment with lactate dehydrogenase. The enzyme resists inactivation by cyanoborohydride in the absence of substrate, but is readily inactivated by this reagent in the presence of phosphatidylserine. Under the conditions of treatment of neutral pH, cyanoborohydride does not react with carbonyl residues at an appreciable rate, but reduces imino groups much more rapidly. This finding, together with demonstrated dependence of the enzyme upon the carbonyl residue of pyruvate for activity, strongly suggests that a Schiff base is formed by addition of the amino group of phosphatidylserine to the pyruvate residue of the enzyme as an essential step in the action of the decarboxylase.     

Crystal structures of isoorotate decarboxylases reveal a novel catalytic mechanism of 5-carboxyl-uracil decarboxylation and shed light on the search for DNA decarboxylase

DNA methylation and demethylation regulate many crucial biological processes in mammals and are linked to many diseases. Active DNA demethylation is believed to be catalyzed by TET proteins and a putative DNA decarboxylase that may share some similarities in sequence, structure and catalytic mechanism with isoorotate decarboxylase (IDCase) that catalyzes decarboxylation of 5caU to U in fungi. We report here the structures of wild-type and mutant IDCases from Cordyceps militaris and Metarhizium anisopliae in apo form or in complexes with 5caU, U, and an inhibitor 5-nitro-uracil. IDCases adopt a typical (β/α)₈ barrel fold of the amidohydrolase superfamily and function as dimers. A Zn²⁺ is bound at the active site and coordinated by four strictly conserved residues, one Asp and three His. The substrate is recognized by several strictly conserved residues. The functional roles of the key residues at the active site are validated by mutagenesis and biochemical studies. Based on the structural and biochemical data, we present for the first time a novel catalytic mechanism of decarboxylation for IDCases, which might also apply to other members of the amidohydrolase superfamily. In addition, our biochemical data show that IDCases can catalyze decarboxylation of 5caC to C albeit with weak activity, which is the first in vitro evidence for direct decarboxylation of 5caC to C by an enzyme. These findings are valuable in the identification of potential DNA decarboxylase in mammals.     

Glutamate decarboxylase side reactions catalyzed by the enzyme

A homogeneous glutamate decarboxylase isolated from pig brain contains 0.8 mol of tightly bound pyridoxal 5-phosphate/enzyme dimer. Upon addition of exogenous pyridoxal 5-phosphate (pyridoxal-5-P), the enzyme acquires maximum catalytic activity. Preincubation of the enzyme with L-glutamate (10 mM) brings about changes in the absorption spectrum of bound pyridoxal-5-P with the concomitant formation of succinic semialdehyde. However, the rate of this slow secondary reaction, i.e. decarboxylative transamination, is 10(-4) times the rate of normal decarboxylation. It is postulated that under physiological conditions enzymatically inactive species of glutamate decarboxylase, generated by the process of decarboxylative transamination, are reconstituted by pyridoxal-5-P produced by the cytosolic enzymes pyridoxal kinase and pyridoxine-5-P oxidase. The catalytic activity of resolved glutamate decarboxylase is recovered by preincubation with phospho-pyridoxyl-ethanolamine phosphate. The experimental evidence is consistent with the interpretation that the resolved enzyme binds the P-pyridoxyl analog, reduces the stability of the covalent bond of the phospho-pyridoxyl moiety, and catalyzes the formation of pyridoxal-5-P.     

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.     

The roles of Tyr(91) and Lys(162) in general acid-base catalysis in the pigeon NADP+-dependent malic enzyme

The role of general acid-base catalysis in the enzymatic mechanism of NADP+-dependent malic enzyme was examined by detailed steady-state kinetic studies through site-directed mutagenesis of the Tyr(91) and Lys(162) residues in the putative catalytic site of the enzyme. Y91F and K162A mutants showed approx. 200- and 27000-fold decreases in k(cat) values respectively, which could be partially recovered with ammonium chloride. Neither mutant had an effect on the partial dehydrogenase activity of the enzyme. However, both Y91F and K162A mutants caused decreases in the k(cat) values of the partial decarboxylase activity of the enzyme by approx. 14- and 3250-fold respectively. The pH-log(k(cat)) profile of K162A was found to be different from the bell-shaped profile pattern of wild-type enzyme as it lacked a basic pK(a) value. Oxaloacetate, in the presence of NADPH, can be converted by malic enzyme into L-malate by reduction and into enolpyruvate by decarboxylation activities. Compared with wild-type, the K162A mutant preferred oxaloacetate reduction to decarboxylation. These results are consistent with the function of Lys(162) as a general acid that protonates the C-3 of enolpyruvate to form pyruvate. The Tyr(91) residue could form a hydrogen bond with Lys(162) to act as a catalytic dyad that contributes a proton to complete the enol-keto tautomerization.     

Anaerobic malonate decarboxylation by Citrobacter diversus

Citrobacter diversus ATCC 27156 was able to grow by decarboxylation of malonate to acetate under strictly anaerobic conditions, in the presence of yeast extract. The growth yield, corrected for growth on yeast extract, was 2.03 g cell dry mass per mol malonate. The addition of malonate to ATP-depleted cell suspensions (less than 0.2 nmol ATP/mg cell protein) resulted in a rapid increase in cellular ATP levels to between 4.5 and 6.0 nmol/mg cell protein. Intact cells decarboxylated malonate at rates of up to 1.5 mumol/min.mg protein. Enzyme assays on malonate-grown cells indicated activation of malonate by an ATP-dependent ligase reaction and by CoA transfer from acetyl-CoA, followed by decarboxylation of malonyl-CoA to acetyl-CoA with subsequent recovery of the invested ATP by substrate level phosphorylation through the activity of acetate kinase. Net ATP synthesis is postulated to be mediated by gradient formation coupled to the decarboxylation of malonyl-CoA. The protonophore CCCP and H(+)-ATPase inhibitor DCCD significantly reduced cellular ATP levels, suggesting a role for proton gradients in the energy metabolism of this strain when growing an malonate. Inhibitors of sodium metabolism or ommission of sodium had no effect on ATP levels or malonate decarboxylation.     

Diethylpyrocarbonate inactivation of NAD-malic enzyme from Ascaris suum

Treatment with diethylpyrocarbonate results in a first-order loss of the malate oxidative decarboxylase activity of NAD-malic enzyme. First-order plots are biphasic, with about 40-50% activity loss in the first phase. The inactivation process is not saturable, and the second-order rate constant is 4.7 M-1 S-1. Malate (250 mM) provides complete protection against inactivation (as measured by a decrease in the inactivation rate), and less malate is required with Mg2+ present. Partial protection (50%) is afforded by either NAD+ (1 mM) or Mg2+ (50 mM). Treatment of modified (inactive) enzyme with hydroxylamine restores activity to 100% of the control when corrected for the effect of hydroxylamine on unmodified enzyme. A total of 10-13 histidine residues/subunit are acylated concomitant with loss of activity while 1-2 tyrosines are modified prior to any activity loss. The presence of Mg2+ and malate at saturating concentrations protect 1-2 histidine residues/subunit. The intrinsic fluorescence of the enzyme decreases with time after addition of diethylpyrocarbonate, but the rate constant for this process is at least 10-fold too low to account for the biphasicity observed in the first order plots. The histidine modified which is responsible for loss of activity has a pK of 8.3 as determined from the pH dependence of the rate of inactivation. The histidine titrated is still modified under conditions where the residue is completely protonated but at a rate 1/100 the rate of the unprotonated histidine. The results suggest that 1-2 histidines are in or near the malate binding site and are required for malate oxidative decarboxylation.     

N-acetylimidazole inactivates renal Na,K-ATPase by disrupting ATP binding to the catalytic site

Treatment of renal Na,K-ATPase with N-acetylimidazole (NAI) results in loss of Na,K-ATPase activity. The inactivation kinetics can be described by a model in which two classes of sites are acetylated by NAI. The class I sites are rapidly reacting, the acetylation is prevented by the presence of ATP (K0.5 congruent to 8 microM), and the inactivation is reversed by incubation with hydroxylamine. These data suggest that the class I sites are tyrosine residues at the ATP binding site. The second class of sites are more slowly reacting, not protected by ATP, nor reversed by hydroxylamine treatment. These are probably lysine residues elsewhere in the protein. The associated K-stimulated p-nitrophenylphosphatase activity is inactivated by acetylation of the class II sites only; thus the tyrosine residues associated with ATP binding to the catalytic center are not essential for phosphatase activity. Inactivated enzyme no longer has high-affinity ATP binding associated with the catalytic site, although low-affinity ATP effects (inhibition of phosphatase and deocclusion of Rb) are still present. The inactivated enzyme can still be phosphorylated by Pi, occlude Rb+ ions, and undergo the major conformational transitions between the E1 Na and E2 K forms of the enzyme. Thus acetylation of the Na,K-ATPase by NAI inhibits high-affinity ATP binding to the catalytic center and produces inactivation.