The Enzyme Complex Citramalate Lyase from Clostridium tetanomorphum

Functional malonate decarboxylase of Klebsiella pneumoniae is an acetyl-S-enzyme with an acetylated phosphoribosyl dephospho-CoA prosthetic group. The mdcH gene product acts as a malonyl-CoA:ACP transacylase and initiates the activation of (deacetyl)malonate decarboxylase by malonyl-transfer to the prosthetic group. The malonyl residue is subsequently decarboxylated to an acetyl residue by the decarboxylase itself. Purified malonate decarboxylase consists of the four subunits MdcA, D, E and C in an apparent 1 : 1 : 1 : 1 stoichiometry. In addition, the preparation contains substoichiometric amounts of MdcH comigrating on SDS/PAGE with MdcD. Malonate decarboxylase isolated from strains with a deletion of the mdcH gene was not activated with malonyl-CoA. Activity could be gained, however, in the additional presence of MdcH that has been synthesized in Escherichia coli and purified from inclusion bodies. Substrates for MdcH are malonyl-CoA or methylmalonyl-CoA but not acetyl-CoA. The enzyme has K m values of 16 mm for both substrates and V max for malonyl-CoA of 190 U´mg ±1 and for methylmalonyl-CoA of 37 U´mg ±1 . Transfer of the methylmalonyl-residue to the prosthetic group proceeds via the covalent methylmalonyl-MdcH intermediate. The transacylase is specifically inhibited by N-ethylmaleimide, and preincubation with malonyl-CoA or methylmalonyl-CoA protects the enzyme from this inhibition.Keywords: enzyme activation; fatty acid biosynthesis; Klebsiella pneumoniae; malonate decarboxylase; malonylCoA:acyl carrier protein transacylase.In spite of the widely used diagnostic criterion of bacterial growth on malonate [1], the enzymic and genetic basis for this behavior was resolved only recently (for a review, see [2]). The key element is a specific malonate decarboxylase that converts malonate directly into acetate and CO 2 . Malonate is chemically rather inert, especially at neutral pH, where both carboxylic residues are dissociated (pK a1 = 2.85, pK a2 = 5.69). The dicarboxylate is therefore activated for the decarboxylation reaction by transiently forming a thioester with the enzyme. The catalytically active enzyme carries an acetyl thioester residue that is exchanged in the first partial reaction by a malonyl thioester residue. This is subsequently decarboxylated with regeneration of the acetyl-S-enzyme (Fig. 1). This malonate decarboxylation mechanism was discovered for the enzyme from Malonomonas rubra, an anaerobic bacterium capable to grow entirely from the free energy of this decarboxylation reaction [3]. Later, aerobic bacteria known to grow on malonate, e.g. Klebsiella pneumoniae [4], Acinetobacter calcoaceticus [5], Pseudomonas putida [6] or Pseudomonas fluorescens [7] were found to dispose of malonate decarboxylases that activate the substrate by the same mechanism forming malonyl-S-enzyme derivates. These aerobic decarboxylases release CO 2 directly from the malonyl-S-enzyme intermediates, forfeiting the free energy of the decarboxylation reaction. In contrast, the M. rubra decarboxylase system catalyze...

Section: Discussionmentioning

confidence: 99%

Formation of catalytically active S‐acetyl(malonate decarboxylase) requires malonyl‐coenzyme A

Hoenke

Dimroth

1999

“…However, any definite conclusion must await a more thorough investigation of the pantothenate content of the enzyme. It should be mentioned in this connection that the enzyme citramalate lyase which is closely related to citrate lyase has been shown to contain 5 rnol pantothenate per mol of enzyme [17].…”

Section: Discussionmentioning

confidence: 99%

Purification and Properties of Citrate Lyase Ligase from Streptococcus diacetilactis

Bowien¹,

Gottschalk²

1977

Citrate lyase ligase (acetate : SH-[acyl-carrier protein] enzyme ligase (AMP)) from Streptococcus diacetilactis was purified 920-fold with a yield of 6.3%. The molecular weight of the enzyme was estimated to be 41 000; the ligase consisted of one polypeptide chain.The acetylation of 1 mol of deacetyl-citrate lyase to enzymatically active citrate lyase required 6 mol ATP. The formation of AMP and pyrophosphate in the acetylation reaction was demonstrated.Citrate lyase ligase was specific for the lyase from S. diacetilactis and did not acetylate lyases from Rhodopseudomonas gelatinosa and Enterobacter aerogenes. The substrates acetate and ATP could be replaced by propionate and dATP, respectively.The reaction rates for ATP, acetate and deacetyl-citrate lyase followed Michaelis-Menten kinetics ( K , values: 26 pM for ATP, 25 mM for acetate and 38 nM for deacetyl-citrate lyase). We have demonstrated an enzyme activity catalyzing a similar reaction in Streptococcus diacetilactis [2]. In contrast to the enzyme from E. aerogenes, it seemed to remain associated with citrate lyase during purification. The separation of both enzymes has now been achieved, and the preparation of pure citrate lyase ligase, the stoichiometry of the reaction and other properties of the enzyme are reported. MATERIALS AND METHODS Preparation of Citrate LyaseStreptococcus diacetilactis DRC 1 was grown as described by Harvey and Collins [3], and citrate lyase

“…Then 0.01 ml20 mM MgClz and stock solutions of 3.4 M potassium acetate, 1 .O M potassium (R,S)-citramalate or 1 .O M sodium pyruvate were added as indicated in Table 1. After reaching equilibrium, the concentration of pyruvate and (S)-citramalate were determined from samples in a single assay using lactate dehydroge-nase followed by citramalate lyase [8]. The concentrations of acetate were calculated according to:…”

Section: Citramalate Lyasementioning

confidence: 99%

“…The equilibrium constants are accurate except for the citramalate lyase equilibrium, since no purified enzyme was available at this time [4]. Citramalate lyase has now been purified to homogeneity [5], and the equilibrium constant for the citramalate formation reaction has been determined. …”

mentioning

confidence: 99%

Equilibrium constants of several reactions involved in the fermentation of glutamate

Miller

1987

Self Cite

The equilibrium constants of the reactions catalysed by (S)-citramalate lyase and (R)-2-hydroxyglutarate dehydrogenase were determined using the purified enzymes from Clostridium tetanomorphum and Acidaminococcus fermentans, respectively. The former constant had to be determined at high ionic strength ( I ) . Therefore it was corrected to I = 0.1 M by applying single-ion activity coefficients estimated from literature data. The result (Kapp = 4.31 f 0.07 M-'; direction of citramalate formation) agreed very well with the constant of the (2R,3S)-2,3-dimethylmalate lyase equilibrium when all optical isomers were taken into account. From these and other data values for the free energies of formation ( A Cf") of (2S,3S)-3-methylaspartate, mesaconate and (S)-citramalate were calculated. The constant of the (R)-Zhydroxyglutarate dehydrogenase equilibrium [Kapp = (1.47 f 0.12)10-'2 M, direction of 2-oxoglutarate formation, Z = 0.1 MI was shown to lie between those for malate and lactate dehydrogenases as expected.Glutamate is fermented by anaerobic bacteria by two different pathways. The first is via 3-methylaspartate, mesaconate and citramalate, whereas the second proceeds via 2-hydroxyglutarate and glutaconate [l, 21. The equilibrium constants for the formation and degradation of these unusual dicarboxylic acids yield new data on the effect of substituents on chemical equilibria, The values for the reactions from glutamate leading to acetate and pyruvate via 3-methylaspartate were measured by Barker and coworkers (for a review see [3]). The equilibrium constants are accurate except for the citramalate lyase equilibrium, since no purified enzyme was available at this time [4]. Citramalate lyase has now been purified to homogeneity [5], and the equilibrium constant for the citramalate formation reaction has been determined. Acetate + pyruvate (S)-CitramalateWe have also determined the equilibrium constant for the 2-hydroxyglutarate dehydrogenase reaction HO