Biosynthesis of the prosthetic group of citrate lyase

Citrate lyase (EC 4.1.3.6) catalyzes the cleavage of citrate to acetate and oxaloacetate and is composed of three subunits (alpha, beta, and gamma). The gamma-subunit serves as an acyl carrier protein (ACP) and contains the prosthetic group 2'-(5' '-phosphoribosyl)-3'-dephospho-CoA, which is attached via a phosphodiester linkage to serine-14 in the enzyme from Klebsiella pneumoniae. In this work, we demonstrate by genetic and biochemical studies with citrate lyase of Escherichia coli and K. pneumoniae that the conversion of apo-ACP into holo-ACP is dependent on the two proteins, CitX (20 kDa) and CitG (33 kDa). In the absence of CitX, only apo-ACP was synthesized in vivo, whereas in the absence of CitG, an adenylylated ACP was produced, with the AMP residue attached to serine-14. The adenylyltransferase activity of CitX could be verified in vitro with purified CitX and apo-ACP plus ATP as substrates. Besides ATP, CTP, GTP, and UTP also served as nucleotidyl donors in vitro, showing that CitX functions as a nucleotidyltransferase. The conversion of apo-ACP into holo-ACP was achieved in vitro by incubation of apo-ACP with CitX, CitG, ATP, and dephospho-CoA. ATP could not be substituted with GTP, CTP, UTP, ADP, or AMP. In the absence of CitG or dephospho-CoA, AMP-ACP was formed. Remarkably, it was not possible to further convert AMP-ACP to holo-ACP by subsequent incubation with CitG and dephospho-CoA. This demonstrates that AMP-ACP is not an intermediate during the conversion of apo- into holo-ACP, but results from a side activity of CitX that becomes effective in the absence of its natural substrate. Our results indicate that holo-ACP formation proceeds as follows. First, a prosthetic group precursor [presumably 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA] is formed from ATP and dephospho-CoA in a reaction catalyzed by CitG. Second, holo-ACP is formed from apo-ACP and the prosthetic group precursor in a reaction catalyzed by CitX.     

Stereochemical course of biotin-independent malonate decarboxylase catalysis.

Malonate decarboxylases, which catalyze the conversion of malonate to acetate, can be classified into biotin-dependent and biotin-independent enzymes. In order to reveal the stereochemical course of the reactions catalyzed by the biotin-independent enzymes from Acinetobacter calcoaceticus and Pseudomonas fluorescens, a chiral substrate, malonate carrying (13)C in one carboxyl group and (3)H at one of the methylene positions, was prepared and used in the reactions catalyzed by these two enzymes. The decarboxylation of (R)-[1-(13)C(1), 2-(3)H]malonate in (2)H(2)O gave a pseudo-racemate of chiral acetate which was converted via acetyl-CoA into malate with malate synthase. From the relative proportions of the isotopomers of malate present, determined by (3)H NMR analysis, it was concluded that in the decarboxylation of malonate by these two biotin-independent enzymes COOH is replaced by H with retention of configuration. The same stereochemical outcome had been previously observed for the reaction catalyzed by the biotin-dependent malonate decarboxylase from Malonomonas rubra (J. Micklefield et al. J. Am. Chem. Soc. 117, 1153-1154, 1995).     

Functions of malonate decarboxylase subunits from Pseudomonas putida

Malonate decarboxylase from Pseudomonasputida is composed of five subunits, alpha, beta, gamma, delta, and epsilon. Two subunits, delta and epsilon, have been identified as an acyl-carrier protein (ACP) and malonyl-CoA:ACP transacylase, respectively. Functions of the other three subunits have not been identified, because recombinant subunits expressed in Escherichia coi formed inclusion bodies. To resolve this problem, we used a coexpression system with GroEL/ES from E. coli, and obtained active recombinant subunits. Enzymatic analysis of the purified recombinant subunits showed that the alpha subunit was an acetyl-S-ACP:malonate ACP transferase and that the betagamma-subunit complex was a malonyl-S-ACP decarboxylase.     

Biosynthesis of malonate in roots of soybean seedlings

Many plants accumulate malonate, but it was shown earlier that malonate does not accumulate as a deadend product of metabolism in soybean (Glycine max v. Hodgson tissues. The metabolism of malonate in the soybean plant at the whole tissue and enzymic level was followed, and the pathway of malonate biosynthesis in young soybean root tissue was shown to be via acetyl-coenzyme A carboxylase.     

Crystal structures of the wild-type, P1A mutant, and inactivated malonate semialdehyde decarboxylase: a structural basis for the decarboxylase and hydratase activities

Malonate semialdehyde decarboxylase (MSAD) from Pseudomonas pavonaceae 170 is a tautomerase superfamily member that converts malonate semialdehyde to acetaldehyde by a mechanism utilizing Pro-1 and Arg-75. Pro-1 and Arg-75 have also been implicated in the hydratase activity of MSAD in which 2-oxo-3-pentynoate is processed to acetopyruvate. Crystal structures of MSAD (1.8 A resolution), the P1A mutant of MSAD (2.7 A resolution), and MSAD inactivated by 3-chloropropiolate (1.6 A resolution), a mechanism-based inhibitor activated by the hydratase activity of MSAD, have been determined. A comparison of the P1A-MSAD and MSAD structures reveals little geometric alteration, indicating that Pro-1 plays an important catalytic role but not a critical structural role. The structures of wild-type MSAD and MSAD covalently modified at Pro-1 by 3-oxopropanoate, the adduct resulting from the incubation of MSAD and 3-chloropropiolate, implicate Asp-37 as the residue that activates a water molecule for attack at C-3 of 3-chloropropiolate to initiate a Michael addition of water. The interactions of Arg-73 and Arg-75 with the C-1 carboxylate group of the adduct suggest these residues polarize the alpha,beta-unsaturated acid and facilitate the addition of water. On the basis of these structures, a mechanism for the inactivation of MSAD by 3-chloropropiolate can be formulated along with mechanisms for the decarboxylase and hydratase activities. The results also provide additional evidence supporting the hypothesis that MSAD and trans-3-chloroacrylic acid dehalogenase, a tautomerase superfamily member preceding MSAD in the trans-1,3-dichloropropene degradation pathway, diverged from a common ancestor but retained the key elements for the conjugate addition of water.     

Studies on the mechanism of formation of the pyruvate prosthetic group of phosphatidylserine decarboxylase from Escherichia coli

Phosphatidylserine decarboxylase from Escherichia coli uses a pyruvate group as the enzyme cofactor (Satre, M., and Kennedy, E. P. (1978) J. Biol. Chem. 253, 479-483). Comparison of the DNA sequence of the psd gene with the partial amino acid sequence of the mature gene product suggests that the two nonidentical subunits of the mature enzyme are formed by cleavage of a proenzyme resulting in the conversion of Ser-254 to an amino-terminal pyruvate residue (Li, Q.-X., and Dowhan, W. (1988) J. Biol. Chem. 263, 11516-11522). The cleavage of the wild-type proenzyme occurs rapidly with a half-time on the order of 2 min. When Ser-254 is changed to cysteine (S254C), threonine (S254T), or alanine (S254A) by site-directed mutagenesis, the rate of processing of the proenzyme and the production of the functional enzyme are drastically affected. Proenzymes with S254C or S254T are cleaved with a half-time of around 2-4 h while the S254A proenzyme does not undergo processing. The reduced processing rate for the mutant proenzymes is consistent with less of the functional enzyme being made. Mutants encoding the S254C and S254T protein produce 16 and 2%, respectively, of the activity of the wild-type allele but can still complement a temperature-sensitive mutant in the psd locus. There is no detectable activity or complementation observed with the S254A protein. These results are consistent with the hydroxyl group of Ser-254 playing a critical role in the cleavage of the peptide bond between Gly-253 and Ser-254 of the prophosphatidylserine decarboxylase and support the mechanism proposed by Snell and coworkers (Recsei and Snell (1984) Annul Rev. Biochem. 53, 357-387) for the formation of the prosthetic group of pyruvate-dependent decarboxylases.     

The biotin protein MadF of the malonate decarboxylase from Malonomonas rubra

The gene for the biotin protein MadF of the Na⁺-pumping malonate decarboxylase from Malonomonas rubra was expressed in Escherichia coli together with the gene for the biotin ligase birA. MadF was partially purified from cell lysates by ammonium sulfate precipitation. Almost pure biotin protein was obtained by subsequent gel chromatography. With recombinant MadF, malonate decarboxylase activity of M. rubra cell extracts previously inactivated by avidin was recovered. Thus, the biological activity of recombinant MadF was proven. Despite the coexpression of birA, MadF was poorly biotinylated. This effect was not caused by an insufficient cofactor supply due to elevated protein levels at constant biotin uptake rates. Attempts to improve the cofactor incorporation were made by site-directed mutagenesis, by coexpression of madK, and by N-terminal elongation of MadF. These measures improved the fraction of MadF containing biotin to maximally 5%. These results might indicate the existence of a biotin ligase in M. rubra with an altered substrate specificity different from that of BirA. Received: 4 June 1998 / Accepted: 7 September 1998     

Cloning and sequencing of genes encoding malonate decarboxylase in Acinetobacter calcoaceticus

Malonate decarboxylase from Acinetobacter calcoaceticus was isolated and characterized (Kim, Y.S., Byun, H.S., J. Biol. Chem. 269 (1994) 29636–29641), and its subunits were reanalyzed recently to be α, β, γ, and δ. The genes for the subunits, MdcA (548 a.a.), B (295 a.a.), C (238 a.a.), and D (102 a.a.), of the enzyme have been cloned by using oligonucleotide primers deduced from amino acid sequences of peptides isolated from the purified enzyme, and sequenced to be clustered in an operon in the order of A-D-B-C. The operon was found to encode more genes than mdcABCD. The Escherichia coli, transformed with the vector containing the insert mdcADBC and about 1.7 kb of an upstream region, expressed the four subunits of the enzyme but the proteins did not show enzyme activity. It indicates that, like the enzymes from Malonomonas rubra and Klebsiella pneumoniae, more genes are needed for the formation of the functional malonate decarboxylase.     

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.     

Biosynthesis of inducible L-ornithine decarboxylase by bacterial of the Vibrionaceae family

The biosynthesis of L-ornithine decarboxylase was investigated in 73 strains of the Vibrionaceae family. V. harveyi 1175 was found to be most active. The maximum accumulation of the enzyme was observed after 4-hour cultivation at pH 5.5 and 33 degrees in the presence of 0.8% L-ornithine HCl used as an inducer without aeration. Under these conditions L-ornithine decarboxylase activity was 3.87 units/mg dry cells.