Cloning and nucleotide sequence of the <i>fabD</i> gene encoding malonyl coenzyme A‐acyl carrier protein transacylase of <i>Escherichia coli</i>

Magnuson, Kelly S.; Oh, W; Larson, Timothy J.; Cronan, John E.

doi:10.1016/0014-5793(92)80128-4

Cited by 50 publications

(38 citation statements)

References 23 publications

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“…Hence, the evolutionary origin for MdcH may be a gene operating in the synthesis of polyacetate compounds. Recently, the fabD gene of E. coli has been cloned [26,27] and the structure of its gene product determined [28]. The active site of the enzyme is related to a/b hydrolases where SerHis and Asp/Glu operate together as a charge relay system.…”

Section: Discussionmentioning

confidence: 99%

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

Hoenke

Dimroth

1999

European Journal of Biochemistry

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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...

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Section: Discussionmentioning

confidence: 99%

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

Hoenke

Dimroth

1999

European Journal of Biochemistry

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“…fatty acid or polyketide synthases has demonstrated that a serine, such as Ser-92 in the E. coli FabD protein (16,22,25), is the active nucleophilic residue of these enzymes. During its transfer from malonyl-CoA to ACP, the malonyl moiety is transiently attached to this serine to form a stable malonyl-serine enzyme intermediate (26).…”

Section: Construction and Activity Of Various Mtfabd Mutant Proteins-mentioning

confidence: 99%

Biochemical Characterization of Acyl Carrier Protein (AcpM) and Malonyl-CoA:AcpM Transacylase (mtFabD), Two Major Components ofMycobacterium tuberculosis Fatty Acid Synthase II

Kremer¹,

Nampoothiri²,

Lesjean³

et al. 2001

Journal of Biological Chemistry

123

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Malonyl coenzyme A (CoA)-acyl carrier protein (ACP) transacylase (MCAT) is an essential enzyme in the biosynthesis of fatty acids in all bacteria, including Mycobacterium tuberculosis. MCAT catalyzes the transacylation of malonate from malonyl-CoA to activated holo-ACP, to generate malonyl-ACP, which is an elongation substrate in fatty acid biosynthesis. To clarify the roles of the mycobacterial acyl carrier protein (AcpM) and MCAT in fatty acid and mycolic acid biosynthesis, we have cloned, expressed, and purified acpM and mtfabD (malonyl-CoA: AcpM transacylase) from M. tuberculosis. According to the culture conditions used, AcpM was produced in Escherichia coli in two or three different forms: apo-AcpM, holo-AcpM, and palmitoylated-AcpM, as revealed by electrospray mass spectrometry. The mtfabD gene encoding a putative MCAT was used to complement a thermosensitive E. coli fabD mutant. Expression and purification of mtFabD resulted in an active enzyme displaying strong MCAT activity in vitro. Enzymatic studies using different ACP substrates established that holo-AcpM constitutes the preferred substrate for mtFabD. In order to provide further insight into the structure-function relationship of mtFabD, different mutant proteins were generated. All mutations (Q9A, R116A, H194A, Q243A, S91T, and S91A) completely abrogated MCAT activity in vitro, thus underlining the importance of these residues in transacylation. The generation and characterization of the AcpM forms and mtFabD opens the way for further studies relating to fatty acid and mycolic acid biosynthesis to be explored in M. tuberculosis. Since a specific type of FabD is found in mycobacterial species, it represents an attractive new drug target waiting to be exploited.

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“…Moreover, acetyl-CoA:ACP and malonyl-CoA:ACP AT activities distinct from the E. coli FabB (KS I) protein (Fig. 3) are reportedly involved in E. coli fatty acid biosynthesis (18)(19)(20)27), and the E. coli FabH (KS III) protein even lacks the conserved GH SXG motif yet exhibits both acetyl-CoA:ACP acetyltransferase…”

Section: Fig 2 Sodium Dodecyl Sulfate-polyacrylamide Gel Electrophomentioning

confidence: 99%

Functional analysis of putative beta-ketoacyl:acyl carrier protein synthase and acyltransferase active site motifs in a type II polyketide synthase of Streptomyces glaucescens

Meurer

Hutchinson

1995

J Bacteriol

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The significance of potential active site motifs for acyltransferase and ␤-ketoacyl:acyl carrier protein synthase regions within the TcmK protein was investigated by determining the effects of mutations in the proposed active sites on the production of tetracenomycins F2 and C. In a Streptomyces glaucescens tcmGHI JKLMNO null mutant, plasmids carrying the S351A mutation produced high amounts of tetracenomycin F2 but plasmids carrying the C173A or C173S mutation or the H350L-S351A double mutation produced no detectable amount of any known intermediate. In a tcmK mutant, plasmids with the S351A mutation restored high production of tetracenomycin C and plasmids carrying the other mutations were able to complement the chromosomal defect to some extent. None of the mutations affected the amount of TcmK produced.

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Cloning and nucleotide sequence of the fabD gene encoding malonyl coenzyme A‐acyl carrier protein transacylase of Escherichia coli

Cited by 50 publications

References 23 publications

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

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

Biochemical Characterization of Acyl Carrier Protein (AcpM) and Malonyl-CoA:AcpM Transacylase (mtFabD), Two Major Components ofMycobacterium tuberculosis Fatty Acid Synthase II

Functional analysis of putative beta-ketoacyl:acyl carrier protein synthase and acyltransferase active site motifs in a type II polyketide synthase of Streptomyces glaucescens

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