The acyl carrier protein of malonate decarboxylase of Malonomonas rubra contains 2'-(5"-phosphoribosyl)-3'-dephosphocoenzyme A as a prosthetic group

Biochemistry. 1996 Apr 16;35(15):4689-96. doi: 10.1021/bi952873p.

Abstract

Malonate decarboxylase of Malonomonas rubra is composed of soluble and membrane-bound components and contains an acetyl residue that is essential for catalytic activity. Upon incubation with hydroxylamine, the acetyl residue is removed, forming an inactive thiol enzyme, which is reactivated by acetylation with ATP, acetate, and a specific ligase. After incubation of the thiol enzyme with iodoacetate in the presence of excess dithioerythritol, the prosthetic group thiol residue was carboxymethylated and reactivation by acetylation was impaired. Radioactive labeling with [1-14C] iodoacetate revealed the site of carboxymethyation on a distinct cytoplasmic protein with the apparent molecular mass of 14 000 Da. The same protein was specifically labeled by enzymic acetylation of the thiol enzyme with [1-14C]acetate and ATP. Malonate decarboxlyation by [14C]acetyl malonate decarboxlyation resulted in the release of the radioactive acetyl residue from the enzyme,indicating that this acetyl residue is exchanged for a malonyl residue during catalysis. The acyl carrier protein has been purified as its [14C]carboxymethylated derivative to apparent homogeneity. The prosthetic group of the acyl carrier protein was isolated after alkaline hydrolysis, and its chemical structure was identified by high-performance liquid chromatography (HPLC) with the corresponding compound from citrate lyase from Klebsiella pneumoniae as reference and by mass spectrometry. Malonate decarboxylase was found to carry the same prosthetic group as citrate lyase, i.e. 2'-(5"-phosphoribosyl)-3'-dephospho-CoA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acyl Carrier Protein / isolation & purification
  • Acyl Carrier Protein / metabolism*
  • Carboxy-Lyases / isolation & purification
  • Carboxy-Lyases / metabolism*
  • Catalysis
  • Chromatography, High Pressure Liquid
  • Chromatography, Thin Layer
  • Coenzyme A / isolation & purification
  • Coenzyme A / metabolism*
  • Gram-Negative Anaerobic Bacteria / enzymology*
  • Mass Spectrometry

Substances

  • Acyl Carrier Protein
  • dephosphocoenzyme A
  • Carboxy-Lyases
  • malonate decarboxylase
  • Coenzyme A