Abstract
Some anaerobic bacteria use biotin-dependent Na+-translocating decarboxylases (Bdc) of β-keto acids or their thioester analogs as key enzymes in their energy metabolism. Glutaconyl-CoA decarboxylase (Gcd), a member of this protein family, drives the endergonic translocation of Na+ across the membrane with the exergonic decarboxylation of glutaconyl-CoA (ΔG0’ ≈−30 kJ/mol) to crotonyl-CoA. Here, we report on the molecular characterization of Gcd from Clostridium symbiosum based on native PAGE, size exclusion chromatography (SEC) and laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS). The obtained molecular mass of ca. 400 kDa fits to the DNA sequence-derived mass of 379 kDa with a subunit composition of 4 GcdA (65 kDa), 2 GcdB (35 kDa), GcdC1 (15 kDa), GcdC2 (14 kDa), and 2 GcdD (10 kDa). Low-resolution structural information was achieved from preliminary electron microscopic (EM) measurements, which resulted in a 3D reconstruction model based on negative-stained particles. The Gcd structure is built up of a membrane-spanning base primarily composed of the GcdB dimer and a solvent-exposed head with the GcdA tetramer as major component. Both globular parts are bridged by a linker presumably built up of segments of GcdC1, GcdC2 and the 2 GcdDs. The structure of the highly mobile Gcd complex represents a template for the global architecture of the Bdc family.
Highlights
The Earth’s biogeochemical carbon cycle involves the microbial decomposition of large quantities of chemically diverse organic compounds primarily derived from carbohydrates, lipids and proteins
Subsequent size exclusion chromatography (SEC) analysis indicated that the Glutaconyl-CoA decarboxylase (Gcd) complexes from F. nucleatum and P. asaccharolyticus aggregated during purification as seen in the elution profile of affinity chromatography and subsequent SEC (Supplementary Figures S1A,B)
Biotin-dependent Na+ translocating decarboxylases play a key role in fermenting bacteria metabolizing β-keto acids and their thioester analogs by using an exergonic decarboxylase reaction and not a redox reaction as usually found in iongradient forming processes of energy conservation
Summary
The Earth’s biogeochemical carbon cycle involves the microbial decomposition of large quantities of chemically diverse organic compounds primarily derived from carbohydrates, lipids and proteins. An example is the family of biotindependent Na+-translocating decarboxylases (Bdc) that use for ion pumping the exergonic decarboxylation of β-keto acids or β-carboxy thioesters and their vinylogues Oxaloacetate decarboxylase (Oad) is mainly found in citrate fermenting enterobacteria (Klebsiella pneumoniae, Salmonella typhimurium, and Vibrio cholerae) (Dimroth, 1980, 1981; Dimroth and Thomer, 1983; Laussermair et al, 1989), whereas (S)-methylmalonylCoA decarboxylase (Mmd) is present in lactate and succinate fermenting anaerobic bacteria (Veillonella parvula, formerly called Veillonella alcalescens, and Propionigenium modestum) (Hilpert and Dimroth, 1983; Huder and Dimroth, 1993; Bott et al, 1997). Na+-translocation coupled to the decarboxylation of the corresponding substrates was demonstrated for Oad (Dimroth, 1980, 1981), Mmd (Hilpert and Dimroth, 1983) and Gcd (Buckel and Semmler, 1982, 1983) with inverted membrane vesicles or purified proteins after incorporation into artificial liposomes
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