Identification and characterization of the tungsten-containing class of benzoyl-coenzyme A reductases.
Aromatic compounds are widely distributed in nature and can only be biomineralized by microorganisms. In anaerobic bacteria, benzoyl-CoA (BCoA) is a central intermediate of aromatic degradation, and serves as substrate for dearomatizing BCoA reductases (BCRs). In facultative anaerobes, the mechanistically difficult reduction of BCoA to cyclohexa-1,5-dienoyl-1-carboxyl-CoA (dienoyl-CoA) is driven by a stoichiometric ATP hydrolysis, catalyzed by a soluble, three [4Fe-4S] cluster-containing BCR. In this work, an in vitro assay for BCR from the obligately anaerobic Geobacter metallireducens was established. It followed the reverse reaction, the formation of BCoA from dienoyl-CoA in the presence of various electron acceptors. The benzoate-induced activity was highly specific for dienoyl-CoA (K(m) = 24 +/- 4 microM). The corresponding oxygen-sensitive enzyme was purified by several chromatographic steps with a 115-fold enrichment and a yield of 18%. The 185-kDa enzyme comprised 73- and 20-kDa subunits, suggesting an alpha(2)beta(2)-composition. MS analysis revealed the subunits as products of the benzoate-induced bamBC genes. The alphabeta unit contained 0.9 W, 15 Fe, and 12.5 acid-labile sulfur. Results from EPR spectroscopy suggest the presence of one [3Fe-4S](0/+1) and three [4Fe-4S](+1/+2) clusters per alphabeta unit; oxidized BamBC exhibited an EPR signal typical for a W(V) species. The FeS clusters and the W- cofactor could only be fully reduced by dienoyl-CoA. BamBC represents the prototype of a previously undescribed class of dearomatizing BCRs that differ completely from the ATP-dependent enzymes from facultative anaerobes.
Kung
Johannes W
JW
Institute of Biochemistry, University of Leipzig, 04103 Leipzig, Germany.
Löffler
Claudia
C
Dörner
Katerina
K
Heintz
Dimitri
D
Gallien
Sébastien
S
Van Dorsselaer
Alain
A
Friedrich
Thorsten
T
Boll
Matthias
M
D000693:Anaerobiosis; D004578:Electron Spin Resonance Spectroscopy; D005798:Genes, Bacterial; D045302:Geobacter / Q000201:enzymology* / Q000235:genetics; D006841:Hydrocarbons, Aromatic / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D005810:Multigene Family; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D021122:Protein Subunits; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry; D014414:Tungsten / Q000737:chemistry*
D006841:Hydrocarbons, Aromatic; D021122:Protein Subunits; D044925:Oxidoreductases Acting on CH-CH Group Donors; C109030:benzoyl-coenzyme A reductase (dearomatizing); D014414:Tungsten