Abstract
The initial enzyme of ethylbenzene metabolism in denitrifying Azoarcus strain EbN1, ethylbenzene dehydrogenase, was purified and characterized. The soluble periplasmic enzyme is the first known enzyme oxidizing a nonactivated hydrocarbon without molecular oxygen as cosubstrate. It is a novel molybdenum/iron-sulfur/heme protein of 155 kDa, which consists of three subunits (96, 43, and 23 kDa) in an alphabetagamma structure. The N-terminal amino acid sequence of the alpha subunit is similar to that of other molybdenum proteins such as selenate reductase from the related species Thauera selenatis. Ethylbenzene dehydrogenase is unique in that it oxidizes the hydrocarbon ethylbenzene, a compound without functional groups, to (S)-1-phenylethanol. Formation of the product was evident by coupling to an enantiomer-specific (S)-1-phenylethanol dehydrogenase from the same organism. The apparent K(m) of the enzyme for ethylbenzene is very low at <2 microm. Oxygen does not affect ethylbenzene dehydrogenase activity in extracts but inactivates the purified enzyme, if the heme b cofactor is in the reduced state. A variant of ethylbenzene dehydrogenase exhibiting significant activity also with the homolog n-propylbenzene was detected in a related Azoarcus strain (PbN1).
Highlights
Three bacterial species capable of anaerobic degradation of the aromatic hydrocarbon ethylbenzene are known to date
The pathway of anaerobic ethylbenzene metabolism is initiated by two consecutive two-electron oxidation steps of ethylbenzene to (S)-1-phenylethanol and further to acetophenone [1,2,3]
We analyzed the first enzyme of the predicted pathway, ethylbenzene dehydrogenase
Summary
Three bacterial species capable of anaerobic degradation of the aromatic hydrocarbon ethylbenzene are known to date All of these are denitrifying bacteria that belong to the genus Azoarcus of the -proteobacteria. It is initiated by a novel biochemical reaction, namely an oxygenindependent oxidation of ethylbenzene to (S)-1-phenylethanol. This intermediate is oxidized further to acetophenone by an alcohol dehydrogenase [1,2,3,4]. The catabolic pathway of n-propylbenzene in strain PbN1 is supposed to be analogous to that of ethylbenzene, yielding benzoyl-CoA and propionyl-CoA as intermediates [2]. We provide evidence that the same enzyme catalyzes anaerobic oxidation of ethylbenzene and n-propylbenzene
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