Identification of a novel gentisate 1,2-dioxygenase from Silicibacter pomeroyi

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Abstract
A 1,125-bp long ORF encoding a novel gentisate 1,2-dioxygenase with two-domain bicupins was cloned from Silicibacter pomeroyi DSS-3 and expressed in Escherichia coli. The resulting product was purified to homogeneity and partially characterized. Non-reductive SDS-PAGE and gel filtration showed that the active recombinant gentisate 1,2-dioxygenase had an estimated molecular mass of 132 kDa, and reductive SDS-PAGE indicated an approximate size of 45 kDa. The enzyme thus appears to be a homotrimeric protein. This is in contrast to the homotetrameric or dimeric protein of the gentisate 1,2-dioxygenases that have been characterized thus far. The K m and K cat/K m for gentisate were 12 μM and 653 × 104 M−1 s−1; the pI was 4.6–4.8. It was optimally active at 40°C and pH 8.0.

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