Identification of functionally important amino acids in a novel indigo-producing oxygenase from Rhodococcus sp. strain T104.

A novel indigo-producing oxygenase gene, designated ipoA (1,197 bp) was characterized from Rhodococcus sp. strain T104. Three indigo-negative mutations (A58V, P59L, and G251D) were obtained through random mutagenesis using an E. coli mutator strain. Subsequent saturation mutagenesis resulted in the identification of nine and three amino acid substitutions that restore activity in the A58V and P59L mutants, respectively. Activity was not restored in the G251D mutation by any other amino acids. Interestingly, activity in the A58V mutant, where a methyl group is only replaced by an isopropyl side chain, is restored by a variety of amino acids, including polar ones. A molecular modeling study suggests that the residues at positions 58, 59, and 251 of the T104 IpoA enzyme are far from the active site, indicating that the mutations must alter the overall structure of the enzyme.

Functional identification of p-cumate operons in the terpene-degrading Rhodococcus sp. strain T104.

We identified a p-cumate degrading gene cluster (designated cmt) with a novel organization in our genomic studies of the terpene-degrading Rhodococcus sp. strain T104. The mutant strain SN140, isolated for the inability to grow on p-cumate, accumulates 2,3-dihydroxy-p-cumate as identified by liquid chromatography-mass spectrometry and 300 MHz proton nuclear magnetic resonance spectroscopy. Reverse transcriptase polymerase chain reaction experiments showed that the cmt genes are operonic and induced specifically by growth on p-cumate. This report is the first example of identifying the genes for p-cumate degradation in a gram-positive organism based on functional data.