Pyrroloquinoline quinone (PQQ) has potential applications in the treatment of Alzheimers disease and jaundice. PQQ is produced and secreted in large amounts by methylotrophic bacteria, and they have potential for commercial PQQ production.
PQQ is the prosthetic group in methanol dehydrogenase of the facultative methylotroph, Methylobacterium extorquens AM1. Previous work had shown that seven genes were required for PQQ production in M.extorquens AM1. Dr. Christina Morris in our lab had sequenced the first two genes, pqqD and pqqG. pqqD encoded a twenty-nine amino acid peptide containing tyrosine and glutamate separated by three amino acids. Tyrosine and glutamate had been shown to be the precursors of PQQ, and this peptide is believed to be the precursor of PQQ biosynthesis in vivo.
This thesis was initiated by mapping the transcriptional start site in front of pqqD. A high abundance 240 base pair transcript containing pqqD and a low abundance 1300 base pair transcript containing both pqqD and the next gene pqqG were detected using RNA-DNA blots. This was supported by data with Tn5lac insertions.
Studies were carried out to understand the regulation of PQQ biosynthesis in more detail. It was found that the products of the regulatory genes moxM, moxD and moxB were required for induction of pqqD on methanol plus methylamine compared to succinate, while the products of the regulatory genes moxQ and moxE were not required. The products of moxM and moxD were also required for high level transcription of pqqD. Measurements of PQQ in the culture supernatants of wild-type and mutants indicated that transcription of pqqD was not the rate limiting step in PQQ biosynthesis, and in addition that regulation of PQQ biosynthesis occurs at more than one step in the process.
Using functional complementation it was found that pqqE of M.extorquens AM1 was equivalent to pqqF of K.pneumoniae. The middle portion of pqqE was sequenced and by aligning PqqE with PqqF of K.pneumoniae, the direction of transcription and size of pqqE was determined. PqqF shows similarity to proteases and this strongly suggests that a protease is involved in PQQ biosynthesis.
