Cloning, sequencing and heterologous expression of pyrogallol-phloroglucinol transhydroxylase from Pelobacter acidigallici

A genomic X-library of Pelobacter acidigallici has been established. Proteo lytic digestion of homogeneous pyrogallol-phloroglucinol transhydroxylase f rom the same microorganism afforded polypeptide fragments whose N-terminal sequences allowed the generation of oligonucleotide primers. Together with primers deduced from the known N-terminal sequences of the two intact subun its these were used in PCR experiments to obtain three amplificates. Screen ing the X-library with the three amplificates led eventually to clones cont aining the whole gene coding for the transhydroxylase. Sequencing the gene revealed two open reading frames coding for 875 and 275 amino acids which correspond to the alpha- and beta-subunits of THL, respe ctively. The two subunits are separated by a 48-bp noncoding region. Compar ison of the sequence with those of other molybdopterin cofactor (MoCo)-enzy mes places THL in the dimethylsulfoxide reductase family. Possible contact sites to the MoCo and to the iron-sulphur clusters were spotted. Using the expression vectors pQE 30 and pT 7-7 three constructs harbouring the THL gene were created. One of them carried a Hiss-tag at the N-terminus of the ol-subunit, another at the C-terminus of the P-subunit. Immunoblot analysis showed high expression of THL, but the inclusion bodies could not be refolded to active enzyme.