Structural Biology of Mutants of Protocatechuate 3,4-Dioxygenase from Pseudomonas putida. Cathleen A. Earhart, Matthew W. Vetting, Marla Burger, & Douglas H. Ohlendorf, Dept. of Biochemistry, University of Minnesota, Minneapolis, MN 55455

Several mutants of the metalloenzyme protocatechuate 3,4-dioxygenase (3,4-PCD; EC 1.13.11.3) from Pseudomonas putida (ATCC 23975) have been produced, crystallized and their structures determined to high resolution. The mutant proteins have also been characterized by kinetic and EPR analysis.

Dioxygenases catalyze the incorporation of both atoms of molecular oxygen into a variety of substrates. This ring-opening reaction is the key step in the degradation pathway for many aromatic compounds found in the environment. Dioxygenases are typically metalloproteins which require non-heme mononuclear iron center as a required cofactor. The prototype intradiol dioxygenase is protocatechuate 3,4-dioxygenase The structure of 3,4-PCD from P. putida has been determined and refined to an R-value of 0.172 to 2.15 Å resolution. The large amount of spectroscopic and kinetic data has led to a proposed mechanism for 3,4-PCD which is being verified through the structural analysis of key complexes. We are also using mutagenesis to further characterize the function of 3,4-PCD with the initial target of Trp 149 which should be important in substrate selection and positioning.

The large amounts of mutant proteins required for biophysical analysis were produced using a new expression vector, pCE110. Previous attempts to obtain high levels of expression of the P. putida protein in both E. coli and in other pseudomonads had been disappointing with only 0.1-0.2 mg of 3,4-PCD per gram of cell paste produced with the best system. This level of expression is about one tenth the level of wild type and poses a significant barrier to the production of mutant proteins. In E. coli, the pCE110 vector produces 2 mg of PCD per gram of cell paste and has been successful in producing large amounts of mutant proteins.