Enzymic Hydrolysis and Synthesis of Carbon-Carbon bonds by Microbial ß-Ketolases

The aim of the project was to produce the ß-ketolase of the orcinol pathway of Pseudomonas putida ORC described by Dagley and Ribbons as well as other ß-ketolases in sufficient amounts for studying their potential for biocatalytic applications (e.g. substrate- regio- and stereo-specificities and reversion of the reaction). This was attempted by optimization of the fermentation- and induction-conditions. A further target of the project was the genetic characterization of the orcinol-pathway. In order to investigate the genetic organization of the orcinol-pathway, clones showing extradiol ring-cleavage activity have been isolated from a gene-library from the DNA of Pseudomonas putida ORC. Two different dioxygenase genes, coding for a class II and a class III extradiol ring-cleaving dioxygenase, were isolated and overexpressed in E. coli. By comparison of protein-characteristics and by substrate specificities of the purified dioxygenases, the class III dioxygenase has been shown to be identical with the orcinol-induced wild-type dioxygenase of P. putida and has therefore been designated as OrcB. The class III dioxygenase and its flanking genes have turned out to be highly homologous to the 3-(3-hydroxyphenyl)-propionic acid (HPP) pathway of E. coli K12. In this way, the orcinol pathway could partially be cloned and characterized. A monooxygenase, a dioxygenase and the first hydrolase, involved in orcinol degradation were found on the cloned DNA-fragment. The gene for the latter enzyme of the orcinol-pathway, the acetylpyruvate hydrolase, was not located on the cloned DNA-fragment, but based on the high homology to the HPP pathway, further conclusions could be drawn. Their verification by cloning is in progress. Furthermore, in order to find an acetylacetone-cleaving enzyme, a strain with the potential to grow with acetylacetone as the sole carbon source was isolated and identified by DSMZ as Acinetobacter johnsonii (ID 98- 849). The primary, acetylacetone cleaving, enzyme of this organism was purified and characterized on a biochemical and genetic level. Contrary to our expectations to find a hydrolytic cleavage reaction, the enzyme cleaves acetylacetone to acetate and methylglyoxal. Concomitantly it incorporated equimolar amounts of molecular oxygen without involvement of prosthetic groups and without exogenous cofactor requirement. Based on these findings the classification of the enzyme as a dioxygenase E.C. 1.13.11.-. was proposed. Furthermore, the gene was isolated and overexpressed in E.coli, producing the active enzyme in good yields. The protein sequences of acetylacetone dioxygenase and of the subsequent open reading frame ORF2 do not show significant similarity to proteins of known function and only few homologies to any sequence data of genes with unknown function. Acetylacetone dioxygenase activity was found to depend on Fe2+. The enzyme accepts 2,4-diketones and derivatives which are uncharged and can be deprotonized, but does not accept any charged substrates. Based on substrate specificities a catalytic mechanism is suggested. The enzyme structure has been elucidated by crystallographic methods (G. Stranzl, C. Kratky, Institute of Physical Chemistry, KFU Graz). The enzyme found is novel, regarding sequence, catalyzed reaction and structure. Acetylacetone oxygenase has proven to be of interest for industrial applications, because of its novel catalytic properties. The outcome of this project has resulted in a patent, in cooperation with the company Biocatalytics, Pasadena, CA (USA). The company wants to produce and sell the enzyme and also studies its use for the production of fine-chemicals. This enzyme will then provide new routes for the production of a-oxoaldehydes.