Table of Contents:
Anaerobic degradation of phenylalanine proceeds via the intermediate phenylacetate to benzoyl-CoA which is the common intermediate in anaerobic degradation of aromatic compounds. Phenylacetate is formed by the oxidation of phenylacetaldehyde. At the beginning of this project it was known that Aromatoleum aromaticum contains on the one hand an aldehyde:ferredoxin oxidoreductase (AOR) that could catalyze this step and on the other hand one or more enzymes that could oxidize phenylacetaldehyde with NAD and NADP as electron acceptors. Consequently, the question arose, which of these enzymes was the key enzyme in anaerobic phenylalanine metabolism. Moreover, AOR is an unusual enzyme especially for a facultative anaerobic bacterium since AOR-type enzymes are exclusively known from obligatory anaerobic, often hyperthermophilic prokaryotes so far. A. aromaticum needs one molybdenum enzyme for denitrifying growth, the nitrate reductase. All AORs known to date contain tungsten which is a metal very similar to molybdenum. This leads to the question which metal the AOR from A. aromaticum contains.
For both questions it was important to enrich and, ideally, purify the phenylacetaldehyde-oxidizing enzymes from cells of A. aromaticum. Sufficient enrichment of all enzyme activities to advance the given tasks was achieved by using a multitude of different chromatographic techniques. Metal analyses of the enriched AOR fractions and accompanying growth experiments with varying molybdate and tungstate concentrations showed that AOR actually contains a tungsten cofactor which could not be replaced by a molybdenum cofactor to form active enzyme. Consequently, A. aromaticum needs to be able to simultaneously produce at least one Mo-enzyme (nitrate reductase) and one W-enzyme (AOR). Most of the enzymes needed for the biosynthesis of the molybdenum cofactor can be shared for biosynthesis of the tungsten cofactor. Within the project at hand several enzymes were identified by bioinformatics analyses that may promote metal specificity of the biosynthesis machinery. These include molybdate- and tungstate-specific transporters and enzymes that transfer the metal to the cofactor. Moreover, another protein from the biosynthetic pathway was found to be encoded in close association to aor genes and may therefore play a role in providing the right cofactor. The enrichment of the NAD(P)-dependent phenylacetaldehyde-oxidizing activities enabled the identification of the corresponding enzyme, an aldehyde dehydrogenase encoded by gene ebA4954. This enzyme (PDH) was heterologously produced, purified with the help of a C-terminal Strep-tag, and biochemically characterized. It is a homotetramer that is very specific for phenylacetaldehyde, shows cooperative substrate-kinetics, is strongly inhibited by phenylacetaldehyde, and reduces both NAD and NADP.
The results summarized in the thesis at hand lead to the new hypothesis that PDH is the key enzyme for phenylalanine metabolism. It was shown that AOR is not essential for denitrifying growth on phenylalanine but oxidizes various aldehydes with similar reaction rates. Therefore it is imaginable that AOR serves as a detoxifying enzyme that oxidizes reactive aldehydes when the metabolism becomes unbalanced. The finding that A. aromaticum simultaneously produces molybdenum and tungsten enzymes leads to many new questions and research approaches.