Dihydrodipicolinate synthase superfamily – on the origin of 5-keto-4-deoxyglucarate dehydratase and 2-keto-3-deoxy-L-arabinonate dehydratase

• Applicant: Professor Dr. Volker Sieber
• Subject Area: Biochemistry
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 469128000

5-Keto-4-deoxyglucarate dehydratase (5-KdgD) catalyzes the dehydration of 5-KDG followed by vinylogous decarboxylation to yield 2-ketoglutarate semialdehyde (KGSA). 2-Keto-3-deoxy-L-arabinonate dehydratase (L-KdaD) catalyzes dehydration of L-KDA to yield the same product, KGSA. Both enzymes belong to the dihydrodipicolinate synthase superfamily (DHDPS), whose members adopt the TIM-barrel fold. Mechanistic studies hypothesized that the two dehydratases share the first steps in these reactions. However, our preliminary study revealed that each enzyme showed no activity toward the other substrate, i.e. neither 5-KdgD is active on L-KDA nor L-KdaD on 5-KDG. Phylogenetic tree analysis of the DHDPS superfamily shows that these two dehydratases are evolutionary related. Thus, it is intriguing to determine at which point in time the common ancestors of these two dehydratases were evolved to be more specific. We propose a “vertical approach” to calculate the most probable ancestors utilizing ancestral sequence reconstruction (ASR) techniques. The predicted ancestors will be resurrected in vitro by means of gene synthesis. We will also approach this hypothesis by rationally designing variants of each dehydratase to interconvert substrate specificity. This approach is aimed to decipher key positions and amino acids that determine their strict substrate specificity. The substitutions obtained from rational design will be compared to the predicted common ancestors prior to gene synthesis. Furthermore, we will test all variants generated to find variants that only dehydrate 5-KDG without concomitant decarboxylation to yield 2,5-dioxoadipic acid (DOA). The sequence of the variants is expected to help find contemporary homologues of non-decarboxylating 5-KdgD. This will also give us access to DOA that is laborious to synthesize for further studies of the compound. Combination of rational design and ASR is a unique approach that can benchmark future studies of evolutionary relationship of protein families.

• DFG Programme: Research Grants
• International Connection: Australia
• Cooperation Partners: Professor Mikael Bodén; Professor Dr. Luke Guddat