Exploring the scope of nickel pincer as enzymatic cofactor: engineering of gluconate epimerase towards the production of rare sugars.

Cofactors provide chemical versatility well beyond the capabilities of enzymes, emphasizing their
potential for biocatalysis. Recently, a tethered niacin-derived nickel pincer cofactor (NPN) has
been discovered in the catalytic site of a lactate racemase for the interconversion of D- and Llactic
acid. Sequence analysis indicated that this fascinating novel cofactor could be widespread in
the prokaryotic world, but has so far only been confirmed to be operative in gluconate 2-
epimerase (G2E). Interestingly, this NPN-dependent G2E was also shown to be active on glucose,
albeit very slowly. Given that most epimerases require activated substrates for activity, this
discovery holds great potential to truly revolutionize the production of rare sugars, which find
applications in the pharmaceutical and food industry. To fully exploit G2E’s potential, it will have
to be made active on other monosaccharides. In this project, enzyme engineering will be used to
(1) decipher the catalytic mechanism of both NPN and G2E as well as (2) create new specificities
towards industrially relevant targets (e.g. galactose to talose conversion). To keep the process
costs as low as possible, in vivo production and utilization of the NPN cofactor will also be assessed
to pave the way for a NPN biofactory. The latter will not only be interesting in light of rare sugar
production but also for other future industrially applicable NPN-dependent reactions.