Engineered polypeptides that exhibit increased catalytic efficiency for unnatural cofactors and uses thereof

An engineered polypeptide having dehydrogenase activity that exhibits catalytic efficiency for a nicotinamide-based unnatural cofactor,

• wherein the engineered polypeptide comprises 1, 2, 3, 4, 5, 6, 7, or 8 introduced amino acid substitutions in comparison to the sequence of a wild-type or parent polypeptide having the polypeptide sequence of SEQ ID NO:1, wherein the introduced amino acid substitutions comprise at least the substitution of I195R in comparison to SEQ ID NO:1,
• wherein the cofactor normally utilized by the wild-type or parent polypeptide is nicotinamide adenine dinucleotide (NAD+) or nicotinamide adenine dinucleotide phosphate (NADP+),
• wherein the nicotinamide-based unnatural cofactor is a cofactor that is not normally utilized by the dehydrogenase encoded by the wild-type or parent polypeptide to catalyze a reaction and which is selected from the group consisting of nicotinamide mononucleotide (NMN+), 1-phenyl-1,4,-dihydronicotinamide (PNA+), 1-benzyl-1,4-dihydronicotinamide (BNA+), 1-(4-hydroxyphenyl) 1,4-dihydronicotinamide (HPNA+), 1-methyl-1,4-dihydronicotinamide (MNA+), nicotinamide flucytosine dinucleotide (NFCD+), and nicotinamide mononucleoside (NR+).