Table 2.

Performance of engineered enzymes with noncanonical cofactors

Enzyme	Uniprot	Strategy	Native Cofactor	Noncanonical Cofactora	Mutations	CSRb	RCEC	RSd	Source
D-lactate dehydrogenase (L. helveticus)	P30901	Bibliography, Saturation, Structure	NAD+	NCD+	V152R-N213E	4.3×10	2.1×10−1	9.0×102	Liu et al., 2020 [5]
					V152R-I177K-N213I	4.2×10	3.1×10−1	8.8×102
Formate dehydrogenase (Pseudomonas sp. 101)	P33160	Bibliography, Saturation, Structure	NAD+	NCD+	V198I-C256I-P260S-E261P-S381N-S383F	1.7×102	3.7×10−2	3.5×103	Guo et al., 2020 [6]
Glucose Dehydrogenase (B. subtilis)	A0A1B2ATD9	Computational, Structure	NADP+	NMN+	Y34Q-A93K-I195R	6.8×10−2	1.8×10−3	4.1×104	Black et al., 2019 [9]
					S17E-Y34Q-A93K-I195R	1.9×10	7.5×10−4	1.1×107
			NAD+	NMN+	Y34Q-A93K-I195R	4.6	2.8×10−3	1.8×106
					S17E-Y34Q-A93K-I195R	5.5×10	1.2×10−3	2.1×107
6-phospho gluconate dehydrogenase (T. maritima)	A0A2N5RL69	Computational, Random, Saturation, Structure	NADP+	NMN+	Mut 5-1e	1.4×10−2	1.2×10−4	4.6×103	Huang et al., 2019 [10]
					Mut 6-1f	1.4×10−2	1.5×10−4	4.4×103
Glucose-6-phosphate dehydrogenase (T. maritima)	A0A2N5RPI0	Computational, Random, Saturation, Structure	NADP+	NMN+	A64S-R65I-T66I	-	-	-
Phosphite dehydrogenase (Ralstonia sp. 4506)	G4XDR8	Bibliography, Computational, Saturation, Structure	NAD+	NCD+	I151R-P176R	2.0×10	1.3×10−1	3.4×102	Liu et al., 2019 [7]
					I151R-P176R-M207A	4.5×10	9.4×10−3	7.8×102
Glucose dehydrogenase (S. solfataricus)	O93715	Bibliography, Saturation, Structure	NAD+	BNA+	I192T-V306G	5.5×10	6.5×10−2	5.5×102	Nowak et al., 2017 [8]
					I192T-V306I	2.9×10−1	1.0×10−1	2.9×10
				P2NA+	I192T-V306G	3.1	3.6×10−2	2.0×102
					I192T-V306I	9.2×10−1	3.2×10−1	6.1×10
				P3NA+	I192T-V306G	3.8×10	2.6×10−1	6.8×102
					I192T-V306I	3.0×10−1	1.1× 10−1	5.3×10
Phosphite dehydrogenase (Ralstonia sp. 4506)	G4XDR8	Bibliography	NAD+	NCD+	I151R	4.3×10	1.4×10−2	5.4×102	Wang et al., 2017 [16]
P450-BM3 (B. megaterium)	P14779	Bibliography, Structure	NADH	BNAH	R966D-W1046S	1.0×10−1	9.6×10	-	Lo et al., 2017 [33]
				MDH	R966D-W1046S	6.4×10−2	6.0×10	-
Alcohol dehydrogenase (P. furiosus)	-	MSAg, Structure	NAD+	NMN+	K249G-H255R	1.6×10−3	4.4×10−4	1.8×102	Campbell et al., 2012 [35]
Malic enzyme (E. coli)	P26616	Computational, MSA, Saturation, Structure	NAD+	NFCD+	L310R	9.8×10	4.1×10−1	1.1×104	Ji et al., 2011 [53]
					L310R-Q401C	2.7×102	4.5×10−1	2.9×104
				NCD+	L310R	1.9×102	7.9×10−1	1.4×104
					L310R-Q401C	4.3×102	7.2×10−1	3.3×104
D-lactate dehydrogenase (L. helveticus)	P30901	MSA, Structure	NAD+	NCFD+	V152R	-	-	-
Malate dehydrogenase (E. coli)	P61889	MSA, Structure	NAD+	NCFD+	L6R	-	-	-
Lactate dehydrogenase (B. stearothermophilus)	P00344	MSA, Saturation, Structure	NAD+	NMN+	C81S-N85R-F16Q	-	-	-	Flores et al., 2005 [34]

^aFull names of the noncanonical cofactors: AmNA⁺, 1-(2-carbamoylmethyl)-1,4-dihydronicotinamide; BNA⁺, 1-benzyl-1,4-dihydronicotinamide; BT⁺, 1-butyl-1,4-dihydronicotinamide; BE⁺, 1-(1-benzyl-1,4-dihydro-3-yl) ethanone; MD⁺, N-4-methoxybenzyl-1,4-dihydronicotinamide; MNA⁺, 1-methyl-1,4-dihydropyridine-3-carboxamide; NCD⁺, Nicotinamide cytosine dinucleotide; NCFD⁺, Nicotinamide flucytosine dinucleotide; NMN⁺, Nicotinamide mononucleotide; P2NA⁺, 1-phenethyl-1,4-dihydropyridine-3-carboxamide; P3NA⁺, 1-(3-phenylpropyl)-1,4-dihydropyridine-3-carboxamide. Reduced cofactor ends with “H”

^bCSR, Cofactor Specificity Ratio (Equation 1)

^CRCE, Relative Catalytic Efficiency (Equation 2)

^dRS, Relative Specificity (Equation 3)

^eMut 5-1 contains A11G-K27R-R33I-T34I-F60Y-D82L-T83L-Q86L-K118N-I120F-D294V-F326S-Y383C-N387S-A447V

^fMut 6-1 contains A11G-K27R-R33I-T34I-F60Y-D82L-T83L-Q86L-K118N-I120F-D251E-D294V-F326S-F329Y-Y383C-N387S-V390G-A447V

^gMSA, Multiple Sequence Alignment