Table 1.

Representative UPLC conversions and (enzyme loadings) for substrates used in substrate walking based mutagenesis of RebH.

Generation	Enzyme	Mutation[a]	Method (substrate)[b]	%Conv. 1 (%Loading)[c]	%Conv. 2 (%Loading)	%Conv. 3 (%Loading)	%Conv. 4 (%Loading)
0	RebH	–	–	53 (0.2)	3 (0.5)	<1 (5)	1 (5)
1	1-PVM	S2P/M71V/K145M	epPCR[d] (1)	43 (0.2)	5 (0.5)	<1 (5)	5 (5)
2	2-T	N467T	epPCR (1)	53 (0.2)	7 (0.5)	<1 (5)	9 (5)
3	3-SS	G112S/N470S	epPCR (2)	22 (0.2)	64 (0.5)	6 (5)	9 (5)
3	3-S	S112G	point mut.(3)[e]	43/5[f] (0.2)	50 (0.5)	29 (5)	39 (5)
4	4-V	A442V	epPCR (3)	39/3[f] (0.2)	43 (0.5)	48 (5)	38 (5)

^[a]Mutations relative to the parent on the previous line.

^[b]Method used to introduce mutations and (substrate used in the screening effort noted).

^[c]Conversions of substrates determined by UPLC and (mol% loadings of enzymes used).

^[d]Error-prone PCR.

^[e]Point mutation introduced via SOE PCR.^[20]

^[f]Second number refers to conversion to dihalogenated product.