Table 4.

Summary of activity data obtained on WT and/or mutated reductases DdahC and MlghC on heptose- and mannose-based substrates

Enzyme	Status	Final AA	Heptose	Man. P4’	Man. P1’	Expectation
GFS	WT	H179 C109	n/a	n/a	Normal substrate	Catalytic dyad serving as general acid and base for epimerization at C3 and C5.
MlghC	WT	R180 Y108	P4γ to P5γ	PI + PIII	PII	Catalytic dyad
DdahC	WT	H180 T110	P4α to P5α	PIII	PII	Catalytic dyad
DdahC	T110C	H180 C110	P4α to P5α ↑↑	PIII =	PII =	GFS-like dyad. Enhanced mannose usage, especially nonepimerized P1’.
DdahC	H180R	R180 T110	P4α to P5α =	PIII =	PII ↑↑↑	Intermediate toward MlghC dyad. Switch of heptose epimer specificity to P4γ and decreased mannose usage.
DdahC	H180R T110Y	R180 Y110	Inactive	PIII =	PII =	MlghC-like dyad. Switch of heptose epimer specificity to P4γ and decreased mannose usage.

↓, modest decrease in enzymatic activity; =, no change in enzymatic activity; ↑ to ↑↑↑, moderate to strong increase in enzymatic activity; Heptose: heptose-based substrates; Man., mannose-based substrates; n/a, not applicable.

Final AA refers to the amino acids present at the site of interest in the WT or mutated enzyme.

Status refers to either WT or mutated enzyme.

Data under columns Heptose, Man. P4’, and Man. P1’ show observed catalytic activity, which differed from original expectations.