Fig 4.

Enzymological properties of mutant LigA. (A) Purified wild-type and mutant LigA visualized by SDS-PAGE and Coomassie staining. (B) Adenylation time course with 10 nM LigA and 1 nM [³²P]NAD⁺. Means ± standard errors of the means are shown for the duplicate wells of a single representative experiment. (C) DNA ligation reactions catalyzed by 1 nM LigA at various NAD⁺ concentrations. Data points indicate reaction rates from a single representative experiment; curves indicate Michaelis-Menten curves as fitted by nonlinear regression analysis. Data for wild-type and mutants are plotted in separate panels because of their differing NAD⁺ concentration scales. RU, relative units. Symbols: ●, LigA; *, LigA:AD (panel B only); △, LigA(Arg⁶¹Ile); ▽, LigA(Ala³⁰³Asp); ◊, LigA(Ala³⁴⁹Val); □, LigA(Ala³⁷³Thr).