Table S1.

Enzyme kinetic parameter estimates for modern-day and ancestral xanthine alkaloid-producing enzymes with selected substrates

Enzyme (+substrate)	KM (µM)	kcat (1/s)	kcat/KM (s−1⋅M−1)
Modern-day enzymes
TcCS1 (X)	95.8	8.37E-05	0.87
TcCS2 (3X)	49.1	9.81E-05	2.00
PcCS1 (X)	95.38	1.52E-03	15.94
PcCS2 (3X)	677	9.33E-04	1.38
CsXMT1 (1X)	657.6	6.32E-04	0.97
CsXMT1 (3X)	1,144	2.99E-04	0.26
CsXMT1 (X)	759	8.74E-05	0.12
Ancestral enzymes
CsAncXMT2 (X)	2,740	2.48E-04	0.09
CsAncXMT2 P25S (X)	1,690	3.97E-04	0.23