Coevolutionary Information Captures Catalytic Functions and Reveals Divergent Roles of Terpene Synthase Interdomain Connections

Inferring the historical
and biophysical causes of diversity within
protein families is a complex puzzle. A key to unraveling this problem
is characterizing the rugged topography of sequence–function
adaptive landscapes. Using biochemical data from a 29 =
512 combinatorial library of tobacco 5-epi-aristolochene
synthase (TEAS) mutants engineered to make the native major product
of Egyptian henbane premnaspirodiene synthase (HPS) and a complementary
512 mutant HPS library, we address the question of how product specificity
is controlled. These data sets reveal that HPS is far more robust
and resistant to mutations than TEAS, where most mutants are promiscuous.
We also combine experimental data with a sequence Potts Hamiltonian
model and direct coupling analysis to quantify mutant fitness. Our
results demonstrate that the Hamiltonian captures variation in product
outputs across both libraries, clusters native family members based
on their substrate specificities, and exposes the divergent catalytic
roles of couplings between the catalytic and noncatalytic domains
of TEAS versus HPS. Specifically, we found that the role of the interdomain
connectivities in specifying product output is more important in TEAS
than connectivities within the catalytic domain. Despite being 75%
identical, this property is not shared by HPS, where connectivities
within the catalytic domain are more important for specificity. By
solving the X-ray crystal structure of HPS, we assessed structural
bases for their interdomain network differences. Last, we calculate
the product profile Shannon entropies of the two libraries, which
showcases that site–site connectivities also play divergent
roles in catalytic accuracy.