Catalytic Promiscuity of Ancestral Esterases and Hydroxynitrile Lyases

Titu Devamani, Alissa M. Rauwerdink, Mark Lunzer, Bryan J. Jones, Joanna L. Mooney, Maxilmilien Alaric O Tan, Zhi Jun Zhang, Jian He Xu, Antony M. Dean, Romas J. Kazlauskas

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Abstract

Catalytic promiscuity is a useful, but accidental, enzyme property, so finding catalytically promiscuous enzymes in nature is inefficient. Some ancestral enzymes were branch points in the evolution of new enzymes and are hypothesized to have been promiscuous. To test the hypothesis that ancestral enzymes were more promiscuous than their modern descendants, we reconstructed ancestral enzymes at four branch points in the divergence hydroxynitrile lyases (HNL's) from esterases ∼100 million years ago. Both enzyme types are α/β-hydrolase-fold enzymes and have the same catalytic triad, but differ in reaction type and mechanism. Esterases catalyze hydrolysis via an acyl enzyme intermediate, while lyases catalyze an elimination without an intermediate. Screening ancestral enzymes and their modern descendants with six esterase substrates and six lyase substrates found higher catalytic promiscuity among the ancestral enzymes (P < 0.01). Ancestral esterases were more likely to catalyze a lyase reaction than modern esterases, and the ancestral HNL was more likely to catalyze ester hydrolysis than modern HNL's. One ancestral enzyme (HNL1) along the path from esterase to hydroxynitrile lyases was especially promiscuous and catalyzed both hydrolysis and lyase reactions with many substrates. A broader screen tested mechanistically related reactions that were not selected for by evolution: decarboxylation, Michael addition, γ-lactam hydrolysis and 1,5-diketone hydrolysis. The ancestral enzymes were more promiscuous than their modern descendants (P = 0.04). Thus, these reconstructed ancestral enzymes are catalytically promiscuous, but HNL1 is especially so.

Original languageEnglish (US)
Pages (from-to)1046-1056
Number of pages11
JournalJournal of the American Chemical Society
Volume138
Issue number3
DOIs
StatePublished - Jan 27 2016

Bibliographical note

Funding Information:
This work was supported by NIH grant 5R01GM102205 and NSF grant 1152804 to A.M.D. and R.J.K. and China Scholarship Council grant to Z.J.Z.

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