Journal
FEBS JOURNAL
Volume 282, Issue 13, Pages 2512-2526Publisher
WILEY
DOI: 10.1111/febs.13293
Keywords
aminotransferase; biocatalysis; directed evolution; substrate specificity; transaminase
Categories
Funding
- Biotechnology and Biological Sciences Research Council (BBSRC) [BB/G016593/1]
- Engineering and Physical Sciences Research Council [GR/S02532/01, 1210475] Funding Source: researchfish
- BBSRC [BB/G016593/1] Funding Source: UKRI
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We have analyzed the natural evolution of transaminase structure and sequence between an -transaminase serine-pyruvate aminotransferase and an -transaminase from Chromobacteriumviolaceum with <20% sequence identity, and identified the active-site regions that are least conserved structurally. We also show that these structural changes correlate strongly with transaminase substrate specificity during evolution and therefore might normally be presumed to be essential determinants of substrate specificity. However, key residues are often conserved spatially during evolution and yet originate from within a different region of the sequence viastructural reorganizations. In the present study, we also show that -transaminase-type serine-pyruvate aminotransferase activity can be engineered into the CV2025 -transaminase scaffold with any one of many possible single-point mutations at three key positions, without the requirement for significant backbone remodeling, or repositioning of the residue from a different region of sequence. This finding has significant implications for enzyme redesign in which solutions to substrate specificity changes may be found more efficiently than is achieved by engineering in all sequence and structure determinants identified by correlation to substrate specificity.
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