Definition of an Index Parameter To Screen Highly Functional Enzymes Derived from a Biochemical and Thermodynamic Analysis of Ancestral meso‐Diaminopimelate Dehydrogenases

Sequence‐based protein design approaches are being adopted to generate highly functional enzymes; however, screening the enzymes remains a time‐consuming task. In this study, by analyzing the enzymatic properties of four ancestral meso‐2,6‐diaminopimelate dehydrogenases (AncDAPDHs), AncDAPDH‐N1, ‐N2, ‐N3, and ‐N4, we attempted to define a new index parameter that is helpful for efficiently screening the enzymes. Biochemical and thermodynamic analyses indicated that only AncDAPDH‐N4 exhibited greater thermal stability than and activity similar to those of native DAPDHs. Structural and sequence comparisons between DAPDH from Corynebacterium glutamicum (CgDAPDH) and the AncDAPDHs suggested that “quality of mutations” is a potential index parameter. In fact, the mutations introduced from CgDAPDH to AncDAPDH‐N4 correlated highly with the mutations accumulated during the evolution process from mesophiles to thermophiles. These results suggest that, although there are several exceptions, the correlation coefficient can be used as an index parameter for screening high‐functioning enzymes from sequence data. Ancestral sequence reconstruction (ASR) is broadly applied to protein engineering; however, screening highly functional enzymes from the designed sequences remains a time‐consuming task. The new index parameter defined in this study (the balance in the bubble) helps to efficiently screen the highly functional ancestral enzymes (represented as “positive bioCATalyst”). Illustration by Yui Kawamura.