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This result reveals a very limited number of routes to increase substrate range.
Introduction of each mutation individually does not expand the substrate range of β-lactamases57.
A.S. supervised protein crystallization and contributed to interpreting mechanism of expanded substrate range.
vCYC/CDK6 complexes furthermore exhibit an extended substrate range and are relatively refractory to inhibition by CDK inhibitors29.
MphB is another homolog also reported to have a narrow substrate range, modifying erythromycin but not azithromycin44.
Therefore, the equivalent residues different between Ancestor 1 (MphI and MphK) and Ancestor 2 (broad substrate range) may be determinants of substrate specificity46.
All variants with an expanded substrate range were identical at three residue positions: Ala40, Glu166, and Leu211 (mutants of MphI Met40, Thr166, Pro211).
For example, combinations of mutations located outside the β-lactam binding pocket of β-lactamases expand substrate range by increasing conformational flexibility50,56,57,58.
Across Nature, their substrate range is vast and exceeds that of other enzymes.
After overexpression in Escherichia coli, the purified protein was shown to have a broad substrate range including adipate.
By contrast, residue 335 does not contact biphenyl but contributes significantly to expansion of the enzyme's substrate range.
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