Deep mutational scanning (DMS) has recently emerged as a powerful method to study protein sequence− function relationships but is not well-explored as a guide to enzyme engineering and identifying of pathways by which their catalytic cycle may be improved. We report such a demonstration in this work using a phenylalanine ammonia-lyase (PAL), which deaminates L-phenylalanine to trans-cinnamic acid and has widespread application in chemoenzymatic synthesis, agriculture, and medicine. In particular, the PAL from Anabaena variabilis (AvPAL*) has garnered significant attention as the active ingredient in Pegvaliase, the only FDA-approved drug for treating classical phenylketonuria (PKU). Although an extensive body of literature exists on the structure, substrate-specificity, and catalytic cycle, protein-wide sequence determinants of function remain unknown, as do intermediate reaction steps that limit turnover frequency, which has hindered the rational engineering of these enzymes. Here, we created a detailed sequence−function landscape of AvPAL* by performing DMS and revealed 112 mutations at 79 functionally relevant sites that affect a positive change in enzyme fitness. Using fitness values and structure−function analysis, we picked a subset of positions for comprehensive single-and multi-site saturation mutagenesis and identified combinations of mutations that led to improved reaction kinetics in cell-free and cellular contexts. We then performed quantum mechanics/molecular mechanics (QM/MM) and molecular dynamics (MD) studies to understand the mechanistic role of the most beneficial mutations and observed that different mutants confer improvements via different mechanisms, including stabilizing transition and intermediate states, improving substrate diffusion into the active site, and decreasing product inhibition. This work demonstrates how DMS can be combined with computational analysis to effectively identify significant mutations that enhance enzyme activity along with the underlying mechanisms by which these mutations confer their benefit.

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(i) Saccharomyces cerevisiae grown in the presence of 1.0 mM l -tryptophan slowly excreted fluorescent material that was chromatographically identifiable as 3-hydroxyanthranilate but did not excrete detectable amounts of anthranilate nor rapidly deplete the medium of l -tryptophan. Under similar growth conditions, Neurospora crassa rapidly excretes anthranilate and rapidly depletes the medium of l -tryptophan. (ii) Chromatographic analysis of crude extracts from yeast revealed a single kynureninase-type enzyme whose synthesis was not measurably affected by the presence of tryptophan in the medium. Previous studies have provided evidence for two kynureninase-type enzymes in N. crassa , an inducible kynureninase and a constitutive hydroxykynureninase. (iii) Kinetic analysis of the partially purified yeast enzyme provided Michaelis constants for l -3-hydroxykynurenine and l -kynurenine of 6.7 × 10 −6 and 5.4 × 10 −4 M, respectively. This and other kinetic properties of the yeast enzyme are comparable to those reported for the constitutive enzyme from N. crassa . (iv) These findings suggest that S. cerevisiae has in common with N. crassa the biosynthetic enzyme hydroxykynureninase but lacks the catabolic enzyme kynureninase. Therefore, it can be predicted that, unlike N. crassa, S. cerevisiae does not carry out the tryptophan-anthranilate cycle. Distinct kynureninase-type enzymes may exist in other microorganisms and in mammals.