Specificity Effects of Amino Acid Substitutions in Promiscuous Hydrolases: Context‐Dependence of Catalytic Residue Contributions to Local Fitness Landscapes in Nearby Sequence Space

Abstract

Catalytic promiscuity can facilitate evolution of enzyme functions—a multifunctional catalyst may act as a springboard for efficient functional adaptation. We test the effect of single mutations on multiple activities in two groups of promiscuous AP superfamily members to probe this hypothesis. We quantify the effect of site‐saturating mutagenesis of an analogous, nucleophile‐flanking residue in two superfamily members: an arylsulfatase (AS) and a phosphonate monoester hydrolase (PMH). Statistical analysis suggests that no one physicochemical characteristic alone explains the mutational effects. Instead, these effects appear to be dominated by their structural context. Likewise, the effect of changing the catalytic nucleophile itself is not reaction‐type‐specific. Mapping of “fitness landscapes” of four activities onto the possible variation of a chosen sequence position revealed tremendous potential for respecialization of AP superfamily members through single‐point mutations, highlighting catalytic promiscuity as a powerful predictor of adaptive potential.

Structural and Mechanistic Analysis of the Choline Sulfatase from Sinorhizobium melliloti: A Class I Sulfatase Specific for an Alkyl Sulfate Ester

“…S1, Table 1 ) was considerably lower than for choline- O -sulfate ( 1a ). Mutations in the active-site residues that are conserved between Sm CS and Sp AS1 [26] , [55] decreased catalytic efficiencies toward 4-nitrophenyl sulfate hydrolysis ( Table 2 ), confirming that Sm CS, and not a contaminating enzyme, is catalyzing the hydrolysis of aryl sulfates. The activity toward sulfate monoester 1b is modest compared to the catalytic efficiencies of many of its family members ( k cat / K M ~ 10 3 –10 7 s − 1 M − 1 ).…”

“…Based on the mutagenesis of the active site residues and their similarity to the active site residues in related ASs and PMHs ( Fig. 3 , Table S7), we propose that the catalytic cycle for Sm CS-catalyzed hydrolysis of choline- O -sulfate is similar to that of the AS-catalyzed hydrolysis of aryl sulfates [26] , [55] , [58] and the Rl PMH-catalyzed hydrolysis of phosphodiesters [52] . The specificity toward choline- O -sulfate is determined by the interaction of a negatively charged glutamate (Glu386) with the positively charged quaternary ammonium group of the substrate ( Fig.…”

“…A defining feature of the phospho- and sulfohydrolases of the AP superfamily is the ability to catalyze multiple hydrolytic reactions with substantial rate accelerations [42] , [81] . The observation of promiscuity has been named “crosswise”; that is, the primary activities of the various family members are also promiscuous reactions in other family members [26] , [41] , [42] , [43] , [44] , [45] , [46] , [47] , [48] , [55] . Sm CS had previously been shown to be active toward phosphoryl choline ( 2a , Fig.…”

Balancing Specificity and Promiscuity in Enzyme Evolution: Multidimensional Activity Transitions in the Alkaline Phosphatase Superfamily

“…If improvements in phosphoesterase activity are selected for, escape from this correlation appears to be possible, but not in all directions, as shown recently for SpAS1. 26 As a result, sulfatases with low phosphodiesterase activity (i.e. enzymes located in the top left corner in Figure 8A) We explored these scenarios based on our new data for correlations of the main activity of PMHs ( Figure 7A1-A4) and for the new ASs ( Figure 7B1-B7).…”

Challenges and advances in the computational modeling of biological phosphate hydrolysis

“…In addition, several important experimental studies recently focused on, e.g., the AP superfamily architecture or interdependence of the catalytic residues. [104][105][106][107] 4 Modeling the evolution of organophosphate hydrolases…”