Evolutionary repurposing of a sulfatase: A new Michaelis complex leads to efficient transition state charge offset

Abstract

The recruitment and evolutionary optimization of promiscuous enzymes is key to the rapid adaptation of organisms to changing environments. Our understanding of the precise mechanisms underlying enzyme repurposing is, however, limited: What are the active-site features that enable the molecular recognition of multiple substrates with contrasting catalytic requirements? To gain insights into the molecular determinants of adaptation in promiscuous enzymes, we performed the laboratory evolution of an arylsulfatase to improve its initially weak phenylphosphonate hydrolase activity. The evolutionary trajectory led to a 100,000-fold enhancement of phenylphosphonate hydrolysis, while the native sulfate and promiscuous phosphate mono- and diester hydrolyses were only marginally affected (≤50-fold). Structural, kinetic, and in silico characterizations of the evolutionary intermediates revealed that two key mutations, T50A and M72V, locally reshaped the active site, improving access to the catalytic machinery for the phosphonate. Measured transition state (TS) charge changes along the trajectory suggest the creation of a new Michaelis complex (E•S, enzyme-substrate), with enhanced leaving group stabilization in the TS for the promiscuous phosphonate ( from -1.08 to -0.42). Rather than altering the catalytic machinery, evolutionary repurposing was achieved by fine-tuning the molecular recognition of the phosphonate in the Michaelis complex, and by extension, also in the TS. This molecular scenario constitutes a mechanistic alternative to adaptation solely based on enzyme flexibility and conformational selection. Instead, rapid functional transitions between distinct chemical reactions rely on the high reactivity of permissive active-site architectures that allow multiple substrate binding modes.

Evolution of new enzymes by gene duplication and divergence

“…These examples show that promiscuous activities that are orders of magnitude less efficient than typical metabolic enzymes can serve as the starting point for evolution of new enzymes in nature. In the laboratory, even less‐efficient promiscuous activities suffice; a promiscuous phenylphosphonatase activity of an arylsulfatase with a k cat / K M of only 0.015 m −1 ·s −1 was improved 10 5 ‐fold by successive rounds of directed evolution [59].…”

“…The finding that optimization is difficult is not limited to natural evolution of new enzymes. Efforts to evolve new enzymes by directed evolution commonly encounter a pattern of diminishing returns in which initial mutations have large effects on efficiency, but later mutations make progressively smaller contributions [59,66,67]. However, multiple rounds of directed evolution sometimes succeed in evolving enzymes with catalytic efficiencies comparable to well‐evolved enzymes.…”

“…Molecular dynamics simulations suggest that Phe68 stacks upon the p ‐nitrophenyl group of the paraoxon, improving its orientation relative to the attacking water. Similarly, a 100 000‐fold improvement of a promiscuous phenylphosphonate hydrolase activity of an aryl sulfatase was achieved by directed evolution, primarily due to two mutations that enlarged the active site while leaving the positions of five catalytic residues unchanged [59]. Molecular dynamics simulations suggest that the substrate in the evolved enzyme binds closer to the active site nucleophile and a Lys that stabilizes the developing negative charge on the leaving group, and is better positioned for attack by the nucleophile.…”

How evolution shapes enzyme selectivity – lessons from aminoacyl‐tRNA synthetases and other amino acid utilizing enzymes

“…However, trade‐offs can be also modest, thus resulting in enzymes with low selectivity as illustrated in the roughly dashed ocher trajectory in Fig. (e.g., a recent directed evolution trajectory indicated 10 5 ‐fold gain in the evolving function at the expense of only 400‐fold loss in the native one ). Further, specialization is a slow process that does not readily lead to high selectivity.…”

Higher-order epistatic networks underlie the evolutionary fitness landscape of a xenobiotic-degrading enzyme

“…The effects of nonproductive binding have previously been described many proteins, including chymotrypsin and an unrelated bacterial phosphotriesterase 30,48 . This is also reminiscent of recent work that demonstrated how laboratory evolution of an arylsulfatase was able to cause a 100,000-fold increase in phosphonate-monoester hydrolase activity by enlarging the active site and repositioning the substrate 49 . In such cases, the structural consequences of mutations can be subtle in the active site, with sub-Ångström distance and angle adjustments resulting in optimized catalytic machinery, causing profound changes in catalytic efficiency and specificity.…”