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

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 (ßleavinggroup 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.

Transition-State Interactions in a Promiscuous Enzyme: Sulfate and Phosphate Monoester Hydrolysis by Pseudomonas aeruginosa Arylsulfatase.

Pseudomonas aeruginosa arylsulfatase (PAS) hydrolyzes sulfate and, promiscuously, phosphate monoesters. Enzyme-catalyzed sulfate transfer is crucial to a wide variety of biological processes, but detailed studies of the mechanistic contributions to its catalysis are lacking. We present linear free energy relationships (LFERs) and kinetic isotope effects (KIEs) of PAS and analyses of active site mutants that suggest a key role for leaving group (LG) stabilization. In LFERs PASWT has a much less negative Brønsted coefficient (ßleaving groupobs-Enz = -0.33) than the uncatalyzed reaction (ßleaving groupobs = -1.81). This situation is diminished when cationic active site groups are exchanged for alanine. The considerable degree of bond breaking during the transition state (TS) is evidenced by an 18Obridge KIE of 1.0088. LFER and KIE data for several active site mutants point to leaving group stabilization by active site K375, in cooperation with H211. 15N KIEs and the increased sensitivity to leaving group ability of the sulfatase activity in neat D2O (Δßleaving groupH-D = +0.06) suggest that the mechanism for S-Obridge bond fission shifts, with decreasing leaving group ability, from charge compensation via Lewis acid interactions toward direct proton donation. 18Ononbridge KIEs indicate that the TS for PAS-catalyzed sulfate monoester hydrolysis has a significantly more associative character compared to the uncatalyzed reaction, while PAS-catalyzed phosphate monoester hydrolysis does not show this shift. This difference in enzyme-catalyzed TSs appears to be the major factor favoring specificity toward sulfate over phosphate esters by this promiscuous hydrolase, since other features are either too similar (uncatalyzed TS) or inherently favor phosphate (charge).

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

Highly proficient, promiscuous enzymes can be springboards for functional evolution, able to avoid loss of function during adaptation by their capacity to promote multiple reactions. We employ a systematic comparative study of structure, sequence, and substrate specificity to track the evolution of specificity and reactivity between promiscuous members of clades of the alkaline phosphatase (AP) superfamily. Construction of a phylogenetic tree of protein sequences maps out the likely transition zone between arylsulfatases (ASs) and phosphonate monoester hydrolases (PMHs). Kinetic analysis shows that all enzymes characterized have four chemically distinct phospho- and sulfoesterase activities, with rate accelerations ranging from 1011- to 1017-fold for their primary and 109- to 1012-fold for their promiscuous reactions, suggesting that catalytic promiscuity is widespread in the AP-superfamily. This functional characterization and crystallography reveal a novel class of ASs that is so similar in sequence to known PMHs that it had not been recognized as having diverged in function. Based on analysis of snapshots of catalytic promiscuity "in transition", we develop possible models that would allow functional evolution and determine scenarios for trade-off between multiple activities. For the new ASs, we observe largely invariant substrate specificity that would facilitate the transition from ASs to PMHs via trade-off-free molecular exaptation, that is, evolution without initial loss of primary activity and specificity toward the original substrate. This ability to bypass low activity generalists provides a molecular solution to avoid adaptive conflict.

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

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.

An efficient, multiply promiscuous hydrolase in the alkaline phosphatase superfamily.

We report a catalytically promiscuous enzyme able to efficiently promote the hydrolysis of six different substrate classes. Originally assigned as a phosphonate monoester hydrolase (PMH) this enzyme exhibits substantial second-order rate accelerations ((k(cat)/K(M))/k(w)), ranging from 10(7) to as high as 10(19), for the hydrolyses of phosphate mono-, di-, and triesters, phosphonate monoesters, sulfate monoesters, and sulfonate monoesters. This substrate collection encompasses a range of substrate charges between 0 and -2, transition states of a different nature, and involves attack at two different reaction centers (P and S). Intrinsic reactivities (half-lives) range from 200 days to 10(5) years under near neutrality. The substantial rate accelerations for a set of relatively difficult reactions suggest that efficient catalysis is not necessarily limited to efficient stabilization of just one transition state. The crystal structure of PMH identifies it as a member of the alkaline phosphatase superfamily. PMH encompasses four of the native activities previously observed in this superfamily and extends its repertoire by two further activities, one of which, sulfonate monoesterase, has not been observed previously for a natural enzyme. PMH is thus one of the most promiscuous hydrolases described to date. The functional links between superfamily activities can be presumed to have played a role in functional evolution by gene duplication.

Examining the promiscuous phosphatase activity of Pseudomonas aeruginosa arylsulfatase: a comparison to analogous phosphatases.

Pseudomonas aeruginosa arylsulfatase (PAS) is a bacterial sulfatase capable of hydrolyzing a range of sulfate esters. Recently, it has been demonstrated to also show very high proficiency for phosphate ester hydrolysis. Such proficient catalytic promiscuity is significant, as promiscuity has been suggested to play an important role in enzyme evolution. Additionally, a comparative study of the hydrolyses of the p-nitrophenyl phosphate and sulfate monoesters in aqueous solution has demonstrated that despite superficial similarities, the two reactions proceed through markedly different transition states with very different solvation effects, indicating that the requirements for the efficient catalysis of the two reactions by an enzyme will also be very different (and yet they are both catalyzed by the same active site). This work explores the promiscuous phosphomonoesterase activity of PAS. Specifically, we have investigated the identity of the most likely base for the initial activation of the unusual formylglycine hydrate nucleophile (which is common to many sulfatases), and demonstrate that a concerted substrate-as-base mechanism is fully consistent with the experimentally observed data. This is very similar to other related systems, and suggests that, as far as the phosphomonoesterase activity of PAS is concerned, the sulfatase behaves like a "classical" phosphatase, despite the fact that such a mechanism is unlikely to be available to the native substrate (based on pK(a) considerations and studies of model systems). Understanding such catalytic versatility can be used to design novel artificial enzymes that are far more proficient than the current generation of designer enzymes.

Vector redesign and in-droplet cell-growth improves enrichment and recovery in live Escherichia coli.

Directed evolution (DE) is a widely used method for improving the function of biomolecules via multiple rounds of mutation and selection. Microfluidic droplets have emerged as an important means to screen the large libraries needed for DE, but this approach was so far partially limited by the need to lyse cells, recover DNA, and retransform into cells for the next round, necessitating the use of a high-copy number plasmid or oversampling. The recently developed live cell recovery avoids some of these limitations by directly regrowing selected cells after sorting. However, repeated sorting cycles used to further enrich the most active variants ultimately resulted in unfavourable recovery of empty plasmid vector-containing cells over those expressing the protein of interest. In this study, we found that engineering of the original expression vector solved the problem of false positives (i.e. plasmids lacking an insert) cells containing empty vectors. Five approaches to measure activity of cell-displayed enzymes in microdroplets were compared. By comparing various cell treatment methods prior to droplet sorting two things were found. Substrate encapsulation from the start, that is prior to expression of enzyme, showed no disadvantage to post-induction substrate addition by pico-injection with respect to recovery of true positive variants. Furthermore in-droplet cell growth prior to induction of enzyme production improves the total amount of cells retrieved (recovery) and proportion of true positive variants (enrichment) after droplet sorting.

Ancestral sequences of a large promiscuous enzyme family correspond to bridges in sequence space in a network representation.

Evolutionary relationships of protein families can be characterized either by networks or by trees. Whereas trees allow for hierarchical grouping and reconstruction of the most likely ancestral sequences, networks lack a time axis but allow for thresholds of pairwise sequence identity to be chosen and, therefore, the clustering of family members with presumably more similar functions. Here, we use the large family of arylsulfatases and phosphonate monoester hydrolases to investigate similarities, strengths and weaknesses in tree and network representations. For varying thresholds of pairwise sequence identity, values of betweenness centrality and clustering coefficients were derived for nodes of the reconstructed ancestors to measure the propensity to act as a bridge in a network. Based on these properties, ancestral protein sequences emerge as bridges in protein sequence networks. Interestingly, many ancestral protein sequences appear close to extant sequences. Therefore, reconstructed ancestor sequences might also be interpreted as yet-to-be-identified homologues. The concept of ancestor reconstruction is compared to consensus sequences, too. It was found that hub sequences in a network, e.g. reconstructed ancestral sequences that are connected to many neighbouring sequences, share closer similarity with derived consensus sequences. Therefore, some reconstructed ancestor sequences can also be interpreted as consensus sequences.

High-Throughput, Lysis-Free Screening for Sulfatase Activity Using Escherichia coli Autodisplay in Microdroplets.

Directed evolution of enzymes toward improved catalytic performance has become a powerful tool in protein engineering. To be effective, a directed evolution campaign requires the use of high-throughput screening. In this study we describe the development of an ultra high-throughput lysis-free procedure to screen for improved sulfatase activity by combining microdroplet-based single-variant activity sorting with E. coli autodisplay. For the first step in a 4-step screening procedure, we quantitatively screened >105 variants of the homodimeric arylsulfatase from Silicibacter pomeroyi (SpAS1), displayed on the E. coli cell surface, for improved sulfatase activity using fluorescence activated droplet sorting. Compartmentalization of the fluorescent reaction product with living E. coli cells autodisplaying the sulfatase variants ensured the continuous linkage of genotype and phenotype during droplet sorting and allowed for direct recovery by simple regrowth of the sorted cells. The use of autodisplay on living cells simplified and reduced the degree of liquid handling during all steps in the screening procedure to the single event of simply mixing substrate and cells. The percentage of apparent improved variants was enriched >10-fold as a result of droplet sorting. We ultimately identified 25 SpAS1 variants with improved performance toward 4-nitrophenyl sulfate (up to 6.2-fold) and/or fluorescein disulfate (up to 30-fold). In SpAS1 variants with improved performance toward the bulky fluorescein disulfate, many of the beneficial mutations occur in residues that form hydrogen bonds between α-helices in the C-terminal oligomerization region, suggesting a previously unknown role for the dimer interface in shaping the substrate binding site of SpAS1.

Inactivation of epoxide hydrolase by catalysis-induced formation of isoaspartate.

Epoxide hydrolases catalyze hydrolytic epoxide ring-opening, most often via formation of a covalent hydroxyalkyl-enzyme intermediate. A mutant of Agrobacterium radiobacter epoxide hydrolase, in which the phenylalanine residue that flanks the invariant catalytic aspartate nucleophile is replaced by a threonine, exhibited inactivation during conversion when the (R)-enantiomer of para-nitrostyrene epoxide was used as substrate. HPLC analysis of tryptic fragments of the epoxide hydrolase, followed by MALDI-TOF and TOF/TOF analysis, indicated that inactivation was due to conversion of the nucleophilic aspartate into isoaspartate, which represents a novel mechanism of catalysis-induced autoinactivation. Inactivation occurred at a lower rate with the (S)-enantiomer of para-nitrostyrene epoxide, indicating that it is related to the structure of the covalent hydroxyalkyl-enzyme intermediate.

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

Hydrolysis of organic sulfate esters proceeds by two distinct mechanisms, water attacking at either sulfur (S-O bond cleavage) or carbon (C-O bond cleavage). In primary and secondary alkyl sulfates, attack at carbon is favored, whereas in aromatic sulfates and sulfated sugars, attack at sulfur is preferred. This mechanistic distinction is mirrored in the classification of enzymes that catalyze sulfate ester hydrolysis: arylsulfatases (ASs) catalyze S-O cleavage in sulfate sugars and arylsulfates, and alkyl sulfatases break the C-O bond of alkyl sulfates. Sinorhizobium meliloti choline sulfatase (SmCS) efficiently catalyzes the hydrolysis of alkyl sulfate choline-O-sulfate (kcat/KM=4.8×103s-1M-1) as well as arylsulfate 4-nitrophenyl sulfate (kcat/KM=12s-1M-1). Its 2.8-Å resolution X-ray structure shows a buried, largely hydrophobic active site in which a conserved glutamate (Glu386) plays a role in recognition of the quaternary ammonium group of the choline substrate. SmCS structurally resembles members of the alkaline phosphatase superfamily, being most closely related to dimeric ASs and tetrameric phosphonate monoester hydrolases. Although >70% of the amino acids between protomers align structurally (RMSDs 1.79-1.99Å), the oligomeric structures show distinctly different packing and protomer-protomer interfaces. The latter also play an important role in active site formation. Mutagenesis of the conserved active site residues typical for ASs, H218O-labeling studies and the observation of catalytically promiscuous behavior toward phosphoesters confirm the close relation to alkaline phosphatase superfamily members and suggest that SmCS is an AS that catalyzes S-O cleavage in alkyl sulfate esters with extreme catalytic proficiency.

Directed evolution of epoxide hydrolase from A. radiobacter toward higher enantioselectivity by error-prone PCR and DNA shuffling.

The enantioselectivity of epoxide hydrolase from Agrobacterium radiobacter (EchA) was improved using error-prone PCR and DNA shuffling. An agar plate assay was used to screen the mutant libraries for activity. Screening for improved enantioselectivity was subsequently done by spectrophotometric progress curve analysis of the conversion of para-nitrophenyl glycidyl ether (pNPGE). Kinetic resolutions showed that eight mutants were obtained with up to 13-fold improved enantioselectivity toward pNPGE and at least three other epoxides. The large enhancements in enantioselectivity toward epichlorohydrin and 1,2-epoxyhexane indicated that pNPGE acts as an epoxyalkane mimic. Active site mutations were found in all shuffled mutants, which can be explained by an interaction of the affected amino acid with the epoxide oxygen or the hydrophobic moiety of the substrate. Several mutations in the shuffled mutants had additive effects.

Catalytic promiscuity in Pseudomonas aeruginosa arylsulfatase as an example of chemistry-driven protein evolution.

In recent years, it has become increasingly clear that many enzymes are catalytically "promiscuous". This can provide a springboard for protein evolution, allowing enzymes to acquire novel functionality without compromising their native activities. We present here a detailed study of Pseudomonas aeruginosa arylsulfatase (PAS), which catalyzes the hydrolysis of a number of chemically distinct substrates, with proficiencies comparable to that towards its native reaction. We demonstrate that the main driving force for the promiscuity is the ability to exploit the electrostatic preorganization of the active site for the native substrate, providing an example of chemistry-driven protein evolution.

A new member of the alkaline phosphatase superfamily with a formylglycine nucleophile: structural and kinetic characterisation of a phosphonate monoester hydrolase/phosphodiesterase from Rhizobium leguminosarum.

The alkaline phosphatase superfamily comprises a large number of hydrolytic metalloenzymes such as phosphatases and sulfatases. We have characterised a new member of this superfamily, a phosphonate monoester hydrolase/phosphodiesterase from Rhizobium leguminosarum (R/PMH) both structurally and kinetically. The 1.42 A crystal structure shows structural homology to arylsulfatases with conservation of the core alpha/beta-fold, the mononuclear active site and most of the active-site residues. Sulfatases use a unique formylglycine nucleophile, formed by posttranslational modification of a cysteine/serine embedded in a signature sequence (C/S)XPXR. We provide mass spectrometric and mutational evidence that R/PMH is the first non-sulfatase enzyme shown to use a formylglycine as the catalytic nucleophile. R/PMH hydrolyses phosphonate monoesters and phosphate diesters with similar efficiency. Burst kinetics suggest that substrate hydrolysis proceeds via a double-displacement mechanism. Kinetic characterisation of active-site mutations establishes the catalytic contributions of individual residues. A mechanism for substrate hydrolysis is proposed on the basis of the kinetic data and structural comparisons with E. coli alkaline phosphatase and Pseudomonas aeruginosa arylsulfatase. R/PMH represents a further example of conservation of the overall structure and mechanism within the alkaline phosphatase superfamily.

Diversity and biocatalytic potential of epoxide hydrolases identified by genome analysis.

Epoxide hydrolases play an important role in the biodegradation of organic compounds and are potentially useful in enantioselective biocatalysis. An analysis of various genomic databases revealed that about 20% of sequenced organisms contain one or more putative epoxide hydrolase genes. They were found in all domains of life, and many fungi and actinobacteria contain several putative epoxide hydrolase-encoding genes. Multiple sequence alignments of epoxide hydrolases with other known and putative alpha/beta-hydrolase fold enzymes that possess a nucleophilic aspartate revealed that these enzymes can be classified into eight phylogenetic groups that all contain putative epoxide hydrolases. To determine their catalytic activities, 10 putative bacterial epoxide hydrolase genes and 2 known bacterial epoxide hydrolase genes were cloned and overexpressed in Escherichia coli. The production of active enzyme was strongly improved by fusion to the maltose binding protein (MalE), which prevented inclusion body formation and facilitated protein purification. Eight of the 12 fusion proteins were active toward one or more of the 21 epoxides that were tested, and they converted both terminal and nonterminal epoxides. Four of the new epoxide hydrolases showed an uncommon enantiopreference for meso-epoxides and/or terminal aromatic epoxides, which made them suitable for the production of enantiopure (S,S)-diols and (R)-epoxides. The results show that the expression of epoxide hydrolase genes that are detected by analyses of genomic databases is a useful strategy for obtaining new biocatalysts.

Substrate specificity engineering of beta-mannosidase and beta-glucosidase from Pyrococcus by exchange of unique active site residues.

A beta-mannosidase gene (PH0501) was identified in the Pyrococcus horikoshii genome and cloned and expressed in E. coli. The purified enzyme (BglB) was most specific for the hydrolysis of p-nitrophenyl-beta-D-mannopyranoside (pNP-Man) (Km: 0.44 mM) with a low turnover rate (kcat: 4.3 s(-1)). The beta-mannosidase has been classified as a member of family 1 of glycoside hydrolases. Sequence alignments and homology modeling showed an apparent conservation of its active site region with, remarkably, two unique active site residues, Gln77 and Asp206. These residues are an arginine and asparagine residue in all other known family 1 enzymes, which interact with the catalytic nucleophile and equatorial C2-hydroxyl group of substrates, respectively. The unique residues of P. horikoshii BglB were introduced in the highly active beta-glucosidase CelB of Pyrococcus furiosus and vice versa, yielding two single and one double mutant for each enzyme. In CelB, both substitutions R77Q and N206D increased the specificity for mannosides and reduced hydrolysis rates 10-fold. In contrast, BglB D206N showed 10-fold increased hydrolysis rates and 35-fold increased affinity for the hydrolysis of glucosides. In combination with inhibitor studies, it was concluded that the substituted residues participate in the ground-state binding of substrates with an equatorial C2-hydroxyl group, but contribute most to transition-state stabilization. The unique activity profile of BglB seems to be caused by an altered interaction between the enzyme and C2-hydroxyl of the substrate and a specifically increased affinity for mannose that results from Asp206.