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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 (ðk cat ∕K M Þ∕ 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.catalytic promiscuity | evolution | mechanism | sulfatase | superfamily E nzymes are usually seen as highly specific catalysts following the classical rule "one enzyme, one activity." This view is challenged by an increasing number of enzymes with broad substrate specificities or side activities indicating that enzymes are catalytically more flexible than originally assumed. Some of these promiscuous enzymes can turn over substrates with different structures while catalyzing the same chemical reaction involving the same transition state (substrate promiscuity). Catalytic promiscuity, by contrast, is the ability of an enzyme to catalyze chemically distinct reactions by stabilization of different transition states (TSs) (1). Catalytic efficiencies (k cat ∕K M ) for the promiscuous substrates are often substantially lower (2 to 9 orders of magnitude) than for the native conversions (1-3). The growing number of examples of this phenomenon (1-4) has engendered excitement on a number of fronts. Catalytic promiscuity provides a functional basis for enzyme evolution by gene duplication. The initial head-start activity of the duplicated gene copy could support an immediate selective advantage (1, 2, 5, 6), to be followed by improvement of the initially weak activity (7). Even low k cat ∕K M values for a promiscuous function can support a significant selective advantage (8). Mimicking this evolutionary shortcut could also provide a more efficient route to changing the function of proteins by directed evolution (5).We describe multiple and efficient catalytic promiscuity in an enzyme from Burkh...

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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 ␣/␤-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.Enantiopure epoxides and vicinal diols are valuable intermediates in the synthesis of a number of pharmaceutical compounds. Epoxide hydrolases (EC 3.3.2.3) catalyze the conversion of epoxides to the corresponding diols. If they are enantioselective, they can be used to produce enantiopure epoxides by means of kinetic resolution (5). In the past, when only epoxide hydrolases from mammalian sources were known (12), the use of epoxide hydrolases in biocatalysis was hampered by their poor availability and insufficient catalytic performance, such as a low turnover rate or poor enantioselectivity. The potential for biocatalytic application of epoxide hydrolases was significantly increased with the discovery of microbial epoxide hydrolases (41), which are easier to produce in large quantities. The cloning and overexpression of several enantioselective epoxide hydrolases, e.g., from Agrobacterium radiobacter (35), Aspergillus niger (3), and potato plants (40), not only facilitated large-scale production of these enzymes but also made it possible to improve their biocatalytic properties by site-directed or random mutagenesis (34,36,43).Since many microbial genome sequences are available in the public domain, it is useful to screen these databases for genes that might encode new enzymes with interesting properties.Novel epoxide hydrolases can be identified by performing a BLAST search of the genomic databases, using amino acid sequences of known epoxide hyd...

a b s t r a c tIn 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.