Structure and Catalytic Mechanism of a Bacterial Friedel–Crafts Acylase

Abstract C−C bond‐forming reactions are key transformations for setting up the carbon frameworks of organic compounds. In this context, Friedel–Crafts acylation is commonly used for the synthesis of aryl ketones, which are common motifs in many fine chemicals and natural products. A bacterial multicomponent acyltransferase from Pseudomonas protegens (PpATase) catalyzes such Friedel–Crafts C‐acylation of phenolic substrates in aqueous solution, reaching up to >99 % conversion without the need for CoA‐activated reagents. We determined X‐ray crystal structures of the native and ligand‐bound complexes. This multimeric enzyme consists of three subunits: PhlA, PhlB, and PhlC, arranged in a Phl(A2C2)2B4 composition. The structure of a reaction intermediate obtained from crystals soaked with the natural substrate 1‐(2,4,6‐trihydroxyphenyl)ethanone together with site‐directed mutagenesis studies revealed that only residues from the PhlC subunits are involved in the acyl transfer reaction, with Cys88 very likely playing a significant role during catalysis. These structural and mechanistic insights form the basis of further enzyme engineering efforts directed towards enhancing the substrate scope of this enzyme.


Introduction
Acyltransferases belong to the EC 2.3 groups of enzymes. This subclass contains enzymest hat transfer an acyl group froma donor compound to the hydroxy,a mino, or mercapto group of an acceptorc ompound, forming an ester,a mide, or thioester. Different acyltransferase classes have been identified in living organismst hat utilize, fori nstance, 1-O-acylglucosides, [1] acylated acyl carrierp roteins, [2] or quinic acid ester [3] as high-energy activateda cyl donors. The most abundant group of acyltransferasesmostly require acyl-CoA derivatives as donor substrates, catalyzing various reactions involved in primary and secondary metabolism. [4] In bacteria, CoA-dependent O-andN -acylation play ak ey role in detoxificationo fa ntibiotics such as chloramphenicol, [5] aminoglycosides, [6] streptothricin, [7] and phosphino-thricin. [8] Acyltransferases are also involved in numerousb iosynthetic pathways, especially in the biosynthesis of membrane phospholipids, [9] of wax esters and triacylglycerols, [10] and of polyketides, [11] and also playar ole in lysozyme resistance. [12] In contrastt oO -a nd N-acylation, natural C-acylation reactions are scarce. So far,o nly ac ouple of acyltransferases, for example, from Pseudomonas protegens (PpATase) and Pseudomonas fluorescens have been reported to catalyzeC ÀCb ond formation,i nt he biosynthesis of the antibiotically active polyketide 1,1'-(2,4,6-trihydroxybenzene-1,3-diyl)bisethanone (diacetylphloroglucinol, DAPG). [13] The biosynthesis of DAPG is regulated by the phlACBDEFGHI gene cluster,w hich is divided into regulatory genes phlEFGHI and the biosynthetic operon phlACBD. [14] For many years, biocatalytic applications were limited to gene phlD, which encodes at ype-III polyketide synthase that was employed for the in vivo production of the DAPG precursor benzene-1,3,5-triol (phloroglucinol, PG) either in Pseudomonas sp. or in Escherichia coli under controlledc onditions in bioreactors. [13b, 15] Very recently,t he scope of applicationsh as been extended to phlACB, which encodes the cofactor-independenta cyltransferase PpATase referred to above. Thisc atalyzes the reversible disproportionation of 1-(2,4,6-trihydroxyphenyl)ethanone (monoacetylphloroglucinol, MAPG) into PG and DAPG in ad ivergent reaction (Scheme 1). PpATase is am ulticomponent enzymea nd is catalytically active without addition of cofactors such as CoA or ATP. Af unctional enzyme is only obtained upon expression of the entire phlABC operon: mixing and incubation of the individually expressed proteins-PhlA,P hlB, and PhlC-with MAPG did not lead to its disproportionation. [13a, 16] CÀCb ond-formingr eactions are key transformations for setting up the carbon frameworks of organic compounds. In this context,F riedel-Crafts acylationi sc ommonly used for the synthesis of aryl ketones,w hich are common motifs in many fine chemicals and natural products. Ab acterial multicomponent acyltransferase from Pseudomonas protegens (PpATase) catalyzes such Friedel-Crafts C-acylation of phenolic substrates in aqueous solution, reaching up to > 99 %c onversion without the need for CoA-activated reagents. We determined X-ray crystal structures of the native and ligand-bound complexes.
This multimeric enzyme consists of three subunits:P hlA, PhlB, and PhlC, arranged in aP hl(A 2 C 2 ) 2 B 4 composition. The structure of ar eactioni ntermediate obtained from crystalss oaked with the naturals ubstrate 1-(2,4,6-trihydroxyphenyl)ethanone together with site-directed mutagenesis studies revealed that only residues from the PhlC subunits are involved in the acyl transfer reaction, with Cys88 very likely playing as ignificant role during catalysis. These structurala nd mechanistici nsights form the basis of further enzymee ngineering efforts directed towardsenhancing the substrate scope of this enzyme.
It was shown that PpATase acceptsv arious C-or O-acyl donors such as isopropenyl acetate and transfersa na cetyl moiety to ap henolic acceptori naFriedel-Crafts-type acetylation. [16b, 17] The enzyme complex also shows chemical reaction promiscuity anda ccepts aniline derivatives as substrates for amide formation. [18] Here we report on the crystal structure determination of PpATase in its substrate-free form, as well as after soaking of the crystalsw ith MAPG. The structuralr esults indicatet hat only one of the three subunits-PhlC-is involved in catalyzing the acylation reaction. Site-directed mutagenesis studies and activity measurements complement the structural data and enable ac atalytic mechanism to be proposed.

Results and Discussion
Overall structure We determinedX -ray crystal structureso faC oA-independent acyltransferase produced by P. protegens DSM 19095 (PpATase) using diffraction data from two different crystal forms at 2.8 and 3.4 resolution, respectively (Table 2i nt he Experimental Sectiona nd Figure S1 in the Supporting Information). The structuresw ere solved by molecular replacementi nvolving extensived ensity modification as wella sa utomated and manual rebuilding( see the Experimental Section). It had been known before that PpATase is am ultimeric enzyme consisting of three subunits-PhlA, PhlB, and PhlC. [19] The final structures showed the hexagonal crystal form (space group P6 1 22) to contain two copies of each of those subunits in the asymmetric unit and the orthorhombic crystal form (space group P2 1 2 1 2 1 )t oc ontain eight copiesofe ach subunit.
In both structures, an analysis of protein-protein interactions within the crystal by using the EBI-Pisa server [20] yielded ah eterododecamer with four copieso fe ach subunit as the most likely biologically active oligomer of PpATase. Thus, the hexagonal crystal form contains half ad odecamer in the asymmetric unit with the other half being generated by ac rystallographic diad. The orthogonal crystals, on the other hand, contain two copies of the full dodecameri nt he asymmetricu nit. Closer inspection of the inter-chain contacts indicated that PhlAa nd PhlC each form strongly interacting homodimers in the crystal ( Figure S2). The composition of the multimeric enzymec omplex is thus best described as Phl(A 2 C 2 ) 2 B 4 ,i nw hich the four copies of PhlB mediate the binding of two PhlA andt wo PhlC dimers ( Figure 1B)t of ormt he complete oligomer ( Figure 1A and B).
Ac avitya nalysis identified four,l arge, contiguous cavities per dodecamer,l ined by residues from one particular copy of PhlA, PhlB, and PhlC. In this heterotrimeric arrangementt he proposed active sites of PhlA andP hlC (see below)a re adjacent to each other on one side of the cavity,w hereas PhlB (especially through al ong loop formed by residues Glu74 to Val87) closes the cavity on the opposite side ( Figure 1C).
Previous sequence analyses had suggested that PhlA could be involved in the first step of DAPG biosynthesis:t hat is, the formation of acetoacetyl-CoA from acetyl-CoA. [25] Inspections of the cavity observed in our structures in the vicinity of PhlA suggestst hat there is indeede nough space fora cetyl-CoA binding. However, key residues necessary to catalyzet his Claisen-type condensation (a cysteine and ag lutamic acid residue) are not presenti nP hlA. Comparison with homologous enzymes shows the crucial cysteiner esidue corresponding to Gly115 in PhlA and the active-site glutamate residue aligning with Cys83 in PhlA. We attempted too btain ac omplex struc-Scheme1.Natural reaction catalyzed by the P. protegens acyltransferase (PpATase) involved in the biosynthesis of DAPG. ture by soaking PpATase with acetyl-CoA, but the soakedc rystal did not diffract sufficiently wellf or structure determination.
Structural analysis of PhlB suggested ac lose similarity to a protein of unknown functionf rom Sulfolobus solfataricus (PDB ID:3 IRB, rmsd 2.6 ,s eq-id:1 8%). [26] This protein fromt he DUF35 family (Pfam PF01796) exhibits at wo-domaina rchitecture consisting of an N-terminal, rubredoxin-like zinc ribbon and aC -terminal oligonucleotide/oligosaccharide-binding (OB) fold domain [27] with an additional N-terminal helical segment possibly involved in protein-protein interactions [26] (Figure S3). For members of this protein family,ageneral role in fatty acid and polyketide biosynthesis as acyl-CoA-binding proteins has been predicted. [26] Althoughasimilar domain organization is observed in PhlB, and the zinc ribbonw ith its Cys 4 metal-ion-binding site is conserved, severe differences are evident in the OB domain and at the Nterminus. Instead of two N-terminal helices,P hlB exhibits an elongated N-terminal tail and as hort, kinked a-helix. In the dodecameric arrangement of PpATase, this region of PhlB (up to Arg28) is not accessible to the solvent but buried within the enzyme complex structure, where it is involved in tight interactions with the adjacent PhlA and PhlC molecules, as well as with the N-terminal tail of an eighboring PhlB molecule (Figure 1B). The same is true for the two long loop regions within the OB domain (residues 73 to 89 and 127 to 136).
Finally,t he sequence of PhlC shows that it belongs to the thiolases uperfamily, [28] although sequence identities to members of this enzyme family are below 45 %. The structure of this subunit exhibits an a/b-hydrolase-type fold and is most similar to structures of the thiolase-like protein ST0096f rom Sulfolobus tokodaii (PDB ID:4 YZO, rmsd 1.6 ,s eq-id:2 6%) and the SCP2 thiolase from Trypanosoma brucei (PDB ID:4 BI9, rmsd 1.7 ,s eq-id:2 4%). [29] In thiolases, ac ysteine and ah istidine residue (corresponding to Cys88 andH is347i nP hlC) are highly conserved and were found to be important for the enzymatic reaction. Beside those two residues,t he active-site cavity of PhlC is lined by amino acid residues His56, Asn87, His144, Trp211, Tyr298, and Ser349, which might play ar olei n substrate binding or catalysis ( Figure S7). Even in the untreated (hexagonal) crystal, residual density was observed at the side chain of Cys88, thus indicating that this residue is at least partially acetylated.
Recently,t he structure of an archaeal acetoacetyl-CoA thiolase/HMG-CoA synthase (HMGCS)c omplex from Methanothermococcus thermolithotrophicus was reported. [30] The arrangement of the individual protein subunits within this complex is very similart ot hat in PpATase:t he HMGCS subunit corresponds to PhlA in PpATase (sequence identity3 8.5 %), whereas the thiolases ubunit corresponds to PhlC (sequence identity 28.9 %). The complex also contains ap rotein from the DUF35 family that is relatedt oP hlB in PpATase (sequence identity 26.7 %). Sequence alignments of the three separates ubunits are shown in Figures S4-S6. In contrast to the PpATase,h owever,t his complex utilizes acetyl-CoA as an acyl donora nd is involved in the mevalonate pathway. The HMGCS subunit is involved in the exergonic condensation of acetoacetyl-CoA and acetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA. The key Cys and Glu residues necessary to catalyzet his Claisen-type condensation are indeed presenti nH MGCS (Cys114 and Glu82),w hereas they are missing in PhlA as mentioneda bove ( Figure S8). The presence of the active Cys residue in the thiolase subunit is preserved in both complexes (Cys88 for PhlC and Cys85 in the thiolase subunit). Soakingo ft he thiolase/ HMGCSc omplex with acetyl-CoA (PDB ID:6 ESQ) revealed the bindingo fC oA at the subunit interface comprised by residues from all three proteins. [30] This region is significantly different from its counterpart in the structure of PpATase (FigureS9), showinga ltered relative positions of individual secondary structure elements, the presence of additional residues in the PhlB region of PpATase, and two additional short b-strands in HMGCS, as well as al ack of CoA-binding residues in PpATase. The overall fold of PhlB is very similar to that in the protein from the DUF35 family in the thiolase/HMGCS complex (Figure S3). The elongated Nterminus of PhlB that is buried within the PpATasec omplex structure, however,i sc ompletely missing in the other structure, andn otable conformational changes are observed in the opposite loop region.

Structure of the complex with MAPG
In order to identify the subunit responsible for the observed transferase activity,w eperformed crystal-soaking experiments. Orthorhombic crystals of PpATase were soaked with the native acetyl donor/acceptor MAPG, and the structure of the complex was solved at 3.4 resolution. Residual density was observed in the vicinity of residue Cys88 in four out of the eight crystallographically independent copies of the PhlC subunit (Figure S10). This density was compatiblew ith an acetylated Cys88 residue and with a( deacetylated) PG molecule bound at this site ( Figure 2). The resulting structurer evealed that only residues from PhlC interact with the bound PG.S pecifically,t he side chains of His56 and of Tyr124 and His347 form hydrogen bonds with the hydroxy groups at positions 1a nd 3, respectively,o ft he aromatic ring ( Figure 2). In contrast, the hydroxy group at position 5i sa pparently not involved in any direct interactions with PhlC. Instead, it points towards as mall cavity lined by mostly hydrophobic residues (such as Phe148, Leu209, and Leu300).
The carbonylo xygen atom of the Cys88-bound acetyl group is positioned in the "oxyanion hole" of the a/b-hydrolase-type fold formingh ydrogen bondsw ith the main-chain amide groups of Cys88 itself and of Gly385.A dditional polar stabilization might be provided by the helix dipole oriented favorably towardsC ys88 (the "nucleophile elbow"p resenti nt his type of fold [31] ). The acetyl group is oriented more or less parallel to the aromatic ring, and its carbonyl carbon atom is appropriately positioned for an electrophilica ttack on the C-6 atom of the substrate, with distances between the two atoms ranging from 3t o3 .5 ( Figure 2). Its methyl group points towards Phe148.
Whereas the conformationso fm ost residues forming the active site of PhlC are similar in the structures, Trp211i np articular stands out because it adopts an open conformation in the untreated crystal and closes the cavity upon substrate binding in the soaked crystal ( Figure 2).

Mutagenesis of PhlC active-siteresidues
The analysis of the PhlC active site identified Cys88, His144, Asn87,H is56,S er349, Tyr124, Tyr298, Asp352,H is347,a nd Trp211a sag roup of mostly polar or charged residues lining the cavity ( Figure S7). Those residues were selected for site-directed mutagenesis, and we analyzed the activity of the corresponding enzyme variants in the acetylation of resorcinol (1) with use of isopropenyl acetate (IPEA) as acetyl donora nd imidazole (see the Experimental Section). The resultsa re shown in Ta ble 1i nt he form of relative amountso ft he reactant 1,t he C-acetylation product 4-acetylresorcinol (2), and the O-acetylation product 2-(3-hydroxyphenyl)acetate (3).

Proposedmechanism of acyl transfer
PpATase catalyzes the reversible acetylation of MAPG into PG and DAPG. [13] It has been shownt hat only am ulticomponent complex consisting of PhlA, PhlB, and PhlC subunits catalyzes the disproportionation of MAPG. [13a, 16] The crystal structures presented here revealed that only residues from PhlC interact with the bound PG, thus strongly suggesting that PhlA and PhlB are not directly involved in the actual acyl transfer step. Instead, these subunitsa re very likely required for the formation of ap roperly folded and functionald odecameric enzyme complex. This finding also supports previous suggestions relating to their possible involvement in preceding steps of DAPG biosynthesis. [25] Combining the structural results with activity data obtained for enzyme variants (Table 1) allows ap lausible catalytic mechanism to be proposed (Scheme 2). The observation of an ace-tylatedC ys88 residue in both crystal structures and the lack of activity measured for the cysteine-to-alanine variant indicates that Cys88 very likely playsasignificant role during catalysis. We suggest that the thiol group of Cys88 attacks the acyl Table 1. Activity of PpATase variants for the Friedel-Crafts acetylationo f benzene-1,3-diol (resorcinol, 1)a nd specific activity for the natural reaction.

PpATase
Acetylation with IPEA/Im [  moiety of the donor and subsequently forms ac ovalenti ntermediate similar to the acyl-enzymei ntermediates formed by serine or cysteineh ydrolases. [32] As in these hydrolases, the nucleophilic attack is facilitated by stabilization of the ensuing negative chargea tt he acyl oxygen atom by polar interactions within the "oxyanion hole". Therei sn oc lear indication of a base, which could activate the thiol by deprotonation, in the vicinity of Cys88. It is reasonable, however,t hat the side chain already be deprotonatedt os ome extent at neutral or slightly basic pH values. Additional stabilization of the thiolate is possible through polar interactions with the OH group of Tyr298 and the imidazole moiety of His347,a lthough the distances to both groups are longer( > 3.6 )t han observed for typical hydrogen bonds. The proposed formation of an acyl-enzyme intermediate is also consistent with our previousf inding that conversionsw ith DAPG/MAPG as acetyl donor did not yield any phenyl acetate derivatives and that the enzyme also catalyzes an intermolecular Fries rearrangement. [16b] The second step of the reaction involves the transfer of the acyl moiety from Cys88 to the aromatic ring of an acceptor molecule. As discussed above,t he carbonyl carbon of the cysteine-bound acetyl group is appropriately positioned to attack the C-6 atom of the bound PG in the complex structure (Scheme 2). Electronic activation of the aromatic ring most likely involves deprotonationo ft he phenolic OH group(s) at C-1 (by His56)a nd/or at C-3 (by the diad His347-Asp352). We have previously shown that, in addition to PG, resorcinol-but not phenol-can act as an acyl acceptor, [16b] thus indicating that at least two OH groups (at positions 1a nd 3) are necessary for substrate binding and/or activity.I nt he structure, the OH group at C-5 of PG does not participate in any polar interactions with the enzyme and it can indeed be replaced by other (alkyl) substituents in PpATase substrates. [16b] The rearomatization step through an intramolecular protont ransfer very likely does not involve any enzymeintervention.
PhlC of PpATase differs from other,m ore common thiolases by the presence of the tryptophan residue Trp211. This residue appearst oh ave al id function, because its conformation significantly changes upon bindingo ft he substrate (Scheme 2). This conformational flexibility couldv ery welli nfluence the activity and above all the substrate specificity of the enzyme. With regard to substitutions at the aromatic ring, the active-site cavity appears to provide more space for attachment at C-4 than at C-5. This is in good agreement with the observedp ref-erence for substituents at C-4 of the resorcinol core structure over those at the C-5 position. [16b]

Conclusion
The presented structural characterization of the multicomponent acytransferase PpATase reveals ac losei nteraction of all three enzyme subunits PhlA, PhlB, and PhlC. However,t he CÀC bond formation withoutt he utilization of CoA is performed through the action only of the PhlC subunit. This structural information provides ab asis for developing libraries of catalysts tailoredf or specific chemical substrates. Our goal is to extend the substrate scope by using established protein engineering techniques.

Experimental Section
Expression and purification:T he CoA-independent acyltransferase from Pseudomonas protegens DSM 19095 (PpATase) was expressed in E. coli as described previously [16b] from ap lasmid containing codon-optimized open reading frames (ORFs) coding for the three subunits of the enzyme:P hlA, PhlB, and PhlC. The nucleotide sequence of the expression plasmid is available from GenBank through the accession number KY173355. Purification of the enzyme was achieved by size-exclusion chromatography as described previously. [16b] Crystallization and soaking:S creening for crystallization conditions was performed with an Oryx8 crystallization robot (Douglas Instruments). Initial trials were set up by employing the sittingdrop vapor-diffusion method in 96-well plates with Index HT (Hampton Research), JCSG + ,a nd Morpheus (Molecular Dimensions) screens. As tock solution of PpATase [12 mg mL À1 in potassium phosphate buffer (pH 7.5, 50 mm)] was used for all crystallization experiments. Both in initial screens and in subsequent optimizations, drops (1 mL) were set with a1 :1 ratio of protein and precipitant solution. The crystallization plates were incubated at 289 K.
Crystal clusters of PpATase were readily obtained under several sets of conditions. Microseed-matrix-screening experiments [33] were set up with initial crystals obtained under conditions Index #2 and #81 as seeding stock Data collection, processing, structure determination, and analysis:D ata were collected at 100 Ko ns ynchrotron beamlines ID23-1 and ID30B (ESRF,G renoble, France) [34] from an untreated crystal (hexagonal, space group P6 1 22) and from ac rystal soaked with MAPG (orthorhombic, space group P2 1 2 1 2 1 ), to crystallographic resolutions of approximately 2.8 and 3.4 ,r espectively.D iffraction data were processed and scaled by using the XDS package. [35] Initial automated molecular replacement attempts with Balbes [36] and the better resolved hexagonal dataset indicated the dimer of 3-hydroxy-3-methylglutaryl-coenzyme As ynthase from Staphylococcus aureus (PDB ID:1 TVZ, 23 %s equence identity) [37] as as uitable search template for PhlA. Ah omology model of PhlC was generated with the aid of the Phyre2 server [19] with the structure of SCP2 thiolase from Leishmania mexicana (PDB ID:3 ZBG, 27 %s equence identity) [29] as template. Molecular replacement was continued within the CCP4 suite [38] by using the program Phaser. [39] By fixing the previously positioned 1TVZ dimer (template for PhlA), two copies of the PhlC homology model could be placed in the asymmetric unit. Density modification based on phases from this partial model by using the program Resolve [40] yielded well-defined electron density for the whole PpATase complex, including density for two missing PhlB molecules. Manual rebuilding of PhlA and PhlC was continued with the aid of the program Coot, [41] and the improved model was then subjected to automated rebuilding with the program Buccaneer. [42] The resulting model containing two copies of each of PhlA, PhlB, and PhlC was completed manually in Coot and refined by using the PHENIX software suite. [43] The structure of the PpATase soaked with MAPG was solved by molecular replacement with ap art of the previously determined hexagonal structure (one copy of each of PhlA, PhlB, and PhlC) as search template. Structure solution resulted in eight copies of this trimeric arrangement in the asymmetric unit of the orthorhombic unit cell. Structure refinement was continued in the same manner as described above, with the programs Coot and PHENIX. Clear difference electron density was observed in all eight chains of PhlC in the vicinity of residue Cys88. In four of those chains we interpreted this density as am olecule of phloroglucinol. Additional density around Cys88 was interpreted as an acetyl group covalently attached to the Sg atom of this amino acid residue.
For both structures, validation was performed with the program MolProbity. [44] Data collection and refinement statistics are summarized in Ta ble 2.
All structure-related figures were generated by using PyMOL (http://www.pymol.org). Structures were superimposed with the SSM Superposition tool [22] as implemented in Coot. Cavities were identified in the final structures by using the LIGSITE algorithm [45] as implemented in the CaSoX plugin for PyMOL. The analysis of the hydrophobicity of these cavities utilized the corresponding function of the program VASCo. [46] Site-directed mutagenesis and activity measurements:V ariants of PpATase were prepared in order to investigate the roles of selected amino acid residues in the enzymatic reaction. Gene mutations were introduced with the aid of the QuickChange II site-directed mutagenesis kit (Agilent Genomics) according to the standard procedure provided by the supplier without modifications. All primer sequences and plasmids used in this study are collated in Ta ble S1. The following variants, each carrying as ingle amino acid exchange within phlC,w ere generated:H 56A, H56S, N87A, C88A, C88S, H144A, H144S, W211A, W211F,Y 298A, Y298V,Y 298F,H 347F, S349A, and D352V.A ll variants were expressed as described for the wild-type enzyme [16b] and were used as cell-free extracts in the activity measurements (Table 1).
The activities of wild-type PpATase and of its variants were tested with the acetylation of benzene-1,3-diol (resorcinol) under the following conditions:R esorcinol (10 mm final concentration in the reaction mixture) was dissolved in potassium phosphate buffer (pH 7.5, 50 mm)a nd preheated to 35 8Cf or 10 min. Cell-free extracts of the recombinant enzymes (30 mg protein) were subsequently added to the preheated mixture. The bioacetylation was started by addition of imidazole (100 mm,a dded from a1m stock solution prepared in the reaction buffer,causing the pH to increase to 8.30), followed by the addition of isopropenyl acetate (IPEA, 100 mm). To ensure proper suspension of the donor in the mixture, Table 2. Crystal structure of PpATase, data collection, andr efinement statistics.

PpATase
PpATase hexagonal [a] orthorhombic (soaked) [ the vessel was manually shaken thoroughly right after starting the reaction. The reaction mixture (1 mL total volume) was horizontally shaken for 1.5 ha t3 5 8Ca nd 750 rpm in an orbital shaker.R eactions were terminated by addition of acetonitrile (1 mL). The precipitated protein was removed by centrifugation (18 407 g,1 0min), and the supernatant (900 mL) was transferred to an Eppendorf tube and left standing for another 40 min. Any residual precipitated protein was once again removed by centrifugation, and the supernatant was directly subjected to HPLC for determination of degree of conversion. The relative amounts of resorcinol, the Cacetylation product 4-acetylresorcinol, and the O-acetylation product 2(3-hydroxyphenyl)acetate were determined by HPLC from standard curves with authentic samples. Each reaction was performed as ad uplicate. Specific activities were measured with aT hermo Scientific Genesys 10 UV Scanning UV/Vis spectrophotometer according to a modified procedure from the literature. [13a] When the disproportionation of MAPG into DAPG and PG is followed spectrophotometrically,a nincrease of absorption due to the formation of DAPG (e = 20 mm À1 cm À1 , l = 370 nm) is recorded. One unit of activity was defined as 1 mmol of product formed by an enzyme in 1min per 1milligram of protein under the following conditions:p otassium phosphate buffer (pH 7.5, 100 mm,9 60 mL) and MAPG (1.2 mmol, 30 mLo fa40 mm stock solution prepared in DMSO) were added to ac uvette and preheated to 35 8C. The reaction (1 mL total volume, 3vol% DMSO) was started by the addition of the enzyme-containing cell-free extract (10 mL). The reaction was followed for 1minute. All reactions were performed in duplicate. The protein concentration was measured with Bradford reagent (e = 0.083 mL mg À1 cm À1 , l = 595 nm), and specific activities were determined as units per mg protein.