Aclacinomycin oxidoreductase (AknOx) from thebiosynthetic pathway of the antibiotic aclacinomycinis an unusual flavoenzyme with a dual active siteIgor Alexeev*, Azmiri Sultana†, Pekka Mantsala*, Jarmo Niemi*‡, and Gunter Schneider†‡

*Department of Biochemistry and Food Chemistry, University of Turku, FIN-20014, Turku, Finland; and †Department of Medical Biochemistryand Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden

Edited by Christopher T. Walsh, Harvard Medical School, Boston, MA, and approved February 16, 2007 (received for review January 22, 2007)

Aclacinomycin (Acl) oxidoreductase (AknOx) catalyzes the last twosteps in the biosynthesis of polyketide antibiotics of the Acl group,the oxidation of the terminal sugar moiety rhodinose to L-aculose.We present the crystal structure of AknOx with bound FAD and theproduct AclY, refined to 1.65-Å resolution. The overall fold ofAknOx identifies the enzyme as a member of the p-cresol methyl-hydroxylase superfamily. The cofactor is bicovalently attached toHis-70 and Cys-130 as 8�-N�1-histidyl, 6-S-cysteinyl FAD. Thepolyketide ligand is bound in a deep cleft in the substrate-bindingdomain, with the tetracyclic ring system close to the enzymesurface and the three-sugar chain extending into the proteininterior. The terminal sugar residue packs against the isoalloxazinering of FAD and positions the carbon atoms that are oxidized closeto the N5 atom of FAD. The structure and site-directed mutagenesisdata presented here are consistent with a mechanism where thetwo different reactions of AknOx are catalyzed in the same activesite but by different active site residues. Tyr-450 is responsible forproton removal from the C-4 hydroxyl group in the first reaction,the oxidation of rhodinose to cinerulose A. Tyr-378 acts as acatalytic base involved in proton abstraction from C3 of cineruloseA in the second reaction, for formation L-aculose. Replacement ofthis residue, however, does not impair the conversion of rhodinoseto cinerulose A.

enzyme mechanism � polyketide antibiotic � protein crystallography �protein structure

Aclacinomycins (Acl) are well studied aromatic polyketides,produced as secondary metabolites by several strains of the

genus Streptomyces (1). They are of considerable medical interestbecause of their potent anticancer activity; for instance, AclA isused for the treatment of acute leukemia and non-Hodgkin lym-phoma (2). Aklavinone, the aglycone moiety of the Acl, is the mostcommon precursor in anthracycline biosynthetic pathways in gen-eral, including the daunorubicin and rhodomycin families (3).

Acl contain a trisaccharide moiety attached to aklavinone at theC-7 position. The first two sugar residues in Acl are rhodosamineand 2-deoxyfucose, although they differ structurally in their thirdsugar component, which is rhodinose in AclN, cinerulose A inAclA, L-aculose in AclY (Fig. 1), or cinerulose B in AclB (4). Acloxidoreductase (AknOx) is a secreted flavin-dependent enzyme,which is involved in the modification of the terminal sugar residuesin the biosynthesis of Acl. It converts the rhodinose moiety of AclNto cinerulose A in AclA by oxidizing the hydroxyl group at carbonC4 to a keto group. In the next biosynthetic step, AknOx convertsAclA to AclY by eliminating two hydrogen atoms from cineruloseA (Fig. 1). AknOx was first isolated and purified from Streptomycesgalilaeus MA144-M1 (ATCC 31133) (5) and requires FAD ascofactor and molecular oxygen as second substrate. SecretedAknOx from S. galilaeus ATCC 31615, which we describe in thispaper, contains 502 amino acid residues with a molecular mass of54.8 kDa. The enzyme is synthesized as a precursor containing asignal sequence of 43 amino acids at the N terminus responsible forextracellular secretion.

This paper focuses on structural and functional features ofAknOx, in particular overall fold, binding of ligands, and relation toother members of this enzyme family. Structural insights from theternary complex with bound FAD and product in combination withthe mutagenesis studies presented here allow a proposal of thecatalytic mechanism of the enzyme. In particular, we show thatAknOx utilizes the same active site to catalyze two FAD-dependentconsecutive reactions in the same biosynthetic pathway. However,the enzyme uses two distinct sets of catalytic residues in the tworeactions, a feature that makes AknOx rather unique amongflavoenzymes.

Results and DiscussionIdentification of AknOx. We observed an AclA oxidizing activity inculture supernatants of ATCC 31615 as previously observed for S.galilaeus ATCC 31133 (5). The N-terminal amino acid sequence ofAknOx from the culture supernatant was found to be DGGAX-GARTALVKVDRVDRRYQDLV. A TBLAST search found asequence containing only three mismatches in the GenBank ac-cession no. AB008466 (Acl biosynthetic cluster from S. galilaeusATCC 31133), starting at nucleotide 18299. In this sequence file,gene aclJ is reported to end at 18104 and a putative oxidoreductaseaclO to begin at 18338 at Val-14 of the observed AknOx sequence.The sequence of the S. galilaeus ATCC 31615 Acl cluster was known

Author contributions: I.A. and A.S. contributed equally to this work; P.M., J.N., and G.S.,designed research; I.A. and A.S. performed research; I.A., A.S., J.N., and G.S. analyzed data;and I.A., A.S., P.M., J.N., and G.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Abbreviations: Acl, aclacinomycin; AknOx, Acl oxidoreductase; GOOX, glucooligosaccha-ride oxidase; PCMH, p-cresol methylhydroxylase.

Data deposition: The crystallographic data reported in this paper have been deposited withthe Protein Data Bank, www.pdb.org (PDB ID code 2IPI).

‡To whom correspondence may be addressed. E-mail: jarnie@utu.fi or gunter.schneider@ki.se.

This article contains supporting information online at www.pnas.org/cgi/content/full/0700579104/DC1.

© 2007 by The National Academy of Sciences of the USA

Fig. 1. Reactions catalyzed by AknOx, the conversion of AclN to AclY.

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up to the position corresponding to nucleotide 18192 (AF257324)(6). Inverse PCR and cloning of selected restriction fragments from�acm15 [a clone partially overlapping �acm12 (6)] lead to sequenc-ing of aknOx, the sequence of which has been added to AF257324[the homologous genes from ATCC 31615 and 31133 have beennamed akn and acl genes, respectively, but unfortunately aknO haspreviously been used for a different gene (6)].

The actual start of aknOx is most likely base 13513 of AF257324.The GTG starting codon is preceded by a putative ribosome-binding site, and the encoded amino acid sequence MFVLNEFT-RRGFLGTAAAVGGTTVVTTALGGAPAAQAAVPEAA be-fore the observed start of the extracellular protein contains thefeatures of a signal sequence for the TAT secretion system (7).

Quality of the Electron-Density Map and the Model. The structure ofthe ternary complex of AknOx with bound FAD and product AclYwas determined by multiwavelength dispersion methods and re-fined to 1.65-Å resolution. The electron density for most of thepolypeptide chain (except a less well defined surface loop regioncomprising residues 259–264) and the bound ligands (Figs. 2a and3a) is of excellent quality, as expected for this resolution. The finalmodel contains 492 amino acid residues of the total 502 residues foreach monomer (in total four polypeptide chains in the asymmetric

unit), four FAD molecules, one bound substrate molecule (chainA), and 1,009 water molecules. The N-terminal his-tag, togetherwith the first nine amino acids and the last C-terminal residue, wasnot visible in the electron density. The Ramachandran plot for thefinal model showed 85.3% residues in the most favored region, withno residues in the disallowed region.

Overall Structure of AknOx. The overall structure of AknOx can bedivided into two distinct domains, the F domain, which binds FAD,and the S domain, which provides most of the residues interactingwith the substrate (Fig. 4). The F domain, comprising residues1–209 and 467–502, can be divided into two � � � subdomains. TheN-terminal subdomain (residues 1–90) consists of a four-strandedmixed �-sheet (B1–B4), flanked on each side by one �-helix, H1 andH2, respectively. The second subdomain comprises residues 91–209and 467–502 that fold into a �-sheet of five antiparallel strands(B5–B9). The sheet packs on one side against four �-helices (H3,H4, H6, and H13) and one 310 helix (�2).

The substrate-binding domain (residue 227–430) is composed ofa seven-stranded antiparallel �-sheet (B10–B16), flanked on oneside by four �-helices (H7, H8, H10, and H11). A deep pocketextends from the protein surface into the interior of the moleculeand binds the trisaccharide chain of the polyketide ligand. Theflavin- and substrate-binding domains are connected by two loopregions containing the residues 210–226 (L1) and 431–466 (L2),respectively.

Quaternary Structure. The asymmetric unit of the AknOx crystalscontains four molecules of the enzyme that form two dimers (AB

Fig. 2. FAD binding in AknOx. (a) Stereoview of the 2Fo�Fc electron densitymap at the FAD-binding site, contoured at 1.0�. The covalently linked residuesHis-70 and Cys-130 are also shown. (b) Stereoview of the FAD-binding site inAknOx. FAD is shown in yellow, and the surrounding amino acids are coloredin green. Hydrogen bonds (distance �3.2 Å) are indicated by dotted lines. Redspheres indicate bound water molecules. For better clarity, b is rotated by�180° relative to a.

Fig. 3. Substrate binding in AknOx. (a) Stereoview of the electron density forthe bound ligand AclY, contoured at 1.0�. (b) Stereoview of the substrate-/product-binding site in AknOx. The product AclY is colored in blue/red, andthe surrounding residues are shown in standard colors. Possible catalyticresidues are colored in light orange. For better clarity, b is rotated by �90°relative to a.

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and CD). The subunits in each dimer are related by a moleculartwo-fold symmetry axis. In each dimer the subunit–subunit inter-face corresponds to a buried surface area of �1,300 Å2, typical forprotein–protein interactions rather than crystal packing. Gel filtra-tion experiments, however, indicate a monomeric species in solu-tion, and dimer formation may be due to the high protein concen-tration in the AknOx crystals.

FAD-Binding Site and Bicovalent Flavinylation in AknOx. The isoallox-azine ring of FAD is mainly bound at the interface of the flavin- andsubstrate-binding domains, whereas the ADP-ribosyl part is packedin a pocket between the two subdomains of the F-domain (Fig. 4).The FAD molecule interacts with the enzyme through a number ofmain- and side-chain hydrogen bonds (Fig. 2b).

The most distinguishing feature in the enzyme–FAD interactionsare the two covalent bonds from the isoalloxazine ring to the sidechains of His-70 and Cys-130, which give 8�-N�1-histidyl, 6-S-cysteinyl FAD (Fig. 2). The N�1 atom of His-70 is covalently boundwith the C8� of the isoalloxazine ring, and the C6 carbon atom ofthe ring is covalently linked to the thiol group of Cys-130. Doublecovalent attachment has so far been observed by crystallography inonly one other case, the structure of glucooligosaccharide oxidase(GOOX) (8). Biochemical studies suggested that S-reticuline oxi-dase (9) and hexose oxidase (10), two members of the same enzymefamily, also contain a similar double covalent linkage between FADand the enzyme. Sequence alignments show that the participatingresidues are conserved in these enzymes. The precise function ofthe covalent attachment remains, however, to be established.

Substrate/Product-Binding Site in AknOx. In one of the molecules inthe asymmetric unit of the crystal, clear electron density was foundin the substrate-binding domain, extending from the enzyme sur-face by a deep cleft into the interior of the protein (Fig. 3a).Although the crystals were grown in the presence of the substrate,AclA, the density best fitted the product, AclY, indicating that thereaction has proceeded during crystallization. The aromatic tetra-cyclic polyketide core is bound mainly by stacking interactions withtwo hydrophobic aromatic residues, Phe-339 and Trp-372, at theentrance of the substrate-binding groove of the S domain (Fig. 3b).The hydroxyl groups of ring A and B of the polyketide moiety areinvolved in hydrogen bonds with the backbone carbonyl oxygen ofThr-408. The trisaccharide chain of the ligand is inserted in thepocket leading to the active site and the atoms of the sugar residuesform a number of van der Waals contacts with several hydrophobicresidues (Phe-29, Phe-72, Phe-334, and Trp-404). The tertiaryamine of rhodosamine forms a hydrogen bond to the side chain of

Thr-408. The L-aculose moiety packs against the isoalloxazine ringof FAD, and positions carbon atoms C2, C3, and C4 close to(distances 3.7–3.9 Å) the N5 nitrogen of FAD, interactions withcatalytic implications (see below). Several other polar residues arein the vicinity of the bound ligand (Fig. 3b). The terminal carbo-hydrate moiety interacts through its keto oxygen atom with the sidechain of Tyr-450. Residues Tyr-378, Ser-376, and Glu-374 form aputative triad, which could be involved in the catalytic reaction(however, see below). Most of the residues that line the ligand-binding site are located in different loop regions, with the exceptionof a few amino acids (Glu-374 and Thr-408), which are positionedin the �-strands. It is of interest to note that the structures of theligand-free molecules (chains B, C, and D) superpose very well withthat of chain A that contains bound product (rmsd values for C�typically 0.4 Å). This observation suggests that the binding ofproduct does not induce large conformational transitions such as,for instance, domain–domain reorientations.

Comparison with Related Proteins. A BLAST search for homologsusing the AknOx sequence returned a number of sequences withamino acid identities �40%. Most of these homologous proteinswere annotated as FAD-dependent oxidoreductases and dehydro-genases. The majority of these homologs originated from differentStreptomyces species, all of unknown structure [supporting infor-mation (SI) Fig. 6]. In these sequences, His-70 is invariant, andCys-130, with one exception, is conserved, suggesting that thepattern of bicovalent attachment of the FAD cofactor is conservedalso in these enzyme species. Several of these enzymes are com-ponents of aromatic polyketide biosynthetic pathways and might beresponsible for sugar modifications similar to that catalyzed byAknOx. For instance, StfE (Q2P9Z3) from Streptomyces steffisbur-gensis (11) has been implicated as an enzyme oxidizing a ringhydroxyl to a keto group, as in the AclN3AclA reaction. SchA26(Q2HR11) participates in the biosynthesis of the angucyclinSch47554 (12). The product contains two L-aculose moieties, sug-gesting that SchA26 could catalyze a similar double oxidation asAknOx. SchA26 also appears to have a TAT signal sequence,pointing to extracellular location.

The Protein Data Bank was searched with the program Dali (13)using the AknOx coordinates. The closest structural homologs arethe flavin-dependent enzymes GOOX (8) (Z score 39.3, rmsd 2.3Å) and 6-hydroxy-D-nicotine oxidase (Z score 36.0, rmsd 2.7 Å)(14), members of the p-cresol methylhydroxylase (PCMH) super-family (15). Structural comparisons of the substrate-binding do-mains among the homologs of AknOx revealed significant devia-tions in primary and tertiary structure for this domain in the family,because of different architectures for the substrate-binding pocketsin these enzymes. The variations in active site topologies naturallyreflect the significant differences in substrate specificity and, to

Table 1. Relative enzymatic activities of AknOx mutants

AknOx mutants

Substrates

AclN AclA

Y450F 0.01–0.02 0.15–0.2Y144F 0.05 0.35H271A 1.0 1.5Y378F 1.0 0.0S376A 0.8–1.0 0.04E374A 0.8–1.0 1.0E374Q 0.8–1.0 1.0Y378F/Y450F 0.04 0.0Y378F/Y144F 0.0 0.0Y450F/Y144F 0.0 0.0Y378F/Y450F/Y144F 0.0 0.0

The enzymatic activity of native recombinant AknOx was set to 1.0.Fig. 4. Stereoview of the overall structure of AknOx. Helices are coloredpink, �-strands blue, and the two loops connecting the F and S domains lightblue. The bound ligands AclA (geen) and FAD (orange) are shown as stickmodels.

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some extent, chemistry in this enzyme family. For example, bothAknOx and GOOX (8) have an open accessible carbohydrate-binding pocket, which allows accommodation of larger oligosac-charides compared with the smaller substrate-binding sites ob-served in PCMH (16) or vanillyl-alcohol oxidase (17).

In Vitro Mutagenesis. Putative active site residues as identified fromthe structure of the ternary complex were probed by site-directedmutagenesis. The observed relative activities of these mutants,determined by the peroxidase-2,2�-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) assay are shown in Table 1. Wild-type AknOxshowed a Km � 8.5 �M and kcat � 0.17 sec�1 in the conversion ofAclA to AclY.

Implications for the Catalytic Mechanism. AknOx is unique in that itcatalyzes two consecutive steps in the biosynthesis of the polyketideantibiotic AclY. The first step is the conversion of the terminal

sugar residue rhodinose to cinerulose A, an oxidation of thehydroxyl at carbon C4 to a keto group. The second reaction is adesaturation, i.e., formally the abstraction of two hydrogen atomsleading to a double bond between carbon atoms C2 and C3 (Fig. 1).In both reactions, the reduced flavin is reoxidized by molecularoxygen, leading to hydrogen peroxide as the second product. Thestructure of AknOx described here is that of the enzyme-FAD-AclY ternary complex, with L-aculose rather than rhodinose as theterminal sugar residue. The structure of the complex provides asuitable template to model bound AclN, the substrate of the firstreaction of AknOx into the active site. In this model, the C4hydroxyl group is within hydrogen-bonding distance to the sidechain of Tyr-450, and the C4 hydrogen points toward the N5nitrogen atom of FAD (distance, 3.7 Å). The model suggests amechanism where Tyr-450 abstracts the proton from the C4hydroxyl group, with hydride transfer from the C4 carbon atom tothe N5 nitrogen atom (Fig. 5a). Hydride transfer rather than acarbanion mechanism is not proven for AknOx but is assumed hereon the basis that most of the FAD-dependent oxidases and dehy-drogenases act by a hydride transfer mechanism (18, 19). Protontransfer from Tyr-450 to the solvent can be facilitated by Tyr-144,which is in the vicinity of the side chain of Tyr-450 and couldparticipate in a proton transfer system (Fig. 5a). Replacement ofTyr-450 by phenylalanine leads to a severely impaired mutant,although there is still some minor residual activity (Table 1). Thisresidual activity could be attributed to either Tyr-378, which wouldbe able to abstract a proton from C3 of rhodinose leading to theproduction of a novel anthracycline, or to Tyr-144 removing theproton from the C4 hydroxyl group instead of Tyr-450. The doublemutant Tyr378Phe/Tyr450Phe showed activity comparable to thesingle mutant of Tyr450Phe, whereas the Tyr144Phe/Tyr450Phedouble mutant was inactive (Table 1). These observations suggestthat Tyr-144 is responsible for the residual activity of the Tyr450Phemutant and is able to substitute for Tyr-450 in proton abstraction.This is also consistent with structural data, i.e., the hydroxyl oxygenof the side chain of Tyr-144 is only 3.9 Å away from the C4 hydroxylof rhodinose.

In the structure of the ternary complex, Tyr-378 is the onlyputative catalytic base suitably positioned to initiate the secondreductive half reaction by abstraction of the proton from the carbonatom C3 of cinerulose A. This proton could be transferred by aproton relay system involving Ser-376 and Glu-374 toward theenzyme surface. Hydride transfer from C2 to FAD results in theformation of a C—C double bond and produces L-aculose (Fig. 5b).In the structure of the enzyme-FAD-AclY complex, the C2 carbonatom of the bound product, AclY, is located above the N5 nitrogenof FAD at a distance of 3.9 Å. Replacement of Tyr-378 byphenylalanine leads to complete loss of activity, consistent with anessential role of this residue in catalysis. It is particularly noteworthythat the Tyr378Phe mutant is completely active in the first reaction,the conversion of rhodinose to cinerulose A (Table 1). The pro-posed pathway for proton transfer from Tyr-378 by Ser-376 toGlu-374 does not require the latter, because replacement of Glu-374 to alanine does not affect catalytic activity. The drop in activityupon replacement of Ser-376 by alanine could reflect either less-efficient transfer of the proton to the solvent channel leading to theenzyme surface or requirement of the hydrogen bond interactionbetween Tyr-378 and Ser-376 for optimal positioning of the cata-lytic base. The inability of the double mutant Tyr144Phe/Tyr450Phe to use AclN as substrate for the second reactionindicates that the keto group of cinerulose A is required to activatethe C–H bond, which is broken in this reaction. The activation ofthis C–H bond thus appears similar to that observed in the �,�dehydrogenation step catalyzed by acyl-CoA dehydrogenases andacyl-CoA oxidases (20). The double mutant further emphasizes theimportance of the hydrogen bond of Tyr-450 to the C2 keto groupof cinerulose A, most likely to ensure an orientation of this moietysuitable for catalysis.

Fig. 5. Proposed catalytic mechanism for AknOx. (a) Reductive half reactionfor the conversion of rhodinose to cinerulose A. (b) Reductive half reaction forthe conversion of cinerulose A to L-aculose. For both reactions, the oxidativehalf reaction is the reoxidation of FAD by molecular oxygen.

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AknOx has the ability to specifically oxidize two separate carbonatoms (C4, followed by C2). Modeling the binding of cinerulose Abased on the structure of the AknOx-FAD-AclY complex positionscarbon atom C2 closest to the N5 atom of FAD, thus explaining thespecific oxidation of this atom in the second reaction. In the caseof rhodinose, both C2 and C4 are close to N5. However, oxidationat C2 in the first reaction is disfavored because of the nonactivatedC–H bond at carbon C3. The regioselectivity of AknOx can thus beunderstood as a result of a combination of conformational differ-ences related to the change in the hybridization of carbon atoms C2,C3, and C4 during the reaction and chemical activation of the C–Hbond at carbon C3 in cinerulose A.

In summary, our data show that AknOx utilizes the same activesite to catalyze two FAD-dependent consecutive reactions in thesame biosynthetic pathway. However, the enzyme uses two distinctsets of catalytic residues in the two reactions, a feature that makesAknOx rather unique among flavoenzymes.

FAD has to be reoxidized by molecular oxygen after the com-pletion of each reductive half reaction catalyzed by AknOx. It ispresently unclear whether the first oxidative half reaction occurswhile the first product, AclA is still bound to the enzyme. Onstructural grounds, there is no need for AclA to dissociate from thecomplex to allow molecular oxygen access to the isoalloxazine ringof FAD. An open channel, located at the interface of the flavin- andsubstrate-binding domains, allows access from the enzyme surfaceto the isoalloxazine ring of FAD for molecular oxygen (SI Fig. 7).However, thin-layer chromatography of extracts from AclN–AknOx reaction mixtures according to the method of Yoshimoto etal. (5) (data not shown) indicates that significant amounts of AclAare also produced, suggesting this intermediate in principle isable to leave the active site before the second catalytic cycle iscompleted.

Mechanistic Relations of AknOx to Other Members of the PCMHSuperfamily. Most of the members of the PCMH superfamily,including AknOx, bind their substrates on the si face of theisoalloxazine ring of FAD. The cofactor in AknOx also adopts theelongated conformation found in the PCMH superfamily, madepossible through the conserved extended pocket between the twosubdomains of the FAD binding domain.

A detailed comparison of the active sites of AknOx, GOOX (8),and PCMH (21) showed that the three enzymes initiate the catalyticreaction by a functionally conserved tyrosine residue Y450, Y429,and Y473, respectively (SI Fig. 8). In GOOX, the proton abstractionstep by Tyr-429 is proposed to be facilitated by Asp-355 by abridging water molecule (8). In AknOx, this aspartic acid residue is

replaced by Tyr-378, which does not interact directly with Tyr-450,the residue corresponding to Tyr-429 in GOOX. Neither doesTyr-378 participate in proton abstraction from the hydroxyl groupof the C4 carbon atom of rhodinose, because its position is too faraway from Tyr-450. The site-directed mutagenesis experimentsfurther demonstrate that Tyr-378 is not involved in this particularreaction. AknOx, however, differs from GOOX and PCMH in thatit catalyzes two different consecutive reactions, the oxidation of ahydroxyl group of the sugar residue and an �,� dehydrogenationreaction leading to desaturation of a carbon–carbon bond in thesame sugar moiety. Tyr-378 is located in an ideal position to initiatethe dehydrogenation step and thus plays a crucial role in catalyzingthe second reaction in AknOx.

Materials and MethodsProtein Production. Native and selenomethionine-substitutedAknOx were produced in Escherichia coli as fusion proteins with anadditional 19 amino acids at the N terminus, including a 7xhis tag,and purified as described (22). When necessary, an additional anionexchange chromatography step was added for further purification.

Mutagenesis. Site-directed mutagenesis was carried out by using thetwo-step four-primer method (23) and wild-type AknOx as tem-plate for single mutants. For double and triple AknOx mutants, thesingle or double mutants, respectively, served as templates. Allmutations were confirmed by DNA sequencing. The pBAD/HisBplasmid was used for protein expression. Mutant enzymes werepurified with the same protocol as recombinant wild-type AknOx.

Enzymatic Activity Assay. AknOx activity in conversion of AclN toAclA and AclA to AclY was determined by the amount ofhydrogen peroxide produced in the reaction, measured by usingthe peroxidase-2,2�-azino-bis(3-ethylbenzthiazoline-6-sulfonicacid) method (24). The purity of the substrates AclN and AclAwas 94% or better, as analyzed by HPLC. AclN was purified froman extract of the HO26 mutant strain of S. galilaeus, lackingAknOx activity, as described (4). AclA was purchased fromCalbiochem (San Diego, CA).

Substrates were used at concentrations ranging from 5.7 to 143�mol/liter. The assay mixture also included 1 �l of horseradishperoxidase (1 units/�l, Sigma, St. Louis, MO), 50 �l of 5 mM2,2�-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), and 10 �l of2,36 �mol/liter AknOx. The reaction volume was increased to atotal of 700 �l by the addition of 0.05 mol/liter Na citrate buffer, pH5.5. A calibration curve was obtained in the same assay formatwithout substrates and enzyme by the addition of known amountsof hydrogen peroxide. The enzymatic activity of AknOx mutantswas measured by using the same assay. The concentration of AknOxwas determined by using the Bradford method.

N-Terminal Sequence of Native Secreted AknOx. S. galilaeus ATCC31615 was cultured in E1 medium (25) for 5 days at 30°C in a shakerflask, and AknOx was purified from the culture supernatant tohomogeneity by ammonium sulfate precipitation as described (5)followed by anion exchange chromatography by using a HiPrep16/10 Q XL column (0–1 mol/liter NaCl gradient in 50 mmol/literTris�HCl, pH 8) and gel exclusion chromatography by using aHiLoad 26/60 Superdex 200 column in an Akta FPLC system (50mmol/liter Tris�HCl, pH 8; all components by Amersham Pharma-cia, Uppsala, Sweden). The N-terminal amino acid sequence wasdetermined by using an Applied Biosystems 477A Protein Se-quencer (Foster City, CA).

Crystallization and Structure Determination. Both native and sel-enomethionine substituted AknOx were cocrystallized with thesubstrate AclA, as described (22). The AknOx crystals werepseudomerohedrally twinned and belong to the space group P21,with cell dimensions a � 68.5 Å, b � 266.2 Å, c � 68.7 Å, and � �

Table 2. Statistics of crystallographic refinement

Parameters

Twin protocol

Two-domain Three-domain

Resolution, Å 20.0–1.65 20.0–1.65Rwork, % 18.5 23.1Rfree, % 24.2 26.5Twin fraction 0.5 0.16, 0.16No. of protein atoms 15,069 15,069No. of ligand atoms 270 270No. of water molecules 1009 994B factor, Å2

Wilson plot 22.3 22.3Protein atoms 17.8 16.8Ligands 15.8 14.9Water molecules 21.8 21.8

Deviations from ideals (rmsd)Bond length, Å 0.007 0.006Bond angles, Å 0.024 0.019

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119°. The analysis of the crystal twinning suggested that the AknOxcrystals are multidomain twins, with a two-fold twin operator alongthe diagonal between a and c and a three-fold twin operator parallelto the crystallographic b axis. The analysis of crystal twinning, theprocedure used for phase determination by MAD and modelbuilding, has been described in detail elsewhere (22).

Crystallographic Refinement. Three- and two-domain twin protocolswere used in refinement carried out with the program SHELXL(26). The statistics for the data set used in refinement to 1.65-Åresolution have been published elsewhere (22). A total of 4%reflections were used for the calculation of Rfree, chosen in thin-resolution shells as recommended for twin refinement. Watermolecules were added manually with the program COOT (27)based on the Fo�Fc map after each round of refinement. Three-domain twin refinement resulted in a final Rfac and Rfree of 23.1%and 26.5%, respectively, with twin fractions of 0.16 for �1 and �2.Two-domain twin protocols resulted in a final Rfac and Rfree valueof 18.5% and 24.2% and a twin fraction of 0.50. The comparableRfree values obtained from two- and three-domain twin operators

suggested the presence of multidomain twinning (coexistence ofboth two- and three-domain twinning) in AknOx. There arepresently no refinement programs for macromolecular crystallog-raphy that allow refinement of two- and three-fold twin operatorsand six twin fractions simultaneously. Details about the refinementprocess and further evidence for multidomain twinning are de-scribed in more detail elsewhere (22). Refinement statistics aregiven in Table 2.

Sequence and Structural Comparisons. Sequence homologs ofAknOx were found by using BLAST (28), and ClustalW (29) wasused for sequence alignment. Structural comparisons were carriedout with DALI (13) and the SSM superimposition option in COOT.Figures were prepared with PyMOL (30) and ESpript (31).

We gratefully acknowledge access to synchrotron radiation at theEuropean Molecular Biology Laboratory outstation, Deutsches Elek-tronen Synchrotron, Hamburg, Germany. This work was supported bygrants from the Swedish Science Council and the Finnish Academy(Grant 210576).

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