a computational study of site mutagenesis in epothilone-B ...

Engineering enzymes for improved catalytic efficiency:a computational study of site mutagenesis inepothilone-B hydroxylase

Akbar Nayeem1,3, Shu-Jen Chiang2, Suo-Win Liu2,Yuhua Sun2, Li You2 and Jonathan Basch2,3

1Computer Aided Drug Design, Division of Molecular Biosciences, Bristol-Myers Squibb Company, PO Box 5400, Princeton, NJ 08543-5400 and2Technical Operations, Bristol-Myers Squibb Company, 6000 ThompsonRoad, East Syracuse, NY 13057-5050, USA

3To whom correspondence should be addressed.E-mail: [email protected] (A.K.); [email protected] (J.B.)

Epothilone F, 21-hydroxyl-epothilone B, is an intermedi-ate in the synthesis of BMS-310705, an antitumor com-pound that has been evaluated in Phase I clinical trials. Abioconversion process utilizing the Gram-positive bacter-ium Amycolatopsis orientalis was used to prepare epothi-lone F from epothilone B. In order to improve the yieldof epothilone F, a mutagenesis program was performedwith the goal of engineering the epothilone-B hydroxylase(EBH) enzyme to improve the yield of epothilone Fthrough oxidative biotransformation. The mutations inEBH increased the yield of epothilone F from 21% in therecombinant expression system to higher than 80% utiliz-ing the best EBH mutants. The studies described hereshow how a homology model of EBH was used to obtainan understanding of the possible mechanism that led toimproved yield of epothilone F in the mutated enzymes.A novel aspect of this study is that it provides someinsight into how mutations distant from the binding sitecan affect enzyme activity.Keywords: biocatalysis/cytochrome P450/epothilone/homology modeling/mutagenesis

Introduction

Epothilones are a class of hybrid polyketide-peptide com-pounds isolated from the myxobacterium Sorangium cellulo-sum. Epothilones exhibit cytotoxic activity in rapidlyproliferating cells by promoting tubulin polymerization(Bollag et al., 1995). Epothilones A and B are the main pro-ducts of the fermentation of S. cellulosum, although 37natural epothilone-related compounds have been isolatedfrom the same fermentation (Hardt et al., 2001). One ofthese compounds identified, epothilone F, is the hydroxy-lation product of epothilone B at the 21 position (Fig. 1).Epothilone F can be produced by the addition of epothiloneB to cultures of S. cellulosum (Gerth et al., 2002),Amycolatopsis orientalis, and Amycolata autotrophica (Liet al., 2000). Epothilone F is used as an intermediate in thesynthesis of the investigational compound, BMS-310705.This compound is more soluble in water than epothilone B,advantageous for formulation and bioavailability and hasdemonstrated antitumor activity (Lee et al., 2002).

Efficient chemical synthesis of highly pure epothilone Fwas challenging due to the required specificity of the

hydroxylation of the starting material, epothilone B, whichhas multiple prospective sites for hydroxylation. Therefore,biotransformation was evaluated as an alternative to chemicalsynthesis. An A. orientalis soil isolate, designated SC15 847,was identified in a screening program to have epothilone Bto epothilone F hydroxylase activity. Genes encoding a cyto-chrome P450 mono-oxygenase enzyme (ebh) and a ferre-doxin iron–sulfur protein were isolated from A. orientalis,cloned into an expression vector and transformed into theGram-positive host, Streptomyces lividans. The resultingrecombinant cultures were found to catalyze the hydroxy-lation of epothilone B to epothilone F (Basch and Chiang,2007). The yields of epothilone F using A. orientalis cultureand the recombinant S. lividans culture were 40% and 21%,respectively, with loss of the remaining epothilone B to anenzyme-associated degradation activity (Fig. 1). Efforts toimprove the yield of epothilone F through bioconversionprocess optimization and A. orientalis strain development didnot result in significant improvements.

Cytochrome P450 mono-oxygenases are found in botheukaryotes and prokaryotes. The catalytic mechanism ofhydroxylation involves molecular oxygen bound to a hemeprosthetic group. With a few exceptions, prokaryotic P450systems are composed of three proteins, a heme containingcytochrome P450, an iron–sulfur electron-transport protein(ferredoxin) and an NADH-dependent ferredoxin reductase.Cytochrome P450 enzymes have been found in prokaryoticbiosynthetic pathways, catalyzing selective hydroxylationand epoxidation reactions. Prokaryotic cytochrome P450systems are also found outside of biosynthetic pathways andfunction as inducible systems to metabolize organic com-pounds. Site-directed and random mutagenesis studies havebeen performed on many cytochrome P450 enzymes to altersubstrate specificity and improve catalytic efficiency (Heet al., 1996; Kim and Guengerich, 2004; Kumar et al., 2005).

In order to improve the yield of epothilone F from epothi-lone B, mutations were introduced into the ebh gene. Thesite-directed mutation of the ebh gene was guided by theexisting literature on mutagenesis of P450 enzymes foraltered specificity, and a homology model of epothilone-Bhydroxylase (EBH). Mutants identified as having increasedyields of the epothilone F were selected and characterized bynucleic acid sequencing.

Mutations were introduced either randomly, with one ortwo mutations per round of mutagenesis, or in a site-directedmanner using the homology model of the EBH protein toguide the choice of mutations. This study focuses on the useof the homology model to understand the mechanism bywhich the introduced mutations resulted in improved yield ofthe desired product (epothilone F), including an attempt torationalize the dramatic effects of mutations distant from thebinding site. While previous studies have reported thatmutations that enhance thermal stability are often found on

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Protein Engineering, Design & Selection vol. 22 no. 4 pp. 257–266, 2009Published online January 28, 2009 doi:10.1093/protein/gzn081

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the protein surface (Hoseki et al., 1999; Zhao and Arnold,1999), explanations for the effects of such mutations onprotein stability are usually offered in terms of electrostaticeffects [e.g. the possibility of introducing a salt bridge invol-ving the mutated residues (Maves and Sligar, 2001)],increased compactness arising from shortening of surfaceloops, increased hydrophobicity at subunit interfaces,decreased flexibility or a combination of such factors. In thepresent study, however, surface mutations far from thebinding site that involve non-polar residues and do not, inany obvious way, affect protein compactness or flexibilitywere seen to have large effects on bioconversion.

Materials and methods

ExperimentalSite-directed mutagenesis The Quikchangew XL Site-Directed Mutagenesis Kit and the Quikchangew MultiSite-Directed Mutagenesis kit (Stratagene) were used tointroduce mutations in the coding region of the ebh gene.The pPCRscript-ebh vector was used as the template formutagenesis. The mutagenesis reactions were transformed toeither XL1-Bluew electrocompetent or XL10-Goldw ultra-competent cells (Stratagene) Escherichia coli strains andplated on Luria broth (LB) plates with ampicillin. After incu-bation 24–48 h at 30–378C, the entire plate was resuspendedin 5 ml of LB and used to inoculate 20–50 ml of LB andgrown 18–24 h at 30–378C. Qiagen midi-preps were per-formed on the cultures to isolate plasmid DNA containingthe desired mutation. Digestion with the restriction enzymesBglII and HindIII was used to excise the mutated expressioncassette for ligation into BglII- and HindIII-digested plasmidpANT849. Screening of the mutants was performed in S.lividans TK24 and Streptomyces rimosus strain R6 593.Streptomyces lividans was transformed as described byHopwood (1985). Streptomyces rimosus was transformedusing the procedure of Pigac and Schrempf (1995).

Random mutagenesis The method of Leung et al. (1989)was used to generate random mutant libraries of the ebhgene. Manganese and/or reduced dATP concentration wasused to control the mutagenesis frequency of the Taq poly-merase. The PCR products were separated on an agarose gel;the desired 1.2 kb fragment was excised and purified. Thefragments were then digested with BglII and HindIIIenzymes and purified using a Qiagen spin column. The puri-fied fragments were then ligated to BglII- and HindIII-digested pANT849 plasmids. Screening of mutants was per-formed in S. rimosus.

Identification of 24-hydroxyl-epothilone B Epothilone B(0.05% in ethanol) was added to S. lividans mutant ebh25-1grown in a shake flask with R2-YE media (Hopwood et al.,1985). The media supernatant was extracted with an equalvolume of 75% butanol and 25% methanol. The butanol/methanol extract (�10 ml) was evaporated to dryness undera nitrogen stream. One milliliter of methanol was added tothe residue (38 mg) and insoluble material was removedby centrifugation. A portion of the supernatant (0.1 ml) wasanalyzed by liquid chromatography/nuclear magnetic reson-ance and the remaining 0.9 ml of supernatant was subjectedto the preparative HPLC (0.2–0.4 ml per injection). Twomajor peaks (A and B) were observed in the supernatant andcollected by preparative HPLC. Peak A eluted between 14and 15 min, while peak B eluted between 16.5 and 17.5 min.Analytical HPLC analysis indicated that peak B was theparent compound epothilone B (Rt 8.5 min), and peak A wasthe biotransformation product (Rt 7.3 min). The peak A frac-tions were pooled and used for MS analysis. The pooled frac-tion was evaporated to a small volume and then lyophilizedto give 3 mg of white solid. The structure of the biotrans-formation product was determined as 24-hydroxyl-epothiloneB, based on MS and NMR data (compared with data ofepothilone B).

Analytical HPLC The organic extract of the biotransform-ation reactions was analyzed by HPLC using a HewlettPackard 1100 Series Liquid Chromatograph with a YMCPacked ODS-AQ column (4.6 mm inner diameter � 15 cmlength). A gradient system of water (solvent A) and aceto-nitrile (solvent B) was used: 20–90% B linear gradient,10 min; 90% to 20% linear gradient, 2 min. The flow ratewas 1 ml/min and UV absorbing peaks were detected at254 nm.

Preparative HPLC The preparative HPLC of 24-hydroxyl-epothilone B was performed using a Varian ProStarSolvent Delivery Module pump (Varian Inc., Palo Alto, CA,USA) with a YMC ODS-A column (30 mm innerdiameter � 100 mm length, 5 mm particle size) and peaksmonitored by absorbance at 210 nm using a GynkotekUVD340S detector. The flow rate was 30 ml/min, with thefollowing elution gradient: (solvent A: water; solvent B:acetonitrile), 20% B, 2 min; 20–60% B linear gradient,18 min; 60% B, 2 min; 60–90% B linear gradient, 1 min;90% B, 3 min; 90% to 20% B linear gradient, 2 min.

Liquid Chromatography/Nuclear Magnetic Resonance Fortymicroliters of sample was injected onto a YMC PackedODS-AQ column (4.6 mm inner diameter � 15 cm length).

Fig. 1. Structure of epothilone B showing the atom numbering. Products ofenzymatic action by EBH on epothilone B are also shown. Thehydroxylation pathway leading to hydroxylation at position 21 (Epo-F) is thedesired pathway, whereas the hydrolysis pathway leads to degradationthrough ring opening (lactone product).

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The column was eluted at 1 ml/min flow rate with a gradientsystem of D2O (solvent A) and acetonitrile-d3 (solvent B):30% B, 1 min; 30–80% B linear gradient, 11 min. The eluentpassed an UV detection cell (monitored at 254 nm) beforeflowing through an F19/H1 NMR probe (60 ml active volume)in a Varian AS-600 NMR spectrometer. The biotransformationproduct was eluted at around 7.5 min and the flow was stoppedmanually to allow the eluent to remain in the NMR probe forNMR data acquisition.

Screening mutant libraries for bioconversion Screening ofthe mutant libraries was performed in S. rimosus transformedhost cells. Streptomyces rimosus colonies were picked fromtransformation plates and inoculated to 96-well plates with2 ml of CRM media with 10 mg/ml thiostrepton and incu-bated 24–48 h at 308C shaking at 200–300 rpm. The cul-tures were crossed with 10% culture volume to a second96-well plate with 2 ml of CRM media with 10 mg/ml thios-trepton and incubated 16–24 h at 308C shaking at 200–300 rpm before the addition of epothilone B dissolved inethanol to a final concentration of 0.05% weight to volume.The 96-well plates were incubated on a shaking platform foran additional 24–48 h. The cultures were extracted with anequal volume of 25% methanol/75% butanol mixture, centri-fuged and the supernatant was analyzed by HPLC.

Evaluation of selected mutants in shake flasks Mutantsselected from the screening experiments were evaluated inErlenmeyer flasks. The bioconversion of epothilone B toepothilone F was performed in S. rimosus host cells trans-formed with expression plasmids containing the ebh geneand its mutants. One hundred microliters of a frozen S.rimosus transformant culture was inoculated to 20 ml CRMmedia with 10 mg/ml thiostrepton and cultivated 16–24 h,308C, 230–300 rpm. Epothilone B in 100% ethanol wasadded to each culture to a final concentration of 0.05%weight/volume. The reaction was typically incubated 20–40 h at 308C, 230–300 rpm. The concentration of epothi-lones B and F was determined by HPLC analysis. Thepercent bioconversion of epothilone B to epothilone F wasdetermined by the final epothilone F concentration calculatedas a percentage of the epothilone B added and adjusted fordilution by feed addition and pH adjustment. These arereported as percentage yield in Table 1.

ComputationalConstruction of the homology model The sequence of EBH,published by Basch and Chiang (2007), appears as accessionnumber ABG22116 in the Entrez protein sequence databaseat NCBI. A sequence similarity search against the proteins inPDB, carried out within the Prime software package (Primeversion 1.5, Schrodinger LLC, Portland, Oregon, USA) pro-duced, as expected, hits to several cytochrome P450 struc-tures. Two structures, 1OG5, which is P450EpoK with abound epothilone B molecule, and 1Z80, which is P450EryFwith bound DEB (6-deoxyerythronolide B), stood out as pro-spective templates. While EBH models were built using bothtemplates, the EBH model built using 1Z80 was preferred onaccount of its higher overall sequence identity (32%) withEBH as well as having more residues in the binding site thatwere similar to EBH (Table II). Although the substrate of

P450EpoK is also epothilone B, P450EpoK hydroxylatesepothilone B at a different position than EBH.

The alignment of the target sequence (EBH) to the template(1Z80) entailed matching similar residues and predicted versusknown secondary structure, a method shown to be more accu-rate than matching similar residues alone (Nayeem et al.,2006). Prior to building the model with 1Z80, which containsDEB, 1Z80 and 1OG5 were structurally aligned so that epothi-lone B from 1OG5 occupied the same space as DEB and couldbe included in the model building process to allow forexcluded volume. Default parameters in Prime were used foraligning the sequences and building the model. The residues in1Q5D that lie within 4 A of the ligand in the binding site areunderlined to facilitate comparison with corresponding resi-dues in EBH. The heme group, a necessary cofactor for cyto-chrome P450 enzymes, was included in the model building.

All side chains within 5 A of epothilone B were refinedused the OPLS2005 force field as implemented in Maestroversion 7.5.116. Three-dimensional models for the mutantswere generated by altering the pertinent amino acids fromthe wild-type (WT) structure and relaxing all side chainswithin 8 A of the mutated residues. Finally, the images forthe protein structures were generated from Maestro.

Characterization of structural pockets andcavities Identification and characterization of surface pocketsand internal cavities in EBH and the mutant model structureswere performed using the MOLCAD module as implementedin the Sybyl software (version 7.1, Tripos Inc., St Louis, MO63144, USA). Since the purpose was to compare relativepocket volumes and shapes rather than absolute values,hydrogen atoms were not added to the proteins prior to thecalculation (this would also introduce an uncertainty in theproper placement of hydrogen atoms). Using a probe radiusof 1.6 A, the Fast Connolly algorithm was used for generat-ing the protein pockets and their corresponding surfaces andvolumes. The program allows rapid identification and calcu-lation of Connolly’s molecular surface and volume(Connolly, 1983b) for all pockets and cavities in a proteinstructure. It ranks the cavities by size, where the largest oneis usually the binding site.

Docking of epothilone-B and estimation of relative bindingenergies GOLD 3.1.1 was used in our docking studies.GOLD gives the best poses by a genetic algorithm (GA)search strategy, and then various molecular features areencoded as a chromosome. A binding site was defined as allatoms of EBH within 10 A of the centroid of the epothiloneB ligand that was placed in the cavity in building the model.The default calculation mode, which provides the most accu-rate docking results, was selected for all calculations. In thestandard calculation mode, by default, the GA run comprised100 000 genetic operations on an initial population of 100members divided into five subpopulations, and the annealingparameters of the fitness function were set at 4.0 for van derWaals and 2.5 for hydrogen bonding. Default parameterswere also selected for others. The number of generated poseswas set to 30, and early termination was turned off. Eachcompound was imported from a Sybyl mol2 file, and thegenerated poses were exported as mol2 files. Sybyl atomtypes for ligands and the protein were set prior to running

Computational analysis of epothilone mutagenesis

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the GOLD calculation. The scoring function of Chemscorewas selected for each run.

A distance constraint, based on the crystal structures1OG5 and 1Z80, was used in reproducing the bindingmodes. In these and other P450 structures, the distance fromthe Fe atom in the porphyrin ring to the carbon atom at thehydroxylation site was measured to lie between 4.4 and4.9 A. Therefore, a distance constraint with a force constantof 1000 between these atoms was used to favor poses thatwere suitable for hydroxylation at the desired position.

Results and discussion

Mutants and yieldBoth site-directed and random mutagenesis were performedto increase the chances of improvement of the EBH enzyme.The first site-directed mutants with improved yields wereebh10-53 and ebh24-16 (Table I). ebh24-16 was used as atemplate for further rounds of site-directed mutagenesis.Beneficial mutations at M176 and E31 were identified byrandom mutagenesis. Site-directed mutagenesis was used toadd mutations at M176 and E31 to the ebh24-16d8 mutant(E31K in Table I). In total, �3000 EBH mutants were gener-ated using site-directed and random mutagenesis andscreened for epothilone B to epothilone F bioconversionactivity. The nucleic acid sequence was determined only forthose mutants that showed an increased epothilone B toepothilone F catalytic activity relative to the WT enzyme (anexception is ebh25-1 as noted below).

Ten EBH mutants with improved yield of epothilone Fand one mutant, ebh25-1, with altered specificity are shownin Table I, along with the site of the mutations and percentyield of the 21-hydroxylation product. The product yieldwith WT EBH in the recombinant system is 21%. Mutantebh25-1 altered the specificity of the reaction, catalyzinghydroxylation at the 24 position of epothilone B instead ofthe 21 position. All other mutants shown in Table I show anincreased yield with respect to the WT protein.

Structure determination of biotransformation productof mutant ebh25-1The structure of the biotransformation product of mutantebh25-1 was determined to be 24-hydroxyl-epothilone B (see

Fig. 1 for atom numbering), based on MS and NMR data. Amolecular weight of 523 was determined for the biotrans-formation product, based upon an M þ Hþ spectra of 524,consistent with a mono-hydroxylation product of epothiloneB. The chemical shift assigned to the C-21 protons of2.66 ppm matches the shift at the C-21 protons of epothiloneB of 2.69 ppm; epothilone F has a resonance at the C-21protons of 4.95 ppm. A shift of 3.81 ppm was assigned to theC-24 protons in the unknown hydroxylation product, consist-ent with hydroxylation at this position.

Homology model of EBHSince an experimental structure for EBH was unavailable, ahomology model of EBH was constructed using as templatethe solved crystal structure of the DEB hydroxylase, EryFenzyme from Saccharopolyspora erytherea (PDB code1Z8O). The sequence alignment used for building the EBHmodel on the 1Z8O template is shown in Fig. 2. Thesequence identity between EBH and EryF is 32% over thelength of the alignment. Residues in the EBH-binding pocketwere inferred by comparing its amino acid sequence withthat of the template using the sequence alignment of Fig. 2,and noting residues in EryF that lie within 5 A of the EryFsubstrate (DEB). Furthermore, since the algorithm for gener-ating the sequence alignment compares known versus com-puted secondary structure in addition to sequencecomparison, the (predicted) and (known) secondary structurestates of EBH and EryF, respectively, are also shown.

In the cytochrome P450 family of enzymes, particularlywell conserved in structure but not in sequence, are 13a-helices (labeled A–L and B0) and 6 b-pleated sheets(labeled 1–6) that surround the buried catalytic site and con-tribute to the overall fold (Graham and Petersen, 1999; Stout,2004; Poulos and Johnson, 2005; Miura et al., 2006;Rupasinghe and Schuler, 2006). The nomenclature for thea-helices and b-strands in the sequence alignment shown inFig. 2 follows that for cytochrome P450EryF (Cupp-Vickeryand Poulos, 1995). Although the overall protein fold is main-tained, several regions that are crucial for defining individualP450 catalytic sites and their substrate-access channels differsubstantially across the available crystal structures. Nearly,all of these variable regions correspond to thesubstrate-recognition site regions originally described by

Table I. Catalytic efficiency of EBH (WT) and its various mutants measured in terms of the percentage yield of the 21-hydroxylation product of epothilone B

Mutant Yield (%) E31 R67 R88 I92V A93 V106 I130 A140 M176 F190 N195 E231 F237 S294 I365

WT 21ebh25-1 0 S Pebh10-53 41 Y Rebh24-16 52 V Aebh-M-18 55 K Vebh24-16d8 75 Q V Aebh24-16c11 75 V G A Tebh24-16-16 75 V A Aebh24-16-74 75 H V Aebh24-16b9 80 Q V T S Aebh24-16g8 85 Q V T A AE31K 90 K Q V A A

Residues predicted to lie within 3.5 A of the bound substrate (epothilone B) are shown in bold font (R67, I92, F237 and S294) and those residues lying beyondthis distance but within 5 A of the substrate (R88 and M176) are shown in italics. The letters in the cells refer to the mutations, e.g. ebh10-53 was the doublemutant F190Y and E231R.

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Gotoh (1992) for members of the vertebrate CYP2 family.The regions most variable in length and backbone configur-ation correspond to the loop region between the B and Chelices (SRS1), the carboxy-terminal end of the F helix(SRS2), part of the FG loop and the N-terminal end of the Ghelix (SRS3) and the b-turn in b-sheet 4 (SRS6) (Baudryet al., 2006). As expected, the sequence divergence betweenEBH and 1Z8O is greatest in these variable regions.

The EBH model built using the sequence alignment toEryF is shown in Fig. 3A. Labeled in the model are the pos-itions of the mutations shown in Table I. The parts of theprotein that move upon substrate binding are also high-lighted. The binding pocket, which is located directly abovethe heme group, is bounded by helices I at the top and Cbelow and further defined by the helices I and H, delimitedby the b-sheet and flanked by the helical connector, the Dand E helices, and the connecting loops as shown in Fig. 3B.

Binding pocketThe analysis of structural pockets and cavities in EBH andits mutants indicates the presence of a buried cavity in thecore of the modeled domain that is significantly smaller thanthe corresponding pocket for the P450 template 1Z8O usedfor constructing the model. This is a consequence of, onaverage, the larger amino acids in EBH that replace thesmaller amino acids in 1Z8O listed in Table II in the courseof model building.

Mapping mutants to structureTable I shows that mutations involving the five residuesnearest to the binding site (R67, R88, I92, M176 and F237)have the highest effect on activity. This is expected since, ingeneral, residues near or within the binding pocket are mostlikely to influence pocket shape and size, and therefore the

Fig. 2. Sequence alignment between EBH and template EryF (1Z80). Also shown are: (i) the predicted and known secondary structure assignments for EBHand EryF, respectively. Positions of residue identity along the sequence alignment are denoted by asterisks (*); (ii) the positions of helices with assignednomenclature (A, B, B0, C etc.) and b-strands (1, 3, 4 etc.); (iii) residues in EBH that, based on alignment with EryF, line the binding site, denoted by bold andunderlined letters. The sequence identity of EBH relative to EryF over the aligned residues is 32%.


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ability of the substrate to interact with the binding site resi-dues. Thus, we see that ebh10-53 consisting of the distantmutations F190Y and E231R increases the product yield1.9-fold relative to the WT, whereas ebh24-16 consisting ofthe mutants I92V and F237A, which are within the bindingsite, causes a relative increase of 2.5-fold. A further mutationinvolving a residue in the binding site, R67Q (ebh24-16d8),causes an even further increase resulting in a product yieldof 3.6-fold relative to WT. All three site mutations R67Q,I92V and F237A besides being mutations in the bindingpocket also involve changes from a larger to a smaller aminoacid, causing an increase in pocket size. The increase inyield could be a result of the increase in pocket size facilitat-ing accommodation of the epothilone B substrate. Theeffects of mutations distant from the active site are moresubtle. An attempt to provide an explanation for some of thedistant site mutations is presented later.

Docking and binding energiesThe EBH model shown in Fig. 3 was used for dockingepothilone B flexibly into the binding pocket using theGOLD 3.1.1 program (Jones et al., 1997; Verdonk et al.,2003) as described above (see the Materials and methodssection). In order to obtain acceptable binding poses relevantto hydroxylation at various sites on epothilone B, appropriatedistance constraints between the Fe atom at the center of theheme group and atoms on epothilone B defining potentialhydroxylation sites were introduced. Binding poses thatmodeled hydroxylation at position 21 in epothilone B(Fig. 1) was generated using a distance range constraint of4.5+ 0.5 A between Fe and C-21. (This distance range wasbased on measured distances between heme iron and carbonhydroxylation sites in several cytochrome P450s.)

Three preferred binding poses are predicted for epothiloneB in the active site of EBH (Fig. 4A–C). The binding pos-ition of epothilone B shown in Fig. 4A displays an orien-tation of epothilone B that has the methyl group of thethiazole ring directed towards the center of the heme ring.This is the binding pose that favors hydroxylation of epothi-lone B at the 21 position to produce epothilone F, which isthe desired product. The other two orientations, in which theFe atom of the heme group is proximal to C-15 of epothiloneB (Fig. 4B), or the center of the thiazole ring (Fig. 4C) are

Fig. 4. Binding models of epothilone B in EBH. In the interest of clarity, only the substrate and the porphyrin group are shown. All carbon atoms are in greyand all non-carbon atoms in black. The three binding poses shown correspond to orientations that tend to favor: (A) hydroxylation at position 21; (B)hydrolysis at position 1; (C) hydrolysis by proton abstraction from the thiazole ring. The product of (A) is the desired product, whereas (B) and (C) areexpected to lead to undesired products and hence degradation. The C-21 position is indicated by an arrow in each case.

Fig. 3. Ribbon diagram of the homology model of EBH showing: (A) thelocations of the mutations described in this study. The porphyrin group isshown in yellow and the bound epothilone B substrate in green. Those partsof the structure that are most prone to movement when a substrate binds tocytochrome P450 are highlighted in red on the ribbon. (B) The helices andb-strands, with their nomenclature, around the binding pocket. Theporphyrin ring is shown as a space fill model.

Table II. Equivalent residues around the binding pocket for P450 enzymes

P450EpoK, EBH and P450EryF

EpoKH (1Q5D) EBH EryF (1Z8O)

R71 R67 P81M90 P86 Y85K92 R88 A87F96 I92 G91A182 M176 I174L183 L177 L175G184 S178 V176G249 F237 L236A250 L238 L240A253 I241 L240A254 A252 A241T258 T246 A245L301 A289 —G304 T292 T258T305 T293 T291V306 S294 T292F327 V315 V314A402 T393 L391F403 I394 L392

Underlined residues are those in EBH that were mutated. The residues inbold are those lying within 5 A of the binding site, whereas those in italicslie in the next shell between 5 and 8 A.

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hypothesized to lead to product degradation through ringopening. The free energies of binding estimated by theGOLD program for the three binding orientations in theWT enzyme and two mutant enzymes (ebh24-16 andebh24-16d8) are shown in Table III. These data show thatthe binding orientation of Fig. 4A is progressively favored inthe mutants catalyzing higher yields of epothilone F, whilethe other two binding orientations (Fig. 4B and C) remainrelatively unchanged or become less favored.

Degradation of the epothilone B nucleus during the bio-transformation is a hydrolysis reaction catalyzed by EBH(Basch and Chiang, 2007). While hydrolysis reactions are nottypically catalyzed by P450 enzymes, the hydrolysis ofepothilone D, suggested to occur via a carbonium ion inter-mediate (Jumaa et al., 2004), presents a possible mechanismfor this reaction. Abstraction of a proton from the thiazolering of epothilone B by the heme-bound oxygen wouldstabilize the carbonium ion intermediate and result inhydrolysis of the lactone ring. Table III shows that thebinding pose in Fig. 4C with the thiazole ring positioned par-allel to the heme ring becomes progressively less favored inthe mutants with the higher yields of epothilone F, while thebinding pose with the lactone oxygen positioned above theheme (Fig. 4B) remains relatively unchanged.

Do the mutants play a structural role?The mutations in Table I map on to various parts of the EBHmodel, as shown in Fig. 3A. The mutations may be classifiedinto those that are (i) proximal to the epothilone B bindingsite (within 5 A), such as S294, M176, R67, I92, E231 andF237. The effects of mutations at these sites can propagatefrom the inner core to the lids forming the canopy of thebinding pocket or can affect pocket size and shape by creat-ing cavities, such as I92V, F237A and M176A; and (ii) distalfrom the binding site (.5 A), such as V106A, I130T andE31K. It is difficult to rationalize the effects of mutationsmade at these sites since they likely affect binding indirectly,for example, by facilitating or obstructing entry of the sub-strate into the binding pocket, or affecting the position of thehelices defining the binding pockets.

In order to understand whether mutations made at the sitesin Table I might play a structural role, we compared thestructures of cytochrome P450s containing a bound substrateor ligand with those of their unbound (apo) counterparts.The results are shown in Fig. 5A–D for four such cases. Ineach case, the complexed form of the protein is structurallysuperimposed on to the apo protein. The plots shown inFig. 5 denote, at each residue position, the deviation in Caposition of the complex with respect to the Ca position inthe apo structure. The Ca-deviation profiles for the fourP450s show a similar pattern, with five positions in thesequence where the deviations are highest. These fiveregions, which occur in roughly the same parts of the fourP450 structures, are denoted by the letters A–E and corre-spond to those parts of the structure where there is mostmovement upon complexation with ligand.

The positions of the mutants in EBH relative to these fiveregions can be seen from Fig. 6. Figure 6A shows the pos-itions of the mutants relative to the five regions in sequence,whereas Fig. 6B shows the positions in the structural model.

Table III. Relative binding free energies calculated using GOLD 3.0 for

different binding poses of epothilone B in WT and mutants of EBH

Mutant Yield(%)

DG (kcal/mol),Fig. 4A

DG (kcal/mol),Fig. 4B

DG (kcal/mol),Fig. 4C

Wild type 21 227.5 238.8 241.1ebh24-16 52 237.0 236.9 237.0ebh24-16d8 75 240.0 237.5 234.2

The different poses are those shown in Fig. 4A–C.

Fig. 5. Effect of substrate binding on structural changes in four P450 enzymes. In each case, the deviation in Ca position (A) of the substrate bound structurewith respect to the apo form (Y-axis) is measured along the residue position in the protein sequence (X-axis). The four cases of substrate bound versus apostructures are: (A) 2C9 (1OG5); (B) EpoK (1PKF); (C) 3A4 (2J0D); and (D) 2B4 (1SU0). The five regions that exhibit the most structural deviation uponsubstrate binding are indicated by the letters A–E.


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Among the thousands of mutants made, only those thataffected the desired hydroxylation (Fig. 1) are shown in Table Iand Fig. 3A. From Figs 3A, 6A and B, we see, significantly,that most of the beneficial EBH mutations lie near thoseregions of cytochrome P450 proteins that ‘open up’ when a

ligand binds, possibly to allow greater ligand access to thebinding site. Consequently, replacing larger amino acids withsmaller (and conservative) ones in these regions should permitgreater access of the ligand to the binding site. This obser-vation provides a hypothesis for how mutations such as A93G,

Fig. 6. (A) Sequence alignment of EBH with respect to EpoK (1PKF). The mutated residues in the EBH sequence (upper) are annotated by the arrows; theresidues in EpoK (lower) that lie in the five regions described in the text are indicated by the letters A–E and are underlined in the sequence. (B) Regions inthe 3D structure of EpoK that correspond to the five regions in Fig. 5 that show the greatest deviation upon binding substrate are indicated by the letters A–E.The positions of these regions in the sequence can be seen from (A).

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V106A and I130T (especially I130T which is a surface mutantfar from the active site) might contribute to better substratebinding leading to increased product yield (vide infra).

Effects of mutations near and distant from active siteThe predicted effect of mutating residues in and around thebinding site on substrate binding was assessed by construct-ing binding models of epothilone B in EBH. A significantresult of these binding models is that, as shown in Figs 4Aand 7A, for constructive binding to occur (i.e. one that gener-ates a binding pose favorable for hydroxylation at the C-21position) the carbonyl oxygen atom joined to C-1 must beanchored (through hydrogen bonding) to the guanidiniumgroup on R67 and the hydroxyl group on S294. Mutation ofeither or both of these residues is expected to free the epothi-lone B substrate of the restriction needed to favor the desiredbinding pose (Atkins and Sligar, 1988). Indeed, we see thatthe S294P mutant ebh25-1 causes complete loss of activity.Furthermore, the guanidinium cation interacts with the aro-matic ring of F296 (Fig. 7A). While the mutation R67Q pre-serves the hydrogen bonding to epothilone B, it destroys thecation-pi interaction with F296 allowing the pocket to openup for substrate entry.

As an example of a mutation involving residues moredistant from the active site, we consider E231R. AsFig. 7B shows, E231 on helix-I forms a salt bridge withK101 on helix-C. The E231R mutation is therefore seennot only as destroying the salt bridge, but possibly alsocausing the two helices to move further apart from eachother because of the repulsive effect of the interaction ofR231 with K101. Since the heme prosthetic group liesbetween helices I and C (Fig. 3B), this relative movementcan open up the pocket allowing easier entry of the sub-strate for reaction.

A final example of the effect of mutations is shown inFig. 7C. This example, which is particularly interesting sinceit shows the effect of long-range coupled-interactions, con-cerns the case where residues around the pocket, i.e. F237 onhelix I and M176 on helix F, are coupled through pi-aromaticinteractions to residues that are outside the pocket (F190 onhelix G and F87 on helix B0). The three phenyl rings of

F237 (helix I), F87 (helix B0), F190 (helix G), and the sulfuratom of M176 (helix F) together form an extended pi inter-action. Non-conservative mutations at one or more of theseresidues are likely to disrupt this interaction causing changesin pocket shape and size.

Effect of mutations on pocket size and shapeWhile we do not claim a rigorous understanding of the effectof the described mutations on product yield, we note that thepocket size and shape change with the favored mutations in amanner so as to allow more room in the pocket for the sub-strate to adjust as necessary for binding in a pose favorablefor reaction. The calculated Connolly surfaces (Connolly,1983a, 1983b) of the binding pockets for the WT EBH struc-ture (21% yield) and the three mutants ebh24-16 (52%yield), ebh24-16d8 (75% yield) and ebh24-16g8 (85% yield)show that, while the sizes of the binding pocket themselvesdo not provide an intuitive picture of the greater ease withwhich the substrate can adjust as needed, the changes inpocket shape with the different mutations allow for morefreedom of movement of the substrate. Such changes inpocket shape could account for the relative changes in freeenergy of binding that tend to favor epothilone B binding inone mutant over another. Such changes are consistent withrecent studies on the ligand-binding modes of P450 3A4inhibitors, which show that protein undergoes conformationalchanges on substrate binding that can affect changes in theactive site volume by more than 80% (Ekroos and Sjogren,2006).

Comparison with P450EpoKCytochrome P450EpoK is found in the epothilone biosyn-thetic pathway of S. cellulosum, catalyzing the C12–C13epoxidation of epothilone D to epothilone B. Using theatomic radii as implemented in the MOLCAD unit of Sybyl7.1 and a solvent probe radius of 1.6 A, the substrate-bindingcavity of P450EpoK is estimated to be �1620 A3, whereasthat for the EBH enzyme estimated to be larger (2240 A3).This increase in pocket size is consistent with the ability ofEBH to accommodate larger substrates than P450EpoK andis mostly due to the fact that, as Table II shows, EBH has a

Fig. 7. Some examples of how residues in/around or distant from the binding pocket might effect size and/or shape characteristics of the binding pocket. (A)Residues around the binding pocket R67 and S294 form hydrogen bonds with substrate epothilone B (dotted lines), anchoring it in position favorable forhydroxylation at position 21. F296, somewhat distant from the binding site, may indirectly influence the binding of epothilone B through a possible cation-piinteraction that the guanidinium group on R67 could make with the aromatic ring of F296. (B) Salt bridge interaction between distant residues E231 (helix I)and K101 (helix C), which could contribute to locking helices I and C together. Mutation of either residue (E231K was done) could destroy this interactionand open up the pocket allowing for increased substrate access. (C) Extended pi interaction involving residues close to (F237 on helix I), around (M176 onhelix F) and far from (F190 on helix G) the binding site. This could help explain why mutations non-conservative mutations at F237, M176 or F190, whichcan disrupt the extended pi interaction, can open up the pocket.


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V315 corresponding to F327 in P450EpoK. This F327, beingbulkier than valine, prevents epothilone B from binding intoP450EpoK in a manner favorable for hydroxylation at the 21position compared with the smaller (and hydrophobic) V315at the equivalent position in EBH. The cavity sizes in themutants ebh-24-16 and ebh-24-16b9 are estimated to be evenlarger, a direct result of replacements I92V, M176A/S andF237A, i.e. to smaller amino acids. The increase in pocketsize in these EBH mutants probably allows for more transla-tional, rotational and conformational freedom for Epo-B,making it more likely to attain the orientation desirable forhydroxylation at the desired position. This is probablyreflected in the increased binding affinity for the two mutantscompared with the WT (Table III).

Final commentsThe point of this study was to retrospectively understandwhy the mutants that led to improved enzyme efficiencycompared with the WT actually worked. Unfortunately,attempts to perform in vitro bioconversion studies with cellextracts were not successful and the enzyme kinetics of theindividual mutants could not be determined, making itimpossible to determine the kinetic contribution to the mech-anism of improved bioconversion. While much of themutation results are rationalized on structural considerations,not all mutations are understood in terms of interactions withthe substrate or binding pocket. Two examples that stand outare E31K and V106A. As discussed above, E31 by virtue ofits proximity to K27 could be involved in a salt bridge withthe latter, and if so, replacing it with K31 will disrupt thesalt bridge. Disruption of this salt bridge between the I andC helices could affect the position of the I helix which couldin turn affect the shape of the binding pocket. Similarly,V106 could be involved in hydrophobic interactions withL235 and L239 on helix I. Replacing V106 with A106 coulddecrease this interaction.

Our modeling studies suggest that (i) mutations thatincrease pocket size, and/or (ii) lie near the five identifiedregions in P450 that are most structurally sensitive to sub-strate binding, positively correlate with increases in bindingefficiency thus leading to improved bioconversion. A clearstep forward to validate this hypothesis would be the sys-tematic design and testing of mutants beyond those discussedin this study. It is our hope that the ideas set forth in thispaper can stimulate further thought and experiment in thisdirection, leading to creative uses of cytochrome P450s andother enzymes for such industrial application.

Acknowledgements

We would like to thank Wenying Li for his assistance in the isolation andidentification of 24-hydroxy epothilone B. The S. rimosus strain R6 593 wasa kind gift of Jasenka Pigac. Further thanks are due to Stanley Krystek andDebbie Loughney for helpful advice and suggestions.

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Received February 5, 2008; revised October 21, 2008;accepted December 1, 2008

Edited by Lars Baltzer

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