Designer enzymes for glycosphingolipid synthesisby directed evolutionSusan M Hancock, Jamie R Rich, Matthew E C Caines, Natalie C J Strynadka & Stephen G Withers

Though glycosphingolipids have great potential as therapeutics for cancer, HIV, neurodegenerative diseases and auto-immunediseases, both extensive study of their biological roles and development as pharmaceuticals are limited by difficulties in theirsynthesis, especially on large scales. Here we addressed this restriction by expanding the synthetic scope of a glycosphingolipid-synthesizing enzyme through a combination of rational mutagenesis and directed evolution with an ELISA-based screeningstrategy. We targeted both a low-level promiscuous substrate activity and the overall catalytic efficiency of the catalyst, andwe identified several mutants with enhanced activities. These new catalysts, which are capable of producing a broad rangeof homogeneous samples, represent a significant advance toward the facile, large-scale synthesis of glycosphingolipids anddemonstrate the general utility of this approach toward the creation of designer glycosphingolipid-synthesizing enzymes.

Over the last two decades, the perceived roles of lipids have expandedbeyond their functioning as static membrane components and energystorage molecules to include roles as highly regulated, bioactivespecies1. One family of lipids, the sphingolipids, is composed ofa sphingoid base that is predominantly N-acylated by a fattyacid, and a polar head group that can be glycosylated (glycosphingo-lipids) or phosphorylated (sphingomyelins). Glycosphingolipidsshow promise as treatments for cancer, malaria and auto-immunediseases by immunomodulation via natural killer T cells2. Theymay also be useful for treating HIV (ref. 3) and neurodegene-rative diseases4. Thus, the ability to synthesize large quantities ofhomogeneous, contaminant-free glycosphingolipids and their deriva-tives could potentially revolutionize therapies for a wide rangeof pathologies.

Although chemical syntheses of glycosphingolipids have beenreported, these remain arduous and target-specific5,6; therefore, ourefforts have focused on the development of biocatalysts. Given thatglucosylceramide synthase, the first enzyme in the natural biosynthes-is of glycosphingolipids, is not yet accessible in useful amounts, wesought to expand the synthetic repertoire of a family of engineeredglycosidases: the glycosynthases7. These mutant glycosidases are ren-dered hydrolytically incompetent by mutation of the nucleophilicglutamate or aspartate. However, when used in combination witha glycosyl fluoride donor of the opposite anomeric configurationto that of the natural substrate, along with the appropriate accep-tor substrate, they have proven valuable for the synthesis of oligo-and polysaccharides8,9.

By applying this approach to the glycosphingolipid-hydrolyz-ing enzyme endo-glycoceramidase II (EGC) from Rhodococcussp. strain M-777 (ref. 10), we previously generated a glyco-synthase that catalyzes the synthesis of lyso-GM1 (1), lyso-GM3 (2)

and lyso-Pk antigen (3) (precursor to the globoside series of gly-cosphingolipids) in greater than 94% yields from D-erythro-sphingosine (4; henceforth referred to as sphingosine) and variousa-configured glycosyl fluorides11 (Fig. 1). Although this was animportant synthetic breakthrough, this first-generation catalyst hadrelatively low activity and restrictively stringent lipid acceptor speci-ficity12, which we aimed to address through enzyme engineering.Specifically, we sought to develop catalysts that could synthesizeconjugates of D-ribo-phytosphingosine (6; hereafter referred to asphytosphingosine) (Fig. 1b).

Although sphingosine and D-erythro-sphinganine (7) are the pre-dominant sphingoid bases, phytosphingosine is synthesized de novoin a number of mammalian cells and can be incorporated into gly-cosphingolipids by glucosylceramide synthase13. Phytosphingosineassimilation into glycosphingolipids is modulated by changesin the hormonal environment14–18, and many bacteria can differen-tially recognize phytosphingosine-containing glycosphingolipids19–22.These observations suggest important roles for phytosphingosine-derived glycosphingolipids that cannot be explored furtherbecause methodologies to efficiently synthesize these molecules arelacking. Preliminary characterization of the EGC glycosynthasesuggested that the kcat/Km for phytosphingosine is 10,000-foldlower than that for sphingosine12. An enzyme with such low,promiscuous activity is an ideal candidate for engineering, becausein addition to the physiological significance of phytosphingo-sine derivatives, use of such an enzyme is in line with one ofthe basic strategies of directed evolution: select for an activity thatalready exists23.

Here we report the use of an ELISA-based screen to selectEGC glycosynthase mutants with enhanced phytosphingo-sine activity and improved catalytic efficiency from mutant

Received 10 November 2008; accepted 15 May 2009; published online 14 June 2009; doi:10.1038/nchembio.191

Department of Chemistry and Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada. Correspondenceshould be addressed to S.G.W. (withers@chem.ubc.ca).

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libraries. We identified and characterized several mutants, one ofwhich increased phytosphingosine activity to almost the samekcat/Km as the natural sphingosine substrate, and we demon-strated its utility for the synthesis of phytosphingosine-containing glycosphingolipids.

RESULTSLibrary formation and screeningAlthough structural information is available for EGC glycosynthase12,our initial attempts at rational engineering of its activity wereunsuccessful. Instead, we decided to implement a directed evolutionapproach to make catalysts ‘to order’. The principal challenge of thisstrategy is that of creating a suitable screen, as illustrated by the fewexamples of directed evolution of enzymes that synthesize glycosidiclinkages24–29: these transformations are difficult to screen in a high-throughput manner. An automated, ELISA-based screen that coulddetect the EGC glycosynthase–catalyzed synthesis of glycosphingolipid1 from a GM1-oligosaccharyl a-fluoride (GM1OSF, 5) donor andmicroplate-bound acceptor 4 in cell lysates was therefore developedand optimized for this purpose30. The screen exploits the GM1 (10)binding specificity of cholera toxin B subunit (CTB), which issubsequently detected by successive antibodies coupled to horseradishperoxidase (HRP) activity (Supplementary Fig. 1 online). Becauseonly the sugar moiety is recognized, the screen is versatile with regardto the lipid screened. A trial screen of a saturation mutagenesis libraryat the nucleophilic residue confirmed that the serine nucleophilemutant (E351S EGC) is the most active glycosynthase30. Using thisas the progenitor sequence, we generated two libraries of EGCglycosynthase mutants from the full-length EGC gene (minus signal

sequence) by error-prone PCR. These libraries, which differed only inthe number of mutations introduced, were cloned into a pET28avector with an N-terminal His6 tag. Subsequent DNA sequencingconfirmed that the two libraries contained 0.8 and 1.5 mutations perkilobase. We inoculated media within 96-well microplates with singlecolonies from these libraries and grew cultures to stationary phasewithout induction, as screen optimization showed that basal-levelexpression of pET28a constructs exhibits sufficient EGC glycosynthaseactivity for detection while avoiding the complications of non-uniform IPTG induction30. Given the level of automation available,we limited our screening process to 10,000 single colonies, and weseparately evaluated each colony for improved ability to use both 4and 6 lipid acceptor substrates for the synthesis of the correspondingGM1 conjugates (Fig. 1).

Second-generation catalyst identificationInitial positive hits were identified as those having a response two tothree s.d. above that of the E351S EGC standards in each microplate.These 130 putative hits were re-screened in triplicate to eliminate falsepositives, and DNA sequencing of the most promising hits identified32 new EGC glycosynthase mutants. On the basis of the plate assays,three of these showed increased phytosphingosine-derived lyso-GM1

(11) synthesis, 27 demonstrated improved synthesis of 1, and twomutants (I276F and D314Y) displayed enhancement in both activities.As expected, we did not identify any mutants in which the nucleo-philic residue mutation of the glycosynthase (E351S) had revertedback to wild type, and although point mutations are mentioned forclarity throughout this manuscript, all mutants also contained theoriginal glycosynthase E351S mutation. To remove false positives

O

NH2

OH6

HO

E351S

OO

E233

HF

O

NH2

OH6

OO

OOO

OH

OHOH

OH

HO

CO2H

HO

OH

OO

O

OH

NHAc

OH

OHO

OH

OH

OH

HOOH

AcHNO

OO

OO

OH

OHOH

HO

HO

CO2H

HO

OH

OO

O

OH

NHAc

OH

OHO

OH

OH

OH

HOOH

AcHN

F

Sphingosine (4) GM1OSF (5) lyso-GM1 (1)

O

NH2

OH6

OO

OOO

OH

OHOH

OH

HO

CO2H

HO

OH

OO

O

OH

NHAc

OH

OHO

OH

OH

OH

HOOH

AcHN

Phytosphingosine-derived lyso-GM1 (11)

O

NH2

OH6

OO

OOO

OH

OHOH

OH

HO

CO2H

HO

OHOHHO

OH

AcHN

Phytosphingosine-derived lyso-GM3 (13)

OO

OOO

OH

OHOH

HO

HO

CO2H

HO

OHOHHO

OH

AcHN

HO

NH2

OH6

D-erythro-Sphinganine (7)

HO

NH2

OH6

OH

HO

NH2

OH6

L-threo-Sphingosine (8)

HO

NH2

OH

D-ribo-Phytosphingosine (6)

(1R,2S)-2-Amino-1-phenylpropan-1,3-diol (9)

F

GM3OSF (14)

a

b c

OH

OH

Figure 1 The synthesis of glycosphingolipids by EGC glycosynthase. (a) The nucleophile mutant (E351S) renders the glycosidase hydrolytically

incompetent, but glycosphingolipids can be synthesized as shown using GM1OSF (5) and sphingosine (4). (b) Various glycosyl fluorides and sphingolipids

used. (c) Glycosphingolipid products synthesized.

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arising only from elevated expression levels, the full complementof mutants identified was expressed, purified and further characterizedby subjecting to an additional round of robotic ELISA screening.The remaining 18 mutants were kinetically characterized by fluoride-selective electrode analysis using a fixed high concentration ofone substrate while varying the concentration of the other (Tables 1and 2). The recently determined crystal structure of EGCglycosynthase in complex with GM3 (12) provided a means ofinterpreting the structural significance of some of the mutations12

(Fig. 2a).

Mutants identified from phytosphingosine screensAs already discussed, the parent glycosynthase was previously shownto have trace activity with (6) (ref. 12), but a more detailed study herehas revealed that this low activity is in addition to, or maybe aconsequence of, the glycosynthase being markedly substrate-inhibitedby this substrate. Typical plots of rate against concentrations from 0.3to 3 mM displayed negative slope, and (due to the poor activity) ratesbelow these concentrations were indistinguishable from the uncata-lyzed hydrolytic background of 5. Using 4 as a non-inhibitory lipidsubstrate and assuming that turnover of 6 was negligible during theassay, the Ki for 6 was estimated to be 0.85 mM. This compares with

a Km value of 0.41 mM for sphingolipid 4. Both Dixon and reciprocalplots gave parallel lines, which are indicative of uncompetitive inhibi-tion (the Dixon plot slope is equal to 1 (Vmax � Ki); SupplementaryFig. 2 online). Uncompetitive inhibition is characteristic of orderedbisubstrate systems; in the EGC glycosynthase reaction, this type ofinhibition could occur if 6 bound in a dead-end fashion with theenzyme-product complex. This could be easily rationalized by asecond molecule of 6 filling the lipid-binding cavity where the fattyacid side chain of the full ceramide normally binds (see Fig. 2b fordetails of the ceramide binding site).

Kinetic analyses were carried out on the five mutants identified ashaving improved phytosphingosine-processing ability (Table 1). Threemutants (V101M K200R, K129N and N148S) showed low-levelactivity (2–3 M–1 s–1) with no observable substrate inhibition (up to3 mM). It was not possible to achieve saturation of 6 with thesemutants due to poor affinity; therefore kcat/Km values were calculatedfrom the gradient of a plot of rate versus concentration. Analysis ofsmall-scale reactions by TLC demonstrated that product 11 wassynthesized by each of the mutants, and confirmed that this lowactivity was genuine. As these mutations were distant from the activesite and resulted in only small changes in activity, the structuralimpact of these point mutations is not discussed further.

The I276F mutation displayed a further fivefold improved kcat/Km

over the previously discussed mutants. This corresponds to a kcat/Km

that is only 30-fold lower than that of the parent glycosynthase with itsfavored lipid substrate 4. We rationalized that the bulky I276Fmutation prevents the substrate inhibition by blocking the bindingof a second lipid chain in the ceramide-binding channel (Fig. 2).Consistent with this, introduction of the I276F mutation into wild-type (hydrolytic) EGC did not affect its ability to process glyco-sphingolipid 1, but caused a 25% loss in activity with GM1 (10), whichhas a full (acylated) ceramide chain (Supplementary Fig. 3 andSupplementary Methods online). The I276F mutation does notappear to affect the affinity for 6, and this poor binding meant thatsubstrate saturation was not possible.

In contrast, the D314Y mutant exhibited a markedly increasedaffinity for 6 (Table 1), with a Km of 0.19 mM, which is twofold

Table 1 Kinetic parameters for phytosphingosine

EGC mutant kcat (s�1) Km (mM) kcat/Km (s�1 M�1)

E351S (parent) — Ki 0.85 Tracea

V101M K200R — — 3.1b

K129N — — 3.1b

N148S — — 2.1b

I276F — — 16b

D314Y 0.07 0.19 358c

Kinetic parameters were determined using phytosphingosine (6) as the lipid acceptorand GM1OSF (5) as donor. Reactions were monitored using a fluoride ion–sensitiveelectrode; see Methods for assay conditions.aE351S EGC is uncompetitively substrate inhibited by 6 (see text); thus kcat/Km cannot beaccurately determined. bWhere saturation of 6 was not achievable, kcat/Km values weredetermined from the gradient of a plot of rate versus substrate concentration. ckcat/Km valuewas calculated from kcat and Km determined from the Michaelis-Menten plot.

Table 2 Kinetic parameters for sphingosine and GM1OSF

GM1OSF (5) Sphingosine (4)

EGC mutant kcat (s�1) Km (mM) kcat/Km (s�1 M�1) kcat (s�1) Km (mM) Kcat/Km (s�1 M�1)

E351S (parent) 0.21 0.77 273 0.19 0.41 463

A81E 0.12 0.97 123 0.12 0.20 600

A153T 0.17 0.42 405 0.20 0.46 435

R177H 0.06 0.32 188 0.06 0.17 353

I183F 0.02 0.18 135 0.09 0.18 527

I276F 0.11 1.3 86 0.11 0.13 846

T257I T416A 0.11 0.37 297 0.18 0.87 206

V269I 0.13a 0.60 217 0.18 0.81 222

V269I G370R 0.17a 0.89 191 0.26 0.48 542

G370E 0.21a 0.85 247 0.28 0.67 418

N334S 0.13a 0.70 186 0.19 0.68 279

D314Y 0.49b 0.58 845c 0.70 1.6 443

P394S 0.22 0.69 319 0.33 0.50 660

A153T D314Yd 0.30b 0.27 1,111c 0.64 1.6 407

A153T D314Y R177Hd 0.28 0.80 350 0.24 0.49 490

Values improved over those of the parent E351S glycosynthase are highlighted in bold. In some cases, kcat reflects a Vmax apparent at 10 mM 4 due to substrate inhibition at 10 mM 4 (a)or nonsaturating conditions due to increased Km of 4 (b). The corresponding kcat/Km values have therefore not been calculated or will be higher than reported at saturating concentrationsof 4 (c). kcat/Km values were calculated from individual values of kcat and Km determined from a Michaelis-Menten plot. Evaluation of combinations of single point mutants (d) made bysite-directed mutagenesis.

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better than the binding of the parent glycosynthase to its ‘natural’substrate, 4. As a result, the D314Y mutant exhibits saturating kineticsand (notably) shows no indication of substrate inhibition (forMichaelis-Menten and Lineweaver-Burk plots see SupplementaryFig. 4 online). In addition, the kcat is elevated from an almostundetectable level in the progenitor glycosynthase to being onlythreefold lower than that for E351S EGC–catalyzed activity with 4.We reason that the D314Y mutation induces movement in residuesArg177 and Asp311, which function to form the tunnel over the lipidhead group upon substrate binding, and through these residues theD314Y mutation influences lipid specificity (Fig. 2b). Regardless ofthe mechanism, this single point mutation transforms activity with 6from trace levels to almost the same kcat/Km as that for 4.

Lipid promiscuity of the D314Y mutantPrevious directed evolution studies have found that selection forchanges in substrate specificity often results in only modest changesto the primary activity, even though no selective pressure was main-tained on that primary activity23. These mutations often give rise tofurther promiscuous activities in the evolved catalysts. The parentE351S EGC glycosynthase was previously shown to have a lipidspecificity ratio (kcat/Km) of 1:0.07:0.0001 for 4:7:6 (ref. 12)(Fig. 1b), so we compared this activity ratio to that of the D314Ymutant (Tables 1 and 2). Indeed, D314Y does maintain its primaryactivity and exhibits a 1:0.81 ratio for 4:6. However, no activity couldbe detected for the D314Y mutant with sphingolipid 7. We alsoevaluated (1R,2S)-2-amino-1-phenylpropan-1,3-diol (APPD, 9) as anonselected substrate. APPD is a sphingosine derivative with stereo-chemistry identical to that of 4 that contains a phenyl group instead ofthe hydrocarbon chain. The kinetic parameters for 9 with the parentE351S glycosynthase were so low that they could not be detected abovethe hydrolytic background of the reaction. In contrast, D314Y showedsaturating kinetics for 9 with a kcat/Km of 65 M–1 s–1. These resultssuggest that while the D314Y mutation does not result in theformation of a truly broad-specificity catalyst (as stereochemistry atC3 is important), it does have broadened specificity for interactions atC4 and beyond. This is consistent with the screen used, as both 4 and6 have identical stereochemistry at C2 and C3. A modifying selectivepressure was therefore only asserted at C4, and here the evolvedenzyme does indeed appear to be promiscuous.

Synthesis of phytosphingosine-derived glycosphingolipidsGlycosphingolipids 1 and 11 could be readily purified from pooledreactions from kinetic analyses, and indeed 10–20 mg quantities wereobtained in that way. To further demonstrate the synthetic utility ofthe evolved D314Y mutant, the synthesis of phytosphingosine-derived

lyso-GM3 (13) was carried out on a 10 mg scale. A 1.5:1 molar ratioof GM3OSF (14):6 was incubated in 25 mM NaOAc, pH 5, 10% (v/v)1,2-dimethoxyethane with a 20 mM final concentration of the D314Ymutant. The reaction showed complete consumption of 6, and gave a54% purified yield, thus demonstrating to our knowledge the firstsynthetic access to phytosphingosine-derived glycosphingolipids.

Mutants identified from sphingosine screensSeveral of the mutants exhibiting improved activity with sphingolipid4 contained mutations that were present in more than one instance(Supplementary Table 1 online). In addition to increasing activitywith lipid acceptor 6, the I276F mutation arose twice within theidentified mutants for improvement with 4—both as a single pointmutant and in combination with the E432K mutation. Preliminaryanalysis indicated that the E432K mutation did not contribute to theI276F E432K phenotype, and so only the single point I276F mutantwas analyzed further. Ile276 resides in the ceramide binding channel ofEGC (Fig. 2), and mutation to a bulkier, hydrophobic residue mayenable more hydrophobic contacts with the lipid substrate, thusrationalizing the observed 0.41 to 0.13 mM improvement in bindingaffinity for 4 (Table 2). In agreement with this, a sequence alignmentwith other endo-glycoceramidases (Supplementary Fig. 5 online)shows that a hydrophobic residue that is generally aliphatic is con-served at this position, except in the Propionibacterium acnes andCyanea nozakii enzymes, which have aromatic residues. The I276Fmutation negatively impacts kcat and Km for the glycosyl fluoridedonor 5; thus the kcat/Km for 4 is increased twofold while activity with5 is reduced overall.

A side effect, presumably arising from the low substrate concentra-tions that were used during the screening process (concentrations oosubstrate Km values), is that we have predominantly evolved forimproved substrate binding: the A81E mutant binds 4 twofold moretightly than the parent glycosynthase; the R177H and I183F mutantsbind both 5 and 4 twofold to fourfold more tightly than the parentglycosynthase; and the T257I T416A mutant binds 5 with improvedaffinity compared to the parent glycosynthase (the first mutation isprobably responsible for this phenotype, based on structural location).This manifestation of one of the general rules of directed evolu-tion23—you get what you screen for—reminds us that in order toselect for increased kcat, it is wise to screen at high substrateconcentrations if at all possible.

Various amino acid substitutions at Gly370, and the point mutationsN334S and V269I, each arose several times during screening (Supple-mentary Table 1), but kinetic analyses were inconsistent withimproved synthetic activity. We tested whether these effects were aconsequence of differential product inhibition by 1 compared to the

a b

Arg177

Ile276

Asp314

Pro394

Asp314

Arg177

Asp311

Gln280

Asn279Glu179

Figure 2 The structural location of the identified

glycosynthase mutants. (a) Surface representation

of the E351S EGC in complex with GM3. Red

areas indicate the sites of mutants displaying

improved activity for the synthesis of lyso-GM1 (1).

(b) Details of the sphingolipid binding site. The

image highlights the potential interplay of the

D314Y mutation with the tunnel-forming residues

Arg177 and Asp311. The distance between

Asp314 and Asp311 (green dashed line) is 6.3 A.

All interactions between E351S EGC and the GM3

lipid moiety are shown in red, water molecules

are shown in cyan, and the C4 of sphingosine,

the site at which it differs from phytosphingosine,

is shown in orange.

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parent glycosynthase (Supplementary Methods and SupplementaryFig. 6 online); however, none of the mutants showed a significantchange in Ki. Either these mutations are false positives, or thesediscrepancies in activity reflect the different assay modes—plate-bound in the initial screen versus solution-based in the validation assay.

The P394S mutation does not affect kinetic parameters withglycosyl fluoride 5, but increases the kcat for sphingolipid 4 from0.19 to 0.33 s–1. It is unclear why only the kcat for 4 is affected. It is alsoambiguous why this mutation, which is near the binding site for thesialic acid (Neu5Ac) residue of GM3 (Fig. 2), affects the distant lipidbinding site, with no apparent effect on the sugar binding, though theeffects are small.

Ala153 is located in a flexible loop that only becomes ordered inthe EGC-GM3 complex crystal structure and makes close contactswith the 4-hydroxyl of galactose in GM3. Thus, to accommodate thesugars present on this hydroxyl in the longer, GM1-based substrates,a shift in the protein conformation or the sugar binding mode isnecessary. The A153T mutation, which was separately identifiedtwice during the screening process, had no effect on the kcat or theKm for the lipid substrate 4, but increased the kcat/Km for 5 througha twofold decrease in Km (Table 2). In order to test the idea thatthis mutation stabilizes the GM1-binding loop conformer, whichmight result in a loss of GM3OSF (14) activity relative to E351SEGC, we measured the kinetic parameters shown in Table 3.Indeed, the A153T mutation appears to switch the 2:1 kcat/Km

preference of 14:5 observed in the E351S EGC glycosynthase, largelythrough effects on Km.

The large enhancement in affinity observed for sphingolipid 6 in theD314Y mutant is at the expense of the Km for the natural lipidsubstrate 4, which rises fourfold to 1.6 mM as a consequence of themutation. However, kcat increases by an equal amount such that kcat/Km

is unchanged. Consequently, it was not possible to achieve saturationof the lipid when probing the kinetic parameters for 5, so the threefoldoverall increase in kcat/Km for that substrate is an underestimate of thetrue value. In the crystal structure of the EGC-GM3 complex, Asp314 islocated approximately 10 A from both the Neu5Ac sugar of GM3 andthe sphingolipid (Fig. 2), which is consistent with the ability of thismutation to affect the kinetic parameters for both the sugar and lipidsubstrates. Given that EGC glycosynthase synthetic reactions aretypically conducted at 10 mM sugar fluoride and sphingolipid con-centrations, the high Km value of this mutant is essentially overridden,and the substantially (4threefold) improved kcat of the D314Y mutantis of great value for synthetic purposes.

Rational design of third-generation catalystsHaving identified several mutations that result in improved activitywith sphingolipid 4, we attempted to rationally combine these muta-tions to see if even more potent enzymes could be created. First, wemade the double point mutant A153T D314Y in an attempt tocombine the improved affinity for the glycosyl fluoride 5 from theA153T mutation with the enhanced kcat from the D314Y mutant.Kinetic analyses (Table 2) showed that this combination successfully

amalgamated the advantageous properties of the individual muta-tions, resulting in a catalyst with a beneficial 3.6 kJ mol–1 reduction inactivation energy for utilization of 5 compared to that of the E351SEGC parent glycosynthase. However, attempts to garner a furtherincrease in kcat by additional incorporation of the P394S mutant wereunsuccessful, as the triple point mutant P394S A153T D314Y wasunstable under the assay conditions. In order to counteract thedetrimental effect of the D314Y mutation on the affinity of sphingo-lipid 4, we chose the R177H mutation from among several pointmutations that had resulted in improved affinity for 4, because thismutation had the least effect on kcat and did not impair affinity forthe sugar substrate (compared to I276F and I183F). As predicted, theKm for 4 was threefold lower for the triple point mutant R177HA153T D314Y compared to the A153T D314Y and D314Y mutants.Unfortunately the kcat value was also lower, and overall the R177HA153T D314Y mutant showed only a modest 0.6 kJ mol–1 reduction inactivation energy compared to the E351S EGC parent glycosynthasefor 5.

DISCUSSIONThrough application of a directed evolution strategy involving arobotic ELISA-based screening process, we have succeeded in convert-ing a glycosphingolipid-synthesizing enzyme with only trace activityfor sphingolipid 6 into one that functions at rates comparable to thoseof the parent enzyme with native lipid substrate 4, without any negativeimpact on kcat/Km for this primary substrate. As is often the case indirected evolution experiments, the mutated residue is not in directcontact with the selected substrate at the point where it differs fromthat of the natural substrate. Rather, the mutation appears to facilitatereorganization of the active site to accept the substrate modification. Asa consequence, and consistent with what has been observed by others23,the mutant identified shows a generally relaxed specificity at the‘selected’ position, with no substantial loss of activity for the primary‘native’ substrate. The mutant does however retain specificity at siteswhere no opposing selective pressure was implemented.

When selection was applied for increased activity with the naturalsubstrate 4, the resulting mutations typically produced catalysts withimproved binding affinities. This outcome is a consequence of theevolutionary pressure that nature has applied to the parent hydrolase,where tight binding of the glycosphingolipid substrate and weakbinding of sugar and sphingolipid products are advantageous pheno-types. Upon conversion of the hydrolase to a glycosynthase and‘reversal’ of the reaction, these properties become unfavorable,although they can be overcome, to some extent, by use of very highsubstrate concentrations. Nonetheless, limitations to this approachexist with substrates of low aqueous solubility such as lipids. Ourselection for improved Km values is thus very valuable. This could alsobe the likely cause of a certain number of false positives, as thescreening was based on surface-displayed lipids of ill-defined concen-trations, whereas validation was performed in a solution phase assay.

By providing catalysts capable of producing well-defined samples ofsphingosine and phytosphingosine-containing glycosphingolipids,

Table 3 Comparison of the kinetic parameters for GM3OSF and GM1OSF with E351S EGC and the A153T EGC mutant

GM3OSF (14) GM1OSF (5)

EGC mutant kcat (s�1) Km (mM) kcat/Km (s�1 M�1) kcat (s�1) Km (mM) kcat/Km (s�1 M�1)

E351S (parent) 0.13 0.29 448 0.21 0.77 273

A153T 0.28 1.3 212 0.17 0.42 405

We used sphingosine (4) as acceptor for this comparison.

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this study provides a more efficient synthetic strategy for the one-step, activation and protecting group-free synthesis of glycosphingo-lipids, and thereby represents a substantial advance toward delineatingthe function of glycosphingolipids in biological phenomena.

METHODSMutant library generation. Two random mutant libraries were generated with

the GeneMorph mutagenesis kit (Stratagene) using 500 ng and 2 mg of pET28a

E351S EGC template, respectively, following the manufacturer’s protocol. The

primers used were: T7for: 5¢-CCCGCGAAATTAATACGACTCACTATAG-3¢;T7rev: 5¢-CGTTTAGAGGCCCCAAGGGGTTATGCTAG-3¢. PCR products

were ligated into pET28a at the NdeI XhoI restriction sites using the EasyClone

(Lucigen) T4 DNA ligase for 2 h and transformed into Escherichia cloni 10G

ELITE electrocompetent cells (Lucigen). Plasmid DNA from individual colo-

nies of each library was sequenced, and the two libraries were shown to have

mutational frequencies of 0.8 and 1.5 mutations per kilobase, respectively.

Subsequently colonies were washed from the agar plates with LB medium (2 �2 ml), and plasmid DNA was prepared and transformed into Tuner (DE3) cells

(Novagen) for screening.

Library screening. A detailed description of the development and optimization

of the screening protocol has been previously reported30. The screens were

performed on a Biomek FX liquid handling workstation (Beckman Coulter)

equipped with a 96-channel pipetting head and an integrated DTX880 plate

reader. Lipids were noncovalently immobilized onto microplates by evaporat-

ing 50 ml solutions in ethanol (0.4 mM 4, 0.1 mM 6 or 0.2 mg ml–1 10) in each

well at 37 1C. The plates were washed and blocked with 1% (w v) bovine serum

albumin, and each well was incubated with 5 (0.1 mM final concentration in

25 mM NaOAc, pH 5 containing 0.2% (v v) Triton X-100) and unclarified cell

lysate from a single E351S EGC mutant library colony. We allowed the enzyme

reaction to proceed for 7 h (4) or 16 h (6). On-plate synthesis of lyso-

glycosphingolipid products 1 and 11 was detected by sequential incubation

with cholera toxin B subunit (2.4 mg ml–1), primary antibody (mouse

monoclonal to cholera toxin B subunit at 1:20,000 dilution) and secondary

antibody (rabbit polyclonal to mouse IgG H&L horseradish peroxidase con-

jugate at 1:16,000 dilution). HRP activity was detected at 405 nm after addition

of 2,2¢-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) substrate (15).

Further assessment of putative hits was carried out in the same manner,

replacing cell lysates with purified mutant EGC (25 mg ml–1).

Expression and purification of EGC mutants. Expression and purification of

EGC mutants was carried out as previously described12. We dialyzed EGC

mutants into 25 mM NaOAc, pH 5 immediately before use. Protein concen-

trations were determined as described previously31 with reference to bovine

serum albumin standards.

Site-directed mutagenesis. Point mutations were introduced using the Quik-

Change methodology (Stratagene) using the manufacturer’s protocol. We

confirmed all the mutations by DNA sequencing.

Fluoride electrode kinetics. We determined the Michaelis-Menten kinetic

parameters for the glycosynthases by varying the concentration of either the

sugar fluoride donor or lipid acceptor while using a fixed, saturating concentra-

tion for the other substrate. Reactions were initiated by lipid addition and

carried out at 37 1C in 25 mM sodium acetate, pH 5.0, containing 8.3% (v/v)

1,2-dimethoxyethane (for discussion of EGC glycosynthase stability see Supple-

mentary Methods). The initial rate (o5% substrate depletion) of fluoride

release was measured using a VWR Symphony fluoride electrode interfaced with

Logger Pro 2.2.1 analysis software (Vernier, Inc.). For lipid acceptor kinetics (at

fixed, high sugar fluoride donor concentration), the rate of spontaneous

hydrolysis of the sugar fluoride was substantial at low lipid concentrations

and was therefore subtracted from all measured enzymatic rates. Michaelis

parameters were determined by nonlinear regression analysis using GraFit 4.0

(Erithacus Software). The standard errors in kcat were 5–10%; in Km they were

10–25%. The difference in Gibbs energy of activation was calculated as follows:

DDGz¼ � RTlnððkcat=KmÞEGC mutant=ðkcat=KmÞE351S EGCÞ

Other methods For all other methods including further details of kinetic

assays and synthetic reactions, see the Supplementary Methods. Glyco-

sphingolipid product characterization data can be found in Supplementary

Tables 2 and 3 onlineand in theSupplementaryMethods. Figures were prepared

using PyMOL32.

Accession codes. Protein Data Bank: The structure of the EGC glycosynthase

(E351S) in complex with GM3 (accession number 2OSX) was deposited as part

of a previous study12.

Note: Supplementary information and chemical compound information is available onthe Nature Chemical Biology website.

ACKNOWLEDGMENTSThe authors thank the Royal Society (UK) and the Government of Canada forpostdoctoral fellowships (S.M.H.), the Natural Sciences and Engineering ResearchCouncil of Canada and Neose Technologies Ltd. for funding, and W. Hol(University of Washington) for providing the recombinant gene for choleratoxin B subunit.

AUTHOR CONTRIBUTIONSS.M.H. and S.G.W. designed the experiments; S.M.H. performed the experiments;S.M.H., M.E.C.C. and S.G.W. analyzed the data; J.R.R. characterized the syntheticproducts; S.M.H. and S.G.W. wrote the manuscript; M.E.C.C. and S.M.H. madethe figures; M.E.C.C. and N.C.J.S. provided feedback on the manuscript.

COMPETING INTERESTS STATEMENTThe authors declare competing financial interests: details accompany the full-textHTML version of the paper at http://www.nature.com/naturechemicalbiology/.

Published online at http://www.nature.com/naturechemicalbiology/

Reprints and permissions information is available online at http://npg.nature.com/

reprintsandpermissions/

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