98
E.D. Goddard-Borger et al. / Carbohydrate Research 435 (2016) 97e99
Fig. 1. Mechanism of EGALC galactosylceramidase and its glycosynthase mutant. (A) The proposed mechanism for the hydrolysis of psychosine by the retaining glycosidase
EGALC. (B) Upon mutating the catalytic nucleophile, the enzyme cannot carry out hydrolysis but can instead mediate glycoside bond formation when supplied with an activated
glycosyl fluoride donor.
bodies. We transitioned to using the secretory pathway in E. coli by
the addition of the PelB signal peptide to target EGALC to the
periplasmic space (Figure S1). After much optimization we could
repeatedly obtain high yields (20 mg lꢀ1) of recombinant EGALC
from the culture supernatant, though prolonged incubation (30 h)
in autoinduction media was absolutely essential for reproducible
yields. Our recombinant EGALC was capable of hydrolyzing psy-
chosine, in line with previous reports on the specificity of this
enzyme [18].
psychosine was relatively low, the ability to produce the glycolipid
in a single step and to readily recover the unused sphingosine
makes the EGALC glycosynthase reaction a viable method for the
synthesis of isotopically labeled psychosine for diagnostic
purposes.
2. Experimental
2.1. Materials
Based upon previous experience in generating other glyco-
synthases, site-directed mutagenesis was performed to produce
three catalytic nucleophile mutants: E341G, E341A, and E341S. The
EGALC E341A/S mutants expressed very well, comparable to the
wild-type enzyme, while the glycine mutant failed to provide any
protein. Neither the E341A nor the E341S mutant had any hydro-
lytic activity on psychosine, as anticipated. Both mutants were
All chemical reagents were purchased from Sigma-Aldrich at
>95% purity unless otherwise stated. 1H and 13C NMR spectra were
recorded using a 400 MHz instrument. All signals were referenced
to solvent peaks (CDCl3: d
7.26 ppm for 1H NMR). TLC analysis used
aluminum backed Merck Silica Gel 60 F254 sheets, detection was
achieved using UV light, 5% H2SO4 in MeOH, or ceric ammonium
molybdate solution with heating as necessary.
evaluated as glycosynthases using sphingosine and
a-D-gal-
actopyranosyl fluoride. Only low activity was observed during the
initial trial reactions, though a small amount of psychosine was
observed by mass spectrometry and thin layer chromatography.
Qualitatively, E341S appeared to be the more active mutant and so
we set about optimizing reaction conditions with this variant.
Two large issues that we had to contend with were the poor
solubility of psychosine and the propensity for EGALC to precipitate
from solution under the reaction conditions. We exhaustively
explored the use of different buffering agents, pH, co-solvents,
detergents, molecular crowding agents and amphiphilic host mol-
ecules to increase substrate and enzyme solubility and to maximize
conversion of substrate to product. Optimized conditions were
2.2. Cloning and expression
A codon-harmonized gene encoding the PelB signal peptide,
residues 23e488 of EGALC (BAF56440.1) and a C-terminal hex-
ahistidine tag was synthesized (BioBasic) and cloned into
pET29b(þ) (Novagen). The sequence of the gene and the protein it
encodes is detailed in Figure S1 (Supplementary Data). Site directed
mutagenesis of EGALC was accomplished using the four-primer
PCR method [20] and the primers detailed in Table S1 (Supple-
mentary Data). Sequence-validated plasmids were transformed
into BL21*(DE3), selecting with kanamycin. Single colonies were
used to inoculate 10 ml of LB media (50 m
g mlꢀ1 Kan), which was
established as: 200 mM
a-D-galactopyranosyl fluoride, 10 mM
incubated overnight at 37 ꢁC. The overnight culture was used to
inoculate two 500 ml cultures of LBE-5052 auto-induction media
sphingosine, 500 mM MOPS (pH 7.5), 20 mM
a
-cyclodextrin and
2.5% methanol. The order of operations was critical for the reaction.
The sphingosine was first dispersed in methanol at 40 ꢁC with the
aid of sonication. The glycosyl fluoride and cyclodextrin were
concurrently dissolved in the MOPS buffer and the resulting solu-
tion was added to the sphingosine in methanol. This mixture was
sonicated at 40 ꢁC for 15 min then cooled to 37 ꢁC and the enzyme
added. The resulting turbid mixture was gently agitated at 37 ꢁC for
3 days or until all the galactosyl fluoride had been depleted. The
psychosine product was extracted using chloroform and purified by
chromatography on silica and C-8 silica to provide pyschosine in
21% yield, along with recovered sphingosine. Although the yield of
(50 m
g mlꢀ1 Kan). These were incubated at 30 ꢁC and shaken at
250 rpm for 36 h. The cultures were centrifuged at 17,000 ꢂ g for
20 min at 4 ꢁC. The culture supernatants were collected and treated
with protease inhibitor cocktail. The supernatant was filtered
through a glass fiber filter then concentrated to a volume of 40 ml
using stirred cells and a membrane with a 10 kDa NMWL. This
concentrate was diluted with 4.5 ml of 8 ꢂ binding buffer (160 mM
Tris, 4 M NaCl, 40 mM ImH, pH 8). The sample was centrifuged at
17,000 ꢂ g for 20 min and filtered before loading on a 1 ml HisTrap
column. The protein was eluted from the column using a gradient of
Goddard-Borger, Ethan D.
