Communications
Appl.Microbiol.Biotechnol. 2001, 55, 519 – 530; g) M. T. Reetz,
Methods Enzymol. 2004, 388, 238 – 256; h) S. Park, K. L. Morley,
G. P. Horsman, M. Holmquist, K. Hult, R. J. Kazlauskas, Chem.
Biol. 2005, 12, 45 – 54.
the catalytic site more accessible to larger substrates, but also
more labile. This argument was supported experimentally by
the analysis of the substrate-specificity profile (Figure 1), in
which it was clear that the active site accommodated longer
fatty acid esters, and by the higher susceptibility of EL1 to
chemical inactivation and denaturation (Figure 2). In addi-
tion, the midpoint of unfolding was 128C lower. To prove the
supposed interaction between D33 and R49, and the fact that
this interaction affects the catalytic activity of the mutant
enzyme toward triacylglycerols, single R49D and R49N
mutant variants of the enzyme were generated by site-
directed mutagenesis. Mutations at R49 produced variants
with no activity on aNL plates, but they did hydrolyze
aNA.[12e] Furthermore, we also introduced a reversed muta-
tion N33R-R49D in R.34 and obtained the lipase phenotype
in both aNL[12f] and rhodamine–triolein plates.[12g] These
results unambiguously confirm that the interaction between
residues 33 and 49 exists, and that it is essential for the
substrate preference of the R.34 enzyme.
In summary, we have provided clear proof that the
substrate specificity of a true carboxylesterase can be
modified toward insoluble substrates, that is, turned into a
true triacylglycerol lipase, without modification of the shape,
size, or hydrophobicity of the substrate-binding sites that are
considered to be essential for chain-length specificity.[1,7,8]
Moreover, minimal changes in the structure are sufficient
for enhancing the acyl chain-length preference of esterases.
More significantly, compared with other lipases,[17,18] the EL1
mutant may constitute an important step toward the synthesis
of structured lipids[19] and may have other lipase applications,
which are under investigation.
[7] For examples, see: a) R. D. Joerger, M. J. Hass, Lipids 1994, 29,
377 – 384; b) H. Atomi, U. Bornscheuer, M. M. Soumanou, H. D.
Beer, G. Wohlfahrt, R. D. Schmid, Microbial Lipases: From
Screening to Design, Vol.1 , Barnes, Bridgwater, 1996, pp. 49 –
50; c) R. R. Klein, G. King, R. A. Moreau, M. J. Haas, Lipids
1997, 32, 123 – 130; d) T. Eggert, G. Pencreacꢀh, I. Douchet, R.
Verger, K.-EJaeger, Eur.J.Biochem. 2000, 267, 6459 – 6469; e) I.
Kauffmann, C. Schmidt-Dannert, Protein Eng. 2001, 14, 919 –
928; f) J. Yang, Y. Koga, H. Nakano, T. Yamane, Protein Eng.
2002, 15, 147 – 152.
[8] J. Pleiss, M. Fischer, R. D. Schmid, Chem.Phys.Lipids 1998, 93,
67 – 80.
[9] R. Verger, Trends Biotechnol. 1997, 15, 32 – 38.
[10] Wild-type esterase was retrieved from the bacteriophage
lambda-based expression library created from DNA extracted
from bovine rumen fluid, after screening in NZY soft agar
containing a-naphthyl acetate (aNA), and expressed from the
pBK-CMV phagemid pBKR.34 in E.coli XLOLR. Sequence
analysis of R.34 is consistent with a 273 amino acid protein of
Mr = 34173.99 Da and an isoelectric point of 5.03. It belongs to
the ester hydrolase of family II of the Arpigny and Jaeger
classification,[11] according to the conserved motif GDS(L); the
catalytic triad was deduced to be formed by Ser137, Asp215, and
His247. For details, see: M. Ferrer, O. V. Golyshina, T. N.
Chernikova, A. N. Khachane, D. Reyes-Duarte, V. A. P. Mar-
tins Dos Santos, C. Strömpl, K. Elborough, G. Jarvis, A. Neef,
M. M. Yakimov, K. N. Timmis, P. N. Golyshin, Environ.Micro-
biol. 2005, in press.
[11] J. L. Arpigny, K.-E. Jaeger, Biochem.J. 1999, 343, 177 – 183.
[12] a) Detailed experimental procedures are available in the Sup-
porting Information; b) The esterase–lipase phenotype of the
EL1 improved variant and wild-type R.34 is shown in the
Supporting Information; c) The optimal pH (7.5), temperature
(508C), and subunit composition (monomer of ꢁ 34 kDa) were
essentially the same for both R.34 and EL1 enzymes (see the
Supporting Information); d) HPLC chromatograms of the
reaction products (from mono- to triglycerides) are shown in
the Supporting Information; e) The esterase–lipase phenotype
of EL1 variants containing R49N and R49D mutations is shown
in the Supporting Information; f) The esterase–lipase phenotype
of R.34 variant containing a reverse mutation N33R-R49D is
shown in the Supporting Information; g) The lipase phenotype
of R.34, EL1, and mutant variants on rhodamine–triolein plates
is shown in the Supporting Information.[15]
Received: July 14, 2005
Revised: September 26, 2005
Published online: October 27, 2005
Keywords: enzymes · esterases · lipases · mutagenesis ·
.
regiospecificity
[1] U. T. Bornscheuer, FEMS Microbiol.Rev. 2002, 26, 73 – 81.
[2] K.-E. Jaeger, T. Eggert, Curr.Opin.Biotechnol. 2002, 13, 390 –
397.
[3] K.-E. Jaeger, B. W. Dijstra, M. T. Reetz, Annu.Rev.Microbiol.
1999, 53, 315 – 351.
[13] V. Khalameyzer, I. Fischer, U. T. Bornscheuer, J. Altenbuchner,
Appl.Environ.Microbiol. 1999, 65, 477 – 482.
[14] a) Sequence alignment of R.34 esterase and other xylanases and
esterases is shown in the Supporting Information; b) The
structure of esterase EST2 from A.acidocaldarius (PDB Acc.
number 1EVQA) was chosen as the most suitable template to
generate a model for R.34. See G. De Simone, S. Galdiero, G.
Manco, D. Lang, M. Rossi, C. Pedone, J.Mol.Biol. 2000, 303,
761 – 771. The degree of sequence identity between these two
proteins is 19%. Ramachandran plots of both model and
template proteins (see the Supporting Information) were
obtained to assess the overall stereochemical quality of the
model. The model is not reliable at the N-terminal part of the
structure (first 32 residues of esterase R.34), where the sequence
similarity between the model and template is very low. However,
the results of threading[16] indicate a consistent structural
similarity in the region starting at residue 33.
[4] P. Fojan, P. H. Jonson, M. T. N. Petersen, S. B. Petersen, Bio-
chimie, 2000, 82, 1033 – 1041.
[5] For examples, see: a) N. Zhang, W. C. Suen, W. Windsor, L.
Xiao, V. Madison, A. Zaks, Protein Eng. 2003, 16, 599 – 605; b) P.
Acharya, E. Rajakumara, R. Sankaranarayanan, N. M. Rao, J.
Mol.Biol. 2004, 341, 1271 – 1281; c) G. Santarossa, P. G. Lafran-
coni, C. Alquati, L. DeGioia, L. Alberghina, P. Fantucci, M.
Lotti, FEBS Lett. 2005, 579, 2383 – 2386.
[6] For examples, see: a) U. T. Bornscheuer, J. Altenbuchner, H. H.
Meyer, Biotechnol.Bioeng. 1998, 58, 554 – 559; b) N. Krebs-
fänger, K. Schierholz, U. T. Bornscheuer, J.Biotechnol. 1998, 60,
105 – 111; c) N. Krebsfänger, F. Zocher, J. Altenbuchner, U. T.
Bornscheuer, Enzyme Microb.Technol. 1998, 22, 641 – 646; d) E.
Henke, U. T. Bornscheuer, Biol.Chem. 1999, 380, 1029 – 1033;
e) K. Liebeton, A. Zonta, K. Schimossek, M. Nardini, D. Lang,
B. W. Dijstra, M. T. Reetz, K.-E. Jaeger, Chem.Biol. 2000, 7,
709 – 718; f) K.-E. Jaeger, T. Eggert, A. Eipper, M. T. Reetz,
[15] G. Kouker, K.-E. Jaeger, Appl.Environ.Microbiol. 1987, 53,
211 – 213.
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Angew. Chem. Int. Ed. 2005, 44, 7553 –7557