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PDBsum entry 1c00
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Obsolete entry |
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PDB id:
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Hydrolase
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Title:
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Thermophylic pnb esterase
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Structure:
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Para-nitrobenzyl esterase. Chain: a. Fragment: a56v, i60v, t73k, l313f, h322y, a343v, m358v, y370f, a400t, g412e, e420g, i437t, t459s. Synonym: pnb esterase. Engineered: yes
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Source:
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Bacillus subtilis. Bacteria. Expressed in: escherichia coli.
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Resolution:
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2.00Å
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R-factor:
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0.203
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R-free:
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0.244
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Authors:
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B.Spiller,A.Gershenson,F.Arnold,R.Stevens
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Key ref:
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B.Spiller
et al.
(1999).
A structural view of evolutionary divergence.
Proc Natl Acad Sci U S A,
96,
12305-12310.
PubMed id:
DOI:
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Date:
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13-Jul-99
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Release date:
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21-Jul-99
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PROCHECK
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Headers
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References
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P37967
(PNBA_BACSU) -
Para-nitrobenzyl esterase from Bacillus subtilis (strain 168)
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Seq:
Struc:
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489 a.a.
483 a.a.*
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Key: |
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PfamA domain |
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Secondary structure |
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CATH domain |
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*
PDB and UniProt seqs differ
at 22 residue positions (black
crosses)
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DOI no:
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Proc Natl Acad Sci U S A
96:12305-12310
(1999)
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PubMed id:
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A structural view of evolutionary divergence.
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B.Spiller,
A.Gershenson,
F.H.Arnold,
R.C.Stevens.
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ABSTRACT
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Two directed evolution experiments on p-nitrobenzyl esterase yielded one enzyme
with a 100-fold increased activity in aqueous-organic solvents and another with
a 17 degrees C increase in thermostability. Structures of the wild type and its
organophilic and thermophilic counterparts are presented at resolutions of 1.5
A, 1.6 A, and 2.0 A, respectively. These structures identify groups of
interacting mutations and demonstrate how directed evolution can traverse
complex fitness landscapes. Early-generation mutations stabilize flexible loops
not visible in the wild-type structure and set the stage for further beneficial
mutations in later generations. The mutations exert their influence on the
esterase structure over large distances, in a manner that would be difficult to
predict. The loops with the largest structural changes generally are not the
sites of mutations. Similarly, none of the seven amino acid substitutions in the
organophile are in the active site, even though the enzyme experiences
significant changes in the organization of this site. In addition to reduction
of surface loop flexibility, thermostability in the evolved esterase results
from altered core packing, helix stabilization, and the acquisition of surface
salt bridges, in agreement with other comparative studies of mesophilic and
thermophilic enzymes. Crystallographic analysis of the wild type and its evolved
counterparts reveals networks of mutations that collectively reorganize the
active site. Interestingly, the changes that led to diversity within the
alpha/beta hydrolase enzyme family and the reorganization seen in this study
result from main-chain movements.
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Selected figure(s)
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Figure 1.
Fig. 1. MOLSCRIPT diagrams (15, 16) of pNB esterases
looking into the active site cavity, showing loops that are not
visible in the electron density as dashed lines and loops that
reorganize most significantly in gold. The catalytic triad is
shown in red and mutations are shown in blue. (A) The WT
structure with secondary structural elements labeled. (B) The
5-6c8 structure. (C) The 8g8 structure, rotated slightly from
the others to clarify the location of the mutations. (D) A
wall-eyed stereo overlay of the C[  ]positions
of the three structures, oriented as in A and B. WT is shown in
blue, organophile 5-6c8 in green, and thermophile 8g8 in purple.
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Figure 3.
Fig. 3. Comparison between the thermophile 8g8 (purple)
and WT (blue). (A) A superposition of the 315-324 and 265-275
loops. The His-322  Tyr
mutation introduces a direct interaction between the loops. The
315-324 loop is pulled closer to the active site to accommodate
the smaller substrate, allowing a 3.5-Å H bond between
Tyr-322 and the main-chain N of Ile-270. Additionally, the new
orientation of 315-324 allows an H bond between the side chains
of Ser-323 and Thr-326, stabilizing helix 12 and the 315-324
loop. (B) A superposition of WT and 8g8 shows the effect of the
Met-358 Val
mutation. In the absence of the His-322 Arg
mutation, the large reorganization of 265-275 is not seen.
Leu-362 and Ile-270 move to fill the cavity created by the
Met-358 Val
mutation.
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Figures were
selected
by an automated process.
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Literature references that cite this PDB file's key reference
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PubMed id
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Reference
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I.Nobeli,
A.D.Favia,
and
J.M.Thornton
(2009).
Protein promiscuity and its implications for biotechnology.
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Nat Biotechnol,
27,
157-167.
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P.A.Romero,
and
F.H.Arnold
(2009).
Exploring protein fitness landscapes by directed evolution.
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Nat Rev Mol Cell Biol,
10,
866-876.
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M.S.Kim,
J.D.Weaver,
and
X.G.Lei
(2008).
Assembly of mutations for improving thermostability of Escherichia coli AppA2 phytase.
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Appl Microbiol Biotechnol,
79,
751-758.
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R.Kourist,
S.Bartsch,
L.Fransson,
K.Hult,
and
U.T.Bornscheuer
(2008).
Understanding promiscuous amidase activity of an esterase from Bacillus subtilis.
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Chembiochem,
9,
67-69.
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T.M.Streit,
A.Borazjani,
S.E.Lentz,
M.Wierdl,
P.M.Potter,
S.R.Gwaltney,
and
M.K.Ross
(2008).
Evaluation of the 'side door' in carboxylesterase-mediated catalysis and inhibition.
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Biol Chem,
389,
149-162.
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W.Li,
J.Ju,
S.R.Rajski,
H.Osada,
and
B.Shen
(2008).
Characterization of the tautomycin biosynthetic gene cluster from Streptomyces spiroverticillatus unveiling new insights into dialkylmaleic anhydride and polyketide biosynthesis.
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J Biol Chem,
283,
28607-28617.
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A.M.Dean,
and
J.W.Thornton
(2007).
Mechanistic approaches to the study of evolution: the functional synthesis.
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Nat Rev Genet,
8,
675-688.
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I.P.Fabrichny,
P.Leone,
G.Sulzenbacher,
D.Comoletti,
M.T.Miller,
P.Taylor,
Y.Bourne,
and
P.Marchot
(2007).
Structural analysis of the synaptic protein neuroligin and its beta-neurexin complex: determinants for folding and cell adhesion.
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Neuron,
56,
979-991.
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PDB codes:
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M.Schmidt,
E.Henke,
B.Heinze,
R.Kourist,
A.Hidalgo,
and
U.T.Bornscheuer
(2007).
A versatile esterase from Bacillus subtilis: cloning, expression, characterization, and its application in biocatalysis.
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Biotechnol J,
2,
249-253.
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P.Liu,
H.E.Ewis,
P.C.Tai,
C.D.Lu,
and
I.T.Weber
(2007).
Crystal structure of the Geobacillus stearothermophilus carboxylesterase Est55 and its activation of prodrug CPT-11.
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J Mol Biol,
367,
212-223.
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PDB codes:
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L.Yuan,
I.Kurek,
J.English,
and
R.Keenan
(2005).
Laboratory-directed protein evolution.
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Microbiol Mol Biol Rev,
69,
373-392.
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S.M.Mnisi,
M.E.Louw,
and
J.Theron
(2005).
Cloning and characterization of a carboxylesterase from Bacillus coagulans 81-11.
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Curr Microbiol,
50,
196-201.
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V.G.Eijsink,
S.Gåseidnes,
T.V.Borchert,
and
B.van den Burg
(2005).
Directed evolution of enzyme stability.
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Biomol Eng,
22,
21-30.
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D.Georlette,
V.Blaise,
T.Collins,
S.D'Amico,
E.Gratia,
A.Hoyoux,
J.C.Marx,
G.Sonan,
G.Feller,
and
C.Gerday
(2004).
Some like it cold: biocatalysis at low temperatures.
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FEMS Microbiol Rev,
28,
25-42.
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E.Henke,
U.T.Bornscheuer,
R.D.Schmid,
and
J.Pleiss
(2003).
A molecular mechanism of enantiorecognition of tertiary alcohols by carboxylesterases.
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Chembiochem,
4,
485-493.
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J.Hoseki,
A.Okamoto,
N.Takada,
A.Suenaga,
N.Futatsugi,
A.Konagaya,
M.Taiji,
T.Yano,
S.Kuramitsu,
and
H.Kagamiyama
(2003).
Increased rigidity of domain structures enhances the stability of a mutant enzyme created by directed evolution.
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Biochemistry,
42,
14469-14475.
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J.Koepke,
E.I.Scharff,
C.Lücke,
H.Rüterjans,
and
G.Fritzsch
(2003).
Statistical analysis of crystallographic data obtained from squid ganglion DFPase at 0.85 A resolution.
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Acta Crystallogr D Biol Crystallogr,
59,
1744-1754.
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PDB code:
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M.A.Ceruso,
A.Grottesi,
and
A.Di Nola
(2003).
Dynamic effects of mutations within two loops of cytochrome c551 from Pseudomonas aeruginosa.
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Proteins,
50,
222-229.
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R.J.Fletcher,
B.E.Bishop,
R.P.Leon,
R.A.Sclafani,
C.M.Ogata,
and
X.S.Chen
(2003).
The structure and function of MCM from archaeal M. Thermoautotrophicum.
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Nat Struct Biol,
10,
160-167.
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PDB code:
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B.van den Burg,
and
V.G.Eijsink
(2002).
Selection of mutations for increased protein stability.
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Curr Opin Biotechnol,
13,
333-337.
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D.N.Bolon,
C.A.Voigt,
and
S.L.Mayo
(2002).
De novo design of biocatalysts.
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Curr Opin Chem Biol,
6,
125-129.
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R.J.Hayes,
J.Bentzien,
M.L.Ary,
M.Y.Hwang,
J.M.Jacinto,
J.Vielmetter,
A.Kundu,
and
B.I.Dahiyat
(2002).
Combining computational and experimental screening for rapid optimization of protein properties.
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Proc Natl Acad Sci U S A,
99,
15926-15931.
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W.F.Bosron,
and
T.D.Hurley
(2002).
Lessons from a bacterial cocaine esterase.
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Nat Struct Biol,
9,
4-5.
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Y.Yamagata,
H.Maeda,
T.Nakajima,
and
E.Ichishima
(2002).
The molecular surface of proteolytic enzymes has an important role in stability of the enzymatic activity in extraordinary environments.
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Eur J Biochem,
269,
4577-4585.
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B.C.Verrelli,
and
W.F.Eanes
(2001).
The functional impact of Pgm amino acid polymorphism on glycogen content in Drosophila melanogaster.
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Genetics,
159,
201-210.
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M.Lehmann,
and
M.Wyss
(2001).
Engineering proteins for thermostability: the use of sequence alignments versus rational design and directed evolution.
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Curr Opin Biotechnol,
12,
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S.Raillard,
A.Krebber,
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J.E.Ness,
E.Bermudez,
R.Trinidad,
R.Fullem,
C.Davis,
M.Welch,
J.Seffernick,
L.P.Wackett,
W.P.Stemmer,
and
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(2001).
Novel enzyme activities and functional plasticity revealed by recombining highly homologous enzymes.
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Chem Biol,
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U.T.Bornscheuer,
and
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Improved biocatalysts by directed evolution and rational protein design.
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Curr Opin Chem Biol,
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I.P.Petrounia,
and
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(2000).
Designed evolution of enzymatic properties.
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Curr Opin Biotechnol,
11,
325-330.
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J.D.Sutherland
(2000).
Evolutionary optimisation of enzymes.
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Curr Opin Chem Biol,
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M.B.Tobin,
C.Gustafsson,
and
G.W.Huisman
(2000).
Directed evolution: the 'rational' basis for 'irrational' design.
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Curr Opin Struct Biol,
10,
421-427.
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M.Chartrain,
P.M.Salmon,
D.K.Robinson,
and
B.C.Buckland
(2000).
Metabolic engineering and directed evolution for the production of pharmaceuticals.
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Curr Opin Biotechnol,
11,
209-214.
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P.C.Babbitt
(2000).
Reengineering the glutathione S-transferase scaffold: a rational design strategy pays off.
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Proc Natl Acad Sci U S A,
97,
10298-10300.
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W.B.Watt,
and
A.M.Dean
(2000).
Molecular-functional studies of adaptive genetic variation in prokaryotes and eukaryotes.
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Annu Rev Genet,
34,
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The most recent references are shown first.
Citation data come partly from CiteXplore and partly
from an automated harvesting procedure. Note that this is likely to be
only a partial list as not all journals are covered by
either method. However, we are continually building up the citation data
so more and more references will be included with time.
Where a reference describes a PDB structure, the PDB
codes are
shown on the right.
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Links
