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PDBsum entry 1dbi
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* Residue conservation analysis
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DOI no:
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J Mol Biol
294:1027-1040
(1999)
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PubMed id:
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Calcium-mediated thermostability in the subtilisin superfamily: the crystal structure of Bacillus Ak.1 protease at 1.8 A resolution.
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C.A.Smith,
H.S.Toogood,
H.M.Baker,
R.M.Daniel,
E.N.Baker.
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ABSTRACT
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Proteins of the subtilisin superfamily (subtilases) are widely distributed
through many living species, where they perform a variety of processing
functions. They are also used extensively in industry. In many of these enzymes,
bound calcium ions play a key role in protecting against autolysis and thermal
denaturation. We have determined the crystal structure of a highly thermostable
protease from Bacillus sp. Ak.1 that is strongly stabilized by calcium. The
crystal structure, determined at 1.8 A resolution (R=0. 182, Rfree=0.247),
reveals the presence of four bound cations, three Ca(2+) and one Na(+). Two of
the Ca(2+) binding sites, Ca-1 and Ca-2, correspond to sites also found in
thermitase and the mesophilic subtilisins. The third calcium ion, however, is at
a novel site that is created by two key amino acid substitutions near Ca-1, and
has not been observed in any other subtilase. This site, acting cooperatively
with Ca-1, appears to give substantially enhanced thermostability, compared with
thermitase. Comparisons with the mesophilic subtilisins also point to the
importance of aromatic clusters, reduced hydrophobic surface and constrained N
and C termini in enhancing the thermostability of thermitase and Ak.1 protease.
The Ak.1 protease also contains an unusual Cys-X-Cys disulfide bridge that
modifies the active site cleft geometry.
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Selected figure(s)
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Figure 4.
Figure 4. Schematic represen-
tation of the topology of Ak.1 pro-
tease. Helices are identified by
circles and b-strands by triangles.
The location of the active site is
indicated with grey shading, with
the location of bound substrate
shown by a molecule with broken
lines.
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Figure 6.
Figure 6. Stereo diagrams showing the Ca
2+
binding sites Ca-1 and Ca-2. (a) Site Ca-1, shown with the equivalent
sites for AkP, thermitase and subtilisin BPN0 superimposed; AkP is shown in blue, thermitase in red and subtilisin
BPN0 in green. (b) Site Ca-2, shown with the equivalent sites for AkP (blue, with cyan spheres for water molecules)
and thermitase (red, with pink spheres for water molecules) superimposed. In both Figures residues are labelled as
for AkP.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(1999,
294,
1027-1040)
copyright 1999.
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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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N.Fakhfakh-Zouari,
N.Hmidet,
A.Haddar,
S.Kanoun,
and
M.Nasri
(2010).
A novel serine metallokeratinase from a newly isolated Bacillus pumilus A1 grown on chicken feather meal: biochemical and molecular characterization.
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Appl Biochem Biotechnol,
162,
329-344.
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R.M.Daniel,
M.E.Peterson,
M.J.Danson,
N.C.Price,
S.M.Kelly,
C.R.Monk,
C.S.Weinberg,
M.L.Oudshoorn,
and
C.K.Lee
(2010).
The molecular basis of the effect of temperature on enzyme activity.
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Biochem J,
425,
353-360.
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R.M.Kennan,
W.Wong,
O.P.Dhungyel,
X.Han,
D.Wong,
D.Parker,
C.J.Rosado,
R.H.Law,
S.McGowan,
S.B.Reeve,
V.Levina,
G.A.Powers,
R.N.Pike,
S.P.Bottomley,
A.I.Smith,
I.Marsh,
R.J.Whittington,
J.C.Whisstock,
C.J.Porter,
and
J.I.Rood
(2010).
The subtilisin-like protease AprV2 is required for virulence and uses a novel disulphide-tethered exosite to bind substrates.
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PLoS Pathog,
6,
e1001210.
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PDB codes:
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T.Foophow,
S.Tanaka,
Y.Koga,
K.Takano,
and
S.Kanaya
(2010).
Subtilisin-like serine protease from hyperthermophilic archaeon Thermococcus kodakaraensis with N- and C-terminal propeptides.
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Protein Eng Des Sel,
23,
347-355.
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W.Wong,
R.M.Kennan,
C.J.Rosado,
J.I.Rood,
J.C.Whisstock,
and
C.J.Porter
(2010).
Crystallization of the virulent and benign subtilisin-like proteases from the ovine footrot pathogen Dichelobacter nodosus.
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Acta Crystallogr Sect F Struct Biol Cryst Commun,
66,
289-293.
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C.Ottmann,
R.Rose,
F.Huttenlocher,
A.Cedzich,
P.Hauske,
M.Kaiser,
R.Huber,
and
A.Schaller
(2009).
Structural basis for Ca2+-independence and activation by homodimerization of tomato subtilase 3.
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Proc Natl Acad Sci U S A,
106,
17223-17228.
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PDB codes:
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G.Cacciapuoti,
I.Peluso,
F.Fuccio,
and
M.Porcelli
(2009).
Purine nucleoside phosphorylases from hyperthermophilic Archaea require a CXC motif for stability and folding.
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FEBS J,
276,
5799-5805.
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G.Cheng,
P.Zhao,
X.F.Tang,
and
B.Tang
(2009).
Identification and characterization of a novel spore-associated subtilase from Thermoactinomyces sp. CDF.
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Microbiology,
155,
3661-3672.
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N.Fakhfakh,
S.Kanoun,
L.Manni,
and
M.Nasri
(2009).
Production and biochemical and molecular characterization of a keratinolytic serine protease from chicken feather-degrading Bacillus licheniformis RPk.
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Can J Microbiol,
55,
427-436.
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O.Almog,
A.González,
N.Godin,
M.de Leeuw,
M.J.Mekel,
D.Klein,
S.Braun,
G.Shoham,
and
R.L.Walter
(2009).
The crystal structures of the psychrophilic subtilisin S41 and the mesophilic subtilisin Sph reveal the same calcium-loaded state.
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Proteins,
74,
489-496.
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PDB codes:
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U.Derewenda,
T.Boczek,
K.L.Gorres,
M.Yu,
L.W.Hung,
D.Cooper,
A.Joachimiak,
R.T.Raines,
and
Z.S.Derewenda
(2009).
Structure and function of Bacillus subtilis YphP, a prokaryotic disulfide isomerase with a CXC catalytic motif .
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Biochemistry,
48,
8664-8671.
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PDB code:
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H.G.Woon,
G.M.Scott,
K.L.Yiu,
D.H.Miles,
and
W.D.Rawlinson
(2008).
Identification of putative functional motifs in viral proteins essential for human cytomegalovirus DNA replication.
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Virus Genes,
37,
193-202.
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M.S.Dodia,
C.M.Rawal,
H.G.Bhimani,
R.H.Joshi,
S.K.Khare,
and
S.P.Singh
(2008).
Purification and stability characteristics of an alkaline serine protease from a newly isolated Haloalkaliphilic bacterium sp. AH-6.
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J Ind Microbiol Biotechnol,
35,
121-131.
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R.R.Thangudu,
M.Manoharan,
N.Srinivasan,
F.Cadet,
R.Sowdhamini,
and
B.Offmann
(2008).
Analysis on conservation of disulphide bonds and their structural features in homologous protein domain families.
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BMC Struct Biol,
8,
55.
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G.Cacciapuoti,
S.Gorassini,
M.F.Mazzeo,
R.A.Siciliano,
V.Carbone,
V.Zappia,
and
M.Porcelli
(2007).
Biochemical and structural characterization of mammalian-like purine nucleoside phosphorylase from the Archaeon Pyrococcus furiosus.
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FEBS J,
274,
2482-2495.
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J.Yang,
L.Liang,
Y.Zhang,
J.Li,
L.Zhang,
F.Ye,
Z.Gan,
and
K.Q.Zhang
(2007).
Purification and cloning of a novel serine protease from the nematode-trapping fungus Dactylellina varietas and its potential roles in infection against nematodes.
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Appl Microbiol Biotechnol,
75,
557-565.
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S.Tanaka,
K.Saito,
H.Chon,
H.Matsumura,
Y.Koga,
K.Takano,
and
S.Kanaya
(2007).
Crystal structure of unautoprocessed precursor of subtilisin from a hyperthermophilic archaeon: evidence for Ca2+-induced folding.
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J Biol Chem,
282,
8246-8255.
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PDB code:
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Y.Zhou,
W.P.Tzeng,
W.Yang,
Y.Zhou,
Y.Ye,
H.W.Lee,
T.K.Frey,
and
J.Yang
(2007).
Identification of a Ca2+-binding domain in the rubella virus nonstructural protease.
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J Virol,
81,
7517-7528.
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M.Pulido,
K.Saito,
S.Tanaka,
Y.Koga,
M.Morikawa,
K.Takano,
and
S.Kanaya
(2006).
Ca2+-dependent maturation of subtilisin from a hyperthermophilic archaeon, Thermococcus kodakaraensis: the propeptide is a potent inhibitor of the mature domain but is not required for its folding.
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Appl Environ Microbiol,
72,
4154-4162.
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S.Tanaka,
K.Saito,
H.Chon,
H.Matsumura,
Y.Koga,
K.Takano,
and
S.Kanaya
(2006).
Crystallization and preliminary X-ray diffraction study of an active-site mutant of pro-Tk-subtilisin from a hyperthermophilic archaeon.
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Acta Crystallogr Sect F Struct Biol Cryst Commun,
62,
902-905.
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G.Cacciapuoti,
S.Forte,
M.A.Moretti,
A.Brio,
V.Zappia,
and
M.Porcelli
(2005).
A novel hyperthermostable 5'-deoxy-5'-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus.
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FEBS J,
272,
1886-1899.
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G.Cacciapuoti,
M.A.Moretti,
S.Forte,
A.Brio,
L.Camardella,
V.Zappia,
and
M.Porcelli
(2004).
Methylthioadenosine phosphorylase from the archaeon Pyrococcus furiosus. Mechanism of the reaction and assignment of disulfide bonds.
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Eur J Biochem,
271,
4834-4844.
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J.Wu,
Y.Bian,
B.Tang,
X.Chen,
P.Shen,
and
Z.Peng
(2004).
Cloning and analysis of WF146 protease, a novel thermophilic subtilisin-like protease with four inserted surface loops.
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FEMS Microbiol Lett,
230,
251-258.
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K.J.Woycechowsky,
and
R.T.Raines
(2003).
The CXC motif: a functional mimic of protein disulfide isomerase.
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Biochemistry,
42,
5387-5394.
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C.Charron,
B.Vitoux,
and
A.Aubry
(2002).
Comparative analysis of thermoadaptation within the archaeal glyceraldehyde-3-phosphate dehydrogenases from mesophilic Methanobacterium bryantii and thermophilic Methanothermus fervidus.
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Biopolymers,
65,
263-273.
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E.Gross,
C.S.Sevier,
A.Vala,
C.A.Kaiser,
and
D.Fass
(2002).
A new FAD-binding fold and intersubunit disulfide shuttle in the thiol oxidase Erv2p.
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Nat Struct Biol,
9,
61-67.
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PDB codes:
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J.Michiels,
C.Xi,
J.Verhaert,
and
J.Vanderleyden
(2002).
The functions of Ca(2+) in bacteria: a role for EF-hand proteins?
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Trends Microbiol,
10,
87-93.
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M.Abou-Hachem,
E.N.Karlsson,
P.J.Simpson,
S.Linse,
P.Sellers,
M.P.Williamson,
S.J.Jamieson,
H.J.Gilbert,
D.N.Bolam,
and
O.Holst
(2002).
Calcium binding and thermostability of carbohydrate binding module CBM4-2 of Xyn10A from Rhodothermus marinus.
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Biochemistry,
41,
5720-5729.
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C.Vieille,
and
G.J.Zeikus
(2001).
Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability.
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Microbiol Mol Biol Rev,
65,
1.
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N.V.Grishin
(2001).
Treble clef finger--a functionally diverse zinc-binding structural motif.
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Nucleic Acids Res,
29,
1703-1714.
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A.A.McCarthy,
D.D.Morris,
P.L.Bergquist,
and
E.N.Baker
(2000).
Structure of XynB, a highly thermostable beta-1,4-xylanase from Dictyoglomus thermophilum Rt46B.1, at 1.8 A resolution.
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Acta Crystallogr D Biol Crystallogr,
56,
1367-1375.
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PDB code:
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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
