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PDBsum entry 5giv
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References listed in PDB file
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Key reference
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Title
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Active site gate of m32 carboxypeptidases illuminated by crystal structure and molecular dynamics simulations.
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Authors
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B.Sharma,
S.N.Jamdar,
B.Ghosh,
P.Yadav,
A.Kumar,
S.Kundu,
V.D.Goyal,
R.D.Makde.
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Ref.
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Biochim Biophys Acta, 2017,
1865,
1406-1415.
[DOI no: ]
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PubMed id
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Abstract
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Enzyme gates are important dynamic features that regulate function. Study of
these features is critical for understanding of enzyme mechanism. In this study,
the active-site gate of M32 carboxypeptidases (M32CP) is illuminated. Only a
handful of members of this family have been structurally and functionally
characterized and various aspects of their activity and mechanism are yet not
clarified. Here, crystal structure of putative M32CP from Deinococcus
radiodurans (M32dr) was solved to 2.4Å resolution. Enzymatic assays confirmed
its identity as a carboxypeptidase. Open and relatively closed conformations
observed in the structure provided supporting evidence for previously
hypothesized hinge motion in this family of enzymes. Molecular dynamics
simulations of 1.5μs displayed distinct open and closed conformations revealing
amplitude of the motion to be beyond what was observed in the crystal structure.
Hinge region and anchoring region of this shell-type gate were identified. A
small displacement of 3Å and a helical tilt of 9° propagated by the hinge
region translates into a 10Å motion at the top of the gate. The dynamics of the
gate was supported by our mutagenesis experiment involving formation of
disulphide bond across helices of the gate. The nearly inactive mutant enzyme
showed 65-fold increase in the enzymatic activity in presence of reducing agent.
Further, while a previously proposed structural basis would have led to its
classification in subfamily II, experimentally observed substrate length
restriction places M32dr in subfamily I of M32CPs.
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