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PDBsum entry 3e2s
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Oxidoreductase
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PDB id
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3e2s
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References listed in PDB file
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Key reference
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Title
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A conserved active site tyrosine residue of proline dehydrogenase helps enforce the preference for proline over hydroxyproline as the substrate.
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Authors
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E.L.Ostrander,
J.D.Larson,
J.P.Schuermann,
J.J.Tanner.
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Ref.
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Biochemistry, 2009,
48,
951-959.
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PubMed id
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Abstract
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Proline dehydrogenase (PRODH) catalyzes the oxidation of l-proline to
Delta-1-pyrroline-5-carboxylate. PRODHs exhibit a pronounced preference for
proline over hydroxyproline (trans-4-hydroxy-l-proline) as the substrate, but
the basis for specificity is unknown. The goal of this study, therefore, is to
gain insight into the structural determinants of substrate specificity of this
class of enzyme, with a focus on understanding how PRODHs discriminate between
the two closely related molecules, proline and hydroxyproline. Two site-directed
mutants of the PRODH domain of Escherichia coli PutA were created: Y540A and
Y540S. Kinetics measurements were performed with both mutants. Crystal
structures of Y540S complexed with hydroxyproline, proline, and the proline
analogue l-tetrahydro-2-furoic acid were determined at resolutions of 1.75,
1.90, and 1.85 A, respectively. Mutation of Tyr540 increases the catalytic
efficiency for hydroxyproline 3-fold and decreases the specificity for proline
by factors of 20 (Y540S) and 50 (Y540A). The structures show that removal of the
large phenol side chain increases the volume of the substrate-binding pocket,
allowing sufficient room for the 4-hydroxyl of hydroxyproline. Furthermore, the
introduced serine residue participates in recognition of hydroxyproline by
forming a hydrogen bond with the 4-hydroxyl. This result has implications for
understanding the substrate specificity of the related enzyme human
hydroxyproline dehydrogenase, which has serine in place of tyrosine at this key
active site position. The kinetic and structural results suggest that Tyr540 is
an important determinant of specificity. Structurally, it serves as a negative
filter for hydroxyproline by clashing with the 4-hydroxyl group of this
potential substrate.
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