PDBsum entry 3e2s

Oxidoreductase

PDB id: 3e2s

Contents

Protein chain

468 a.a. *

Ligands

PRO

FAD

1PE ×2

Waters ×147

* Residue conservation analysis

PDB id:

3e2s

Name:

Oxidoreductase

Title:

Crystal structure reduced puta86-630 mutant y540s complexed with l- proline

Structure:

Proline dehydrogenase. Chain: a. Fragment: unp residues 86-630. Synonym: proline oxidase. Engineered: yes. Mutation: yes

Source:

Escherichia coli. Organism_taxid: 83333. Strain: k12. Gene: puta, poaa. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.00Å R-factor: 0.215 R-free: 0.251

Authors:

J.J.Tanner

Key ref:

E.L.Ostrander et al. (2009). A conserved active site tyrosine residue of proline dehydrogenase helps enforce the preference for proline over hydroxyproline as the substrate. Biochemistry, 48, 951-959. PubMed id: 19140736

Date:

06-Aug-08 Release date: 03-Feb-09

Protein chain

P09546  (PUTA_ECOLI) -  Bifunctional protein PutA from Escherichia coli (strain K12)

Seq: 1320 a.a.

Struc: 468 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class 1: E.C.1.2.1.88 - L-glutamate gamma-semialdehyde dehydrogenase.
• Reaction: L-glutamate 5-semialdehyde + NAD⁺ + H2O = L-glutamate + NADH + 2 H⁺

L-glutamate 5-semialdehyde + NAD(+) (Bound ligand (Het Group name = PRO) matches with 88.89% similarity) + H2O (Bound ligand (Het Group name = FAD) matches with 76.36% similarity) = L-glutamate + NADH + 2 × H(+)

• Enzyme class 2: E.C.1.5.5.2 - proline dehydrogenase.
• Reaction: L-proline + a quinone = (S)-1-pyrroline-5-carboxylate + a quinol + H⁺

L-proline + quinone (Bound ligand (Het Group name = PRO) corresponds exactly) = (S)-1-pyrroline-5-carboxylate + quinol + H(+)

• Cofactor: FAD

FAD (Bound ligand (Het Group name = FAD) corresponds exactly)

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

Biochemistry 48:951-959 (2009)

PubMed id: 19140736

A conserved active site tyrosine residue of proline dehydrogenase helps enforce the preference for proline over hydroxyproline as the substrate.

E.L.Ostrander, J.D.Larson, J.P.Schuermann, J.J.Tanner.

ABSTRACT

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.