PDBsum entry 2a7n

Oxidoreductase

PDB id: 2a7n

Contents

Protein chain

353 a.a.

Ligands

FMN

MES

Waters ×322

References listed in PDB file

Key reference

Title

Structures of the g81a mutant form of the active chimera of (s)-Mandelate dehydrogenase and its complex with two of its substrates.

Authors

N.Sukumar, A.Dewanti, A.Merli, G.L.Rossi, B.Mitra, F.S.Mathews.

Ref.

Acta Crystallogr D Biol Crystallogr, 2009, 65, 543-552. [DOI no: 10.1107/S0907444909010270]

PubMed id

19465768

Abstract

(S)-Mandelate dehydrogenase (MDH) from Pseudomonas putida, a membrane-associated flavoenzyme, catalyzes the oxidation of (S)-mandelate to benzoylformate. Previously, the structure of a catalytically similar chimera, MDH-GOX2, rendered soluble by the replacement of its membrane-binding segment with the corresponding segment of glycolate oxidase (GOX), was determined and found to be highly similar to that of GOX except within the substituted segments. Subsequent attempts to cocrystallize MDH-GOX2 with substrate proved unsuccessful. However, the G81A mutants of MDH and of MDH-GOX2 displayed approximately 100-fold lower reactivity with substrate and a modestly higher reactivity towards molecular oxygen. In order to understand the effect of the mutation and to identify the mode of substrate binding in MDH-GOX2, a crystallographic investigation of the G81A mutant of the MDH-GOX2 enzyme was initiated. The structures of ligand-free G81A mutant MDH-GOX2 and of its complexes with the substrates 2-hydroxyoctanoate and 2-hydroxy-3-indolelactate were determined at 1.6, 2.5 and 2.2 A resolution, respectively. In the ligand-free G81A mutant protein, a sulfate anion previously found at the active site is displaced by the alanine side chain introduced by the mutation. 2-Hydroxyoctanoate binds in an apparently productive mode for subsequent reaction, while 2-hydroxy-3-indolelactate is bound to the enzyme in an apparently unproductive mode. The results of this investigation suggest that a lowering of the polarity of the flavin environment resulting from the displacement of nearby water molecules caused by the glycine-to-alanine mutation may account for the lowered catalytic activity of the mutant enzyme, which is consistent with the 30 mV lower flavin redox potential. Furthermore, the altered binding mode of the indolelactate substrate may account for its reduced activity compared with octanoate, as observed in the crystalline state.

Figure 5.
Figure 5 Schematic diagram of water arrangements in the active sites of the reduced native and G81A mutant form of MDH-GOX2. Hydrogen-bonding distances are in Å. (a) Native reduced enzyme. The network of three water molecules (Wat169^WT, Wat196^WT, Wat142^WT) and one sulfate ion are shown along with their interactions with each other, with nearby side chains and with two other waters, Wat110^WT and Wat375^WT. (b) G81A mutant enzyme. The three waters (Wat282^GA, Wat283^GA and Wat284^GA) displace the native sulfate ion and three-water network and take up new positions. Wat54^GA is in the same position as Wat97^WT of the native enzyme. R is a ribityl phosphate group and R' is a Leu side chain.

Figure 8.
Figure 8 Schematic diagram of the active-site structures of the 2-hydroxyoctanoate and 3-indolelactate complexes of the G81A mutant form of MDH-GOX2. Hydrogen-bonding interactions between the carboxylate and hydroxyl O atoms and nearby side chains or water molecules are shown as dashed lines and the distances are in Å. Residues making hydrophobic contact to the ligand are indicated as shown at the bottom right. C atoms are black, O atoms red and N atoms cyan. Covalent bonds within the ligand are drawn with pink lines, while those within the protein are drawn with orange lines. (a) The 2-hydroxyoctanoate-G81A complex. The ligand is labeled `Octanoate'. (b) The (D,L)-2-hydroxy-3-indolelactate-G81A complex. The ligand is labeled `ILAC'. This diagram was prepared using the program LIGPLOT (Wallace et al., 1995[Wallace, A. C., Laskowski, R. A. & Thornton, J. M. (1995). Protein Eng. 8, 127-134.]).

The above figures are reprinted by permission from the IUCr: Acta Crystallogr D Biol Crystallogr (2009, 65, 543-552) copyright 2009.

Secondary reference #1

Title

High resolution structures of an oxidized and reduced flavoprotein. The water switch in a soluble form of (s)-Mandelate dehydrogenase.

Authors

N.Sukumar, A.R.Dewanti, B.Mitra, F.S.Mathews.

Ref.

J Biol Chem, 2004, 279, 3749-3757. [DOI no: 10.1074/jbc.M310049200]

PubMed id

14604988

Figure 4.
FIG. 4. Schematic diagram of the protein environment of FMN in the oxidized form of MDH. Hydrogen bond distances are in Å. Residues making hydrophobic contact to FMN are indicated as shown in the lower right. Carbon atoms are black, oxygen atoms red, and nitrogen atoms cyan. This diagram was prepared using the program LIgplot (33)

Figure 6.
FIG. 6. Comparison of the oxidized and reduced forms of MDH-GOX2 at the enzyme active site. The side chains of Tyr26, Tyr131, backbone of Gly81, the flavin ring, the sulfate anion, and 7 waters of the oxidized protein are shown in atom colors (carbon yellow, oxygen red, nitrogen light blue, and sulfur green). Those atoms of Tyr26 and the 6 waters that show significant movement, as well as of the sulfate anion in the reduced enzyme, are shown in dark blue. This diagram was made using TURBO-FRODO (21).

The above figures are reproduced from the cited reference with permission from the ASBMB

Secondary reference #2

Title

Structure of an active soluble mutant of the membrane-Associated (s)-Mandelate dehydrogenase.

Authors

N.Sukumar, Y.Xu, D.L.Gatti, B.Mitra, F.S.Mathews.

Ref.

Biochemistry, 2001, 40, 9870-9878. [DOI no: 10.1021/bi010938k]

PubMed id

11502180