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Structure of the monotopic membrane protein (S)-mandelate dehydrogenase at 2.2?A resolution.

ABSTRACT: The x-ray structure of the monotopic membrane protein (S)-mandelate dehydrogenase (MDH) from Pseudomonas putida reveals an inherent flexibility of its membrane binding segment that might be important for its biological activity. The surface of MDH exhibits a concentration of the positive charges on one side and the negative charges on the other side. The putative membrane binding surface of MDH has a concentric circular ridge, formed by positively charged residues, which projects away from the protein surface by ?4?Å; this is an unique structural feature and not observed in other monotopic membrane proteins to our knowledge. There are three ?-helixes in the membrane binding region. Based on the structure of MDH, it is possible to propose that the interaction of MDH with the membrane is stabilized by coplanar electrostatic interactions, between the positively charged concentric circular ridge and the negatively charged head-groups of the phospholipid bilayer, along with three ?-helixes that provide additional stability by inserting into the membrane. The structure reveals the possible orientation of these helixes along with possible roles for the individual residues which form those helixes. These ?-helixes may play a role in the enzyme's mobility. A detergent molecule, N-Dodecyl-?-maltoside, is inserted between the membrane binding region and rest of the molecule and may provide structural stability to intra-protein regions by forming hydrogen bonds and close contacts. From the average B-factor of the MDH structure, it is likely that MDH is highly mobile, which might be essential for its interaction in membrane and non-membrane environments, as its substrate (S)-mandelate, is from the cytoplasm, while its electron acceptor is a component of the membrane electron transport chain.

SUBMITTER: Sukumar N

PROVIDER: S-EPMC6204087 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Structure of the monotopic membrane protein (S)-mandelate dehydrogenase at 2.2 Å resolution.

Sukumar N N Liu S S Li W W Mathews F S FS Mitra B B Kandavelu P P

Biochimie 20180730

The x-ray structure of the monotopic membrane protein (S)-mandelate dehydrogenase (MDH) from Pseudomonas putida reveals an inherent flexibility of its membrane binding segment that might be important for its biological activity. The surface of MDH exhibits a concentration of the positive charges on one side and the negative charges on the other side. The putative membrane binding surface of MDH has a concentric circular ridge, formed by positively charged residues, which projects away from the p ...[more]

PMID: 30071260

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Project description:Mandelate racemase (MR, EC 5.1.2.2) from Pseudomonas putida catalyzes the Mg(2+)-dependent interconversion of the enantiomers of mandelate, stabilizing the altered substrate in the transition state by 26 kcal/mol relative to the substrate in the ground state. To understand the origins of this binding discrimination, we determined the X-ray crystal structures of wild-type MR complexed with two analogues of the putative aci-carboxylate intermediate, benzohydroxamate and Cupferron, to 2.2-Å resolution. Benzohydroxamate is shown to be a reasonable mimic of the transition state and/or intermediate because its binding affinity for 21 MR variants correlates well with changes in the free energy of transition state stabilization afforded by these variants. Both benzohydroxamate and Cupferron chelate the active site divalent metal ion and are bound in a conformation with the phenyl ring coplanar with the hydroxamate and diazeniumdiolate moieties, respectively. Structural overlays of MR complexed with benzohydroxamate, Cupferron, and the ground state analogue (S)-atrolactate reveal that the para carbon of the substrate phenyl ring moves by 0.8-1.2 Å between the ground state and intermediate state, consistent with the proposal that the phenyl ring moves during MR catalysis while the polar groups remain relatively fixed. Although the overall protein structure of MR with bound intermediate analogues is very similar to that of MR with bound (S)-atrolactate, the intermediate-Mg(2+) distance becomes shorter, suggesting a tighter complex with the catalytic Mg(2+). In addition, Tyr 54 moves closer to the phenyl ring of the bound intermediate analogues, contributing to an overall constriction of the active site cavity. However, site-directed mutagenesis experiments revealed that the role of Tyr 54 in MR catalysis is relatively minor, suggesting that alterations in enzyme structure that contribute to discrimination between the altered substrate in the transition state and the ground state by this proficient enzyme are extremely subtle.

Dataset Information

Structure of the monotopic membrane protein (S)-mandelate dehydrogenase at 2.2?A resolution.

Publications

Structure of the monotopic membrane protein (S)-mandelate dehydrogenase at 2.2 Å resolution.

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Dataset Information

Structure of the monotopic membrane protein (S)-mandelate dehydrogenase at 2.2?A resolution.

Publications

Structure of the monotopic membrane protein (S)-mandelate dehydrogenase at 2.2 Å resolution.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Structure of the monotopic membrane protein (S)-mandelate dehydrogenase at 2.2 Å resolution.