D-LDH

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Introduction

Some respiratory proteins contain integral membrane subunits (such as fumarate reductase, succinate dehydrogenase), while others are peripheral membrane proteins, such as D-lactate dehydrogenase (D-LDH). These respiratory enzymes can oxidize various organic substrates (such as formate, lactate, succinate), and then transfer electrons to various oxidants (such as oxygen, nitrate, fumarate). The free energy gained from these reactions can be used by other membrane proteins, such as solute transport or ATP synthesis. E. coli uses D-lactate as a carbon source to transfer two electrons and two protons to the electron transfer chain. The electron acceptor is quinone, and all reactions occur on the cytoplasmic surface of the inner membrane.

The separation and purification of D-LDH require detergents, and a variety of detergents and lipids can enhance enzyme activity. Studies have shown that D-LDH can be eluted from E. coli membranes with 0.6 M guanidine hydrochloride, and its amino acid composition is not particularly non-polar. This indicates that D-LDH is peripheral like some other primary dehydrogenases involved in electron transfer in E. coli, is a peripheral protein rather than an integral membrane protein. D-LDH consists of 571 amino acid residues and is a 65 kDa single protein chain. The reduction of D-LDH by the substrate D-lactate exhibits two stages. The fast stage represents the rapid formation of the enzyme-substrate complex, and the slow stage represents the slow release of the product pyruvate from the enzyme. An anionic D-LDH semiquinone (one electron is reduced) has been observed and identified by using optical and electron paramagnetic resonance spectroscopy during the oxidation of the reduced enzyme by nitroxide-spin labels.

D-LDH belongs to the recently discovered family of FAD-dependent protein, and they share conserved residues in their FAD-binding domains. This family includes proteins such as p-cresol methylhydroxylase (PCMH), vanillylalcohol oxidase, and UDP-N-acetylenolpyruvylglucosamine (MurB). These proteins are found both in eukaryotes and eubacteria, and catalyze a variety of redox reactions in a variety of organisms. The most significant structural difference between D-LDH and the other proteins in this FAD family lies in the membrane-binding domain. Based on these structural differences and comparisons with the structures of other peripheral membrane proteins, scientists have proposed a model for D-LDH association with the membranes.

Figure 1. Cartoon of D-LDH associating with the membrane

Comparison with other members of the FAD family

The D-LDH structure consists of three discrete domains, namely the FAD binding domain (residues 1-268 and 520-571), the cap domain (residues 269-310, 388-425 and 450-519), and the membrane-binding domain (residues 311-387 and 426-449, of which residues 329-376 are located in the disordered region). The FAD binding domain is composed of two α+β subdomains. The cap domain consists of a 7-strand antiparallel β-sheet, with α helix on each side. The membrane-binding domain is composed of four α helices. There are hydrophobic and polar interactions at the contact interface between the FAD binding and the cap domain, as well as two salt bridges (Arg-40-Asp-498 and Arg-136-Glu-271) at the extremities of the surface. The only interaction between Leu-327 and Phe-39 exists at the interface between FAD and membrane-binding domain.

After screening the database with the D-LDH sequence using BLAST profile method and multiple alignment program, it is found that D-LDH belongs to the recently discovered FAD-dependent protein family, which is characterized by sharing a conserved FAD-binding domain. Although the sequence identity between D-LDH and its family members (such as MurB, PCMH, etc.) is less than 15% in the entire alignment, they share some conserved residues in the N-terminal region, including some FAD binding domain. Some of these conserved residues are involved in the binding of FAD cofactors, and some are located near the binding site. As a result, as shown by the program DALI, the structure of the FAD binding domain is homologous in all these proteins and is distinguished from the Rossmann fold. Although there is no obvious sequence similarity, this structural similarity is observed to a lesser extent in the cap domain.

Figure 2. Comparison of the three individual domains of D-LDH with the corresponding domains of PCMH and MurB