Total Chemical Synthesis of an Integral Membrane Enzyme: Diacylglycerol Kinase from <i>Escherichia coli</i>

Lahiri, Sunanda; Brehs, Manuel; Olschewski, Diana; Becker, Christian F. W.

doi:10.1002/anie.201006686

Cited by 64 publications

(52 citation statements)

References 35 publications

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“…On another technical front the Glaubitz lab has recently initiated studies of DAGK in lipid bilayers using solid-state NMR, illustrating that another orthogonal method is positioned to provide structural and mechanistic insight (57). In addition, Becker and coworkers have recently published the total chemical synthesis of DAGK, resulting in functional enzyme(45). Access to a robust synthetic scheme should allow for residue-specific manipulation beyond what is readily feasible using biological methods, possibly providing the basis for new structural and mechanistic insight.…”

Section: Structure Of Dagkmentioning

confidence: 99%

Prokaryotic Diacylglycerol Kinase and Undecaprenol Kinase

Horn¹,

Sanders

2012

Annu. Rev. Biophys.

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Prokaryotic diacylglycerol kinase (DAGK) and undecaprenol kinase (UDPK) are the lone members of a family of multispan membrane enzymes that are very small, lack relationships to any other family of proteins—including water soluble kinases, and that exhibit an unusual structure and active site architecture. Escherichia coli DAGK plays an important role in recycling diacylglycerol produced as a byproduct of biosynthesis of molecules located in the periplasmic space. UDPK seems to play an analogous role in Gram-positive bacteria, where its importance is evident by the fact that UDPK is essential for biofilm formation by the oral pathogen Streptococcus mutans. DAGK has also long served as a model system for studies of membrane protein biocatalysis, folding, stability, and structure. This review explores our current understanding of the microbial physiology, enzymology, structural biology, and folding of the prokaryotic diacylglycerol kinase family, which is based on over 40 years of studies.

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Section: Structure Of Dagkmentioning

confidence: 99%

Prokaryotic Diacylglycerol Kinase and Undecaprenol Kinase

Horn¹,

Sanders

2012

Annu. Rev. Biophys.

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“…The target protein can originate from native membranes, from the membranes or inclusion bodies of recombinant organisms, from cell-free expression 3,4 and from chemical synthesis. 5 …”

Section: Introductionmentioning

confidence: 99%

Membrane Protein Structure Determination Using Crystallography and Lipidic Mesophases: Recent Advances and Successes

2012

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The crystal structure of the β2-adrenergic receptor in complex with an agonist and its cognate G protein has just recently been solved. It is now possible to explore in molecular detail the means by which this paradigmatic transmembrane receptor binds agonist, communicates the impulse or signalling event across the membrane and sets in motion a series of G protein-directed intracellular responses. The structure was determined using crystals of the ternary complex grown in a rationally designed lipidic mesophase by the so-called in meso method. The method is proving to be particularly useful in the G protein-coupled receptor field where the structures of thirteen distinct receptor types have been solved in the past five years. In addition to receptors, the method has proven useful with a wide variety of integral membrane protein classes that include bacterial and eukaryotic rhodopsins, a light harvesting complex II (LHII), photosynthetic reaction centers, cytochrome oxidases, β-barrels, an exchanger, and an integral membrane peptide. This attests to the versatility and range of the method and supports the view that the in meso method should be included in the arsenal of the serious membrane structural biologist. For this to happen however, the reluctance in adopting it attributable, in part, to the anticipated difficulties associated with handling the sticky, viscous cubic mesophase in which crystals grow must be overcome. Harvesting and collecting diffraction data with the mesophase-grown crystals is also viewed with some trepidation. It is acknowledged that there are challenges associated with the method. Over the years, we have endeavored to establish how the method works at a molecular level and to make it user-friendly. To these ends, tools for handling the mesophase in the pico- to nano-liter volume range have been developed for highly efficient crystallization screening in manual and robotic modes. Methods have been implemented for evaluating the functional activity of membrane proteins reconstituted into the bilayer of the cubic phase as a prelude to crystallogenesis. Glass crystallization plates have been built that provide unparalleled optical quality and sensitivity to nascent crystals. Lipid and precipitant screens have been designed for a more rational approach to crystallogenesis such that the method can now be applied to an even wider variety of membrane protein types. In this Current Topics article, these assorted advances are outlined along with a summary of the membrane proteins that have yielded to the method. The prospects for and the challenges that must be overcome to further develop the method are described.

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“…For half a century, this 121-residue kinase has served as a paradigm for investigating membrane protein enzymology 1,3-7 , folding 8,9 , assembly 10-13 , and stability 1,14 . Here, we present crystal structures for three functional forms of this unique and paradigmatic kinase, one of which is wild type (WT).…”

mentioning

confidence: 99%

Crystal structure of the integral membrane diacylglycerol kinase

Lyons

Pye

et al. 2013

Nature

162

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Diacylglycerol kinase (DgkA) catalyzes the ATP-dependent phosphorylation of diacylglycerol to phosphatidic acid for use in shuttling water-soluble components to membrane derived oligosaccharide and lipopolysaccharide in the cell envelope of Gram-negative bacteria1. For half a century, this 121-residue kinase has served as a paradigm for investigating membrane protein enzymology1,3-7, folding8,9, assembly10-13, and stability1,14. Here, we present crystal structures for three functional forms of this unique and paradigmatic kinase, one of which is wild type (WT). These reveal a homo-trimeric enzyme with three transmembrane helices and an N-terminal amphiphilic helix per monomer. Bound lipid substrate and docked ATP identify the putative active site which is of the composite, shared site type. The crystal structures rationalize extensive biochemical and biophysical data on the enzyme. They are however at variance with a published solution NMR model2 in that domain swapping, a key feature of the solution form, is not observed in the crystal structures.

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Total Chemical Synthesis of an Integral Membrane Enzyme: Diacylglycerol Kinase from Escherichia coli

Cited by 64 publications

References 35 publications

Prokaryotic Diacylglycerol Kinase and Undecaprenol Kinase

Prokaryotic Diacylglycerol Kinase and Undecaprenol Kinase

Membrane Protein Structure Determination Using Crystallography and Lipidic Mesophases: Recent Advances and Successes

Crystal structure of the integral membrane diacylglycerol kinase

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