Probing the reaction mechanism of IspH protein by x-ray structure analysis

Gräwert, Tobias; Span, Ingrid; Eisenreich, Wolfgang; Rohdich, Felix; Eppinger, Jörg; Bacher, Adelbert; Groll, M.

doi:10.1073/pnas.0913045107

Cited by 102 publications

(264 citation statements)

References 18 publications

Supporting

Mentioning

235

Contrasting

Unclassified

Order By: Relevance

“…The robots demonstrated in this article were all used under ambient conditions at 20 °C. However, there are proteins and projects that require non-ambient temperatures, controlled light 11,12 and an oxidizing or a reducing environment 13 . All of these can be catered for, with relative ease, when a crystallization robot is used.…”

Section: Discussionmentioning

confidence: 99%

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Boland

Walsh

et al. 2012

JoVE

View full text Add to dashboard Cite

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural & Functional Biology (MS&FB) Group [1][2][3] . JoVE video articles describing the method are available 1,4 . The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline.In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&FB Group 5,6 . The other two have recently become available and are included here for completeness.An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 °C) under ambient conditions. Video LinkThe video component of this article can be found at https://www.jove.com/video/4000/ Protocol Preparing the Crystallization PlateSetting up to do a crystallization trial robotically begins with the preparation of the base-plate of the glass sandwich crystallization plate (Figure 2), described in detail in Reference 2. The base-plate must first be silanized and the perforated double-stick spacer that creates the wells, must be applied to the plate. The materials and supplies needed for this are itemized under Materials.1. Place the plate on a paper towel, apply a few drops of silanizing solution and distribute it evenly over the plate surf...

show abstract

Section: Discussionmentioning

confidence: 99%

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Boland

Walsh

et al. 2012

JoVE

View full text Add to dashboard Cite

show abstract

“…These two residues are absolutely conserved, but their functions may have evolved in plants. In bacterial IspH, His-41 is involved in substrate binding, whereas His-124 is required for delivering H + to the key residue Glu-126 and the substrate HMBPP during catalysis (Rekittke et al, 2008;Gräwert et al, 2009Gräwert et al, , 2010Wang et al, 2010). In E. coli IspH, the His-41Asn mutant did not have detrimental effects, but the activity of IspH was undetectable in the His124Asn mutant (Gräwert et al, 2009).…”

Section: Structure and Enzymatic Mechanism Of Hdr: Similarity And Difmentioning

confidence: 99%

“…amino acid residues 111 to 466, encompassing the bacterial IspH) shares approximately 25% identity and approximately 42% similarity with the E. coli protein. Many amino acid residues found to be critical for E. coli and A. aeolicus IspH (Gräwert et al, 2004(Gräwert et al, , 2009(Gräwert et al, , 2010Rekittke et al, 2008, Wang et al, 2010 were also conserved in purple bacteria, cyanobacteria, green algae, and land plants (Fig. 2).…”

Section: Sequence Analysis Of Plant Hdr and Bacterial Isphmentioning

confidence: 99%

“…In E. coli and A. aeolicus IspH, Glu-126 is a key catalytic residue that delivers H + to the active site, and Thr-167, Ser-225, and Asn-227 are involved in substrate binding (Gräwert et al, 2009(Gräwert et al, , 2010Wang et al, 2010). These amino acids are conserved in cyanobacteria, green algae, and land plants.…”

Section: Structure and Enzymatic Mechanism Of Hdr: Similarity And Difmentioning

confidence: 99%

“…Studies from Aquifex aeolicus and E. coli indicate that IspH has a trefoil-like structure with a central Fe 4 S 4 cluster, which is coordinated by the Cys residues located in each of the three folding domains (Rekittke et al, 2008;Gräwert et al, 2009). The central cavity is the active site, and the iron-sulfur cluster serves as an electrontransfer cofactor in the active site (Gräwert et al, 2004(Gräwert et al, , 2009(Gräwert et al, , 2010Rekittke et al, 2008;Wang et al, 2010). The IspH mechanism of action is still under debate, but there is considerable evidence in support of the organometallic hypothesis (Wang et al, 2010Span et al, 2012a;Xu et al, 2012;Li et al, 2013).…”

mentioning

confidence: 99%

See 2 more Smart Citations

Functional Evidence for the Critical Amino-Terminal Conserved Domain and Key Amino Acids of Arabidopsis 4-HYDROXY-3-METHYLBUT-2-ENYL DIPHOSPHATE REDUCTASE

et al. 2014

View full text Add to dashboard Cite

The plant 4-HYDROXY-3-METHYLBUT-2-ENYL DIPHOSPHATE REDUCTASE (HDR) catalyzes the last step of the methylerythritol phosphate pathway to synthesize isopentenyl diphosphate and its allyl isomer dimethylallyl diphosphate, which are common precursors for the synthesis of plastid isoprenoids. The Arabidopsis (Arabidopsis thaliana) genomic HDR transgene-induced gene-silencing lines are albino, variegated, or pale green, confirming that HDR is essential for plants. We used Escherichia coli isoprenoid synthesis H (Protein Data Bank code 3F7T) as a template for homology modeling to identify key amino acids of Arabidopsis HDR. The predicted model reveals that cysteine (Cys)-122, Cys-213, and Cys-350 are involved in iron-sulfur cluster formation and that histidine (His)-152, His-241, glutamate (Glu)-242, Glu-243, threonine (Thr)-244, Thr-312, serine-379, and asparagine-381 are related to substrate binding or catalysis. Glu-242 and Thr-244 are conserved only in cyanobacteria, green algae, and land plants, whereas the other key amino acids are absolutely conserved from bacteria to plants. We used sitedirected mutagenesis and complementation assay to confirm that these amino acids, except His-152 and His-241, were critical for Arabidopsis HDR function. Furthermore, the Arabidopsis HDR contains an extra amino-terminal domain following the transit peptide that is highly conserved from cyanobacteria, and green algae to land plants but not existing in the other bacteria. We demonstrated that the amino-terminal conserved domain was essential for Arabidopsis and cyanobacterial HDR function. Further analysis of conserved amino acids in the amino-terminal conserved domain revealed that the tyrosine-72 residue was critical for Arabidopsis HDR. These results suggest that the structure and reaction mechanism of HDR evolution have become specific for oxygen-evolving photosynthesis organisms and that HDR probably evolved independently in cyanobacteria versus other prokaryotes.

show abstract

Radical Enzymes

Golding

2012

Encyclopedia of Radicals in Chemistry, Biology and Materials

View full text Add to dashboard Cite

Radical enzymes catalyze reactions with intermediate radicals, which are not free but bound to the enzyme. The catalytic reactions comprise generation of a radical species that initiates formation of the substrate‐derived radical, followed by conversion to the product‐related radical, and either recycling or decomposition of the initiating radical with release of the product. These characteristics are illustrated with specific radical enzymes. Ribonucleotide reductases (RNRs) use a thiyl radical to enable a common reaction mechanism for the reduction of ribose. There are three classes of RNRs that differ in the manner of thiyl radical generation: use of coenzyme B 12 , S ‐adenosylmethionine (SAM), or dioxygen with bi‐transition metal centers. Coenzyme B 12 ‐dependent eliminases and mutases are like SAM radical enzymes in that they apply the same 5′‐deoxyadenosyl radical as initiating radical, which is also used by coenzyme B 12 ‐ or SAM‐dependent RNRs. The SAM radical enzymes, which multiply in number, are involved in the maturation of enzymes and transfer of ribonucleic acids, as well as in the biosynthesis of many vitamins, coenzymes, and antibiotics. The 2‐ and 4‐hydroxyacyl‐coenzyme A (CoA) dehydratases contain a [4Fe–4S] cluster and utilize intermediate ketyl radicals, which are formed either by reduction or by combined oxidation/deprotonation of the substrates. Similar radical species participate in the reduction of benzoyl‐CoA and other aromatic compounds as well as in photolyases that repair DNA. Finally, flavin radical enzymes and two [4Fe–4S] cluster enzymes of the nonmevalonate pathway of isoprenoid biosynthesis are described. In conclusion, biotechnological applications and evolutionary aspects are discussed.

show abstract

Probing the reaction mechanism of IspH protein by x-ray structure analysis

Cited by 102 publications

References 18 publications

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Functional Evidence for the Critical Amino-Terminal Conserved Domain and Key Amino Acids of Arabidopsis 4-HYDROXY-3-METHYLBUT-2-ENYL DIPHOSPHATE REDUCTASE

Radical Enzymes

Contact Info

Product

Resources

About