Heavy Atom Detergent/Lipid Combined X-ray Crystallography for Elucidating the Structure-Function Relationships of Membrane Proteins.

Membrane proteins reside in the lipid bilayer of biomembranes and the structure and function of these proteins are closely related to their interactions with lipid molecules. Structural analyses of interactions between membrane proteins and lipids or detergents that constitute biological or artificial model membranes are important for understanding the functions and physicochemical properties of membrane proteins and biomembranes. Determination of membrane protein structures is much more difficult when compared with that of soluble proteins, but the development of various new technologies has accelerated the elucidation of the structure-function relationship of membrane proteins. This review summarizes the development of heavy atom derivative detergents and lipids that can be used for structural analysis of membrane proteins and their interactions with detergents/lipids, including their application with X-ray free-electron laser crystallography

X-ray structure of Galdieria Rubisco complexed with one sulfate ion per active site

AbstractRibulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the reactions of carboxylation and oxygenation of ribulose-1,5-bisphosphate. These reactions require that the active site should be closed by a flexible loop (loop 6) of the large subunit. Rubisco from a red alga, Galdieria partita, has the highest specificity for carboxylation reaction among the Rubiscos hitherto reported. The crystal structure of unactivated Galdieria Rubisco has been determined at 2.6 Å resolution. The electron density map reveals that a sulfate binds only to the P1 anion-binding site of the active site and the loop 6 is closed. Galdieria Rubisco has a unique hydrogen bond between the main chain oxygen of Val332 on the loop 6 and the ϵ-amino group of Gln386 of the same large subunit. This interaction is likely to be crucial to understanding for stabilizing the loop 6 in the closed state and to making a higher affinity for anionic ligands

An isomorphous replacement method for efficient de novo phasing for serial femtosecond crystallography.

SACLAのX線自由電子レーザーを用いた新規タンパク質立体構造決定に世界で初めて成功. 京都大学プレスリリース. 2015-09-14.Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) holds great potential for structure determination of challenging proteins that are not amenable to producing large well diffracting crystals. Efficient de novo phasing methods are highly demanding and as such most SFX structures have been determined by molecular replacement methods. Here we employed single isomorphous replacement with anomalous scattering (SIRAS) for phasing and demonstrate successful application to SFX de novo phasing. Only about 20,000 patterns in total were needed for SIRAS phasing while single wavelength anomalous dispersion (SAD) phasing was unsuccessful with more than 80,000 patterns of derivative crystals. We employed high energy X-rays from SACLA (12.6 keV) to take advantage of the large anomalous enhancement near the LIII absorption edge of Hg, which is one of the most widely used heavy atoms for phasing in conventional protein crystallography. Hard XFEL is of benefit for de novo phasing in the use of routinely used heavy atoms and high resolution data collection

Substrate Specificity of an Aminopropyltransferase and the Biosynthesis Pathway of Polyamines in the Hyperthermophilic Crenarchaeon Pyrobaculum calidifontis

The facultative anaerobic hyperthermophilic crenarchaeon Pyrobaculum calidifontis possesses norspermine (333), norspermidine (33), and spermidine (34) as intracellular polyamines (where the number in parentheses represents the number of methylene CH2 chain units between NH2, or NH). In this study, the polyamine biosynthesis pathway of P. calidifontis was predicted on the basis of the enzymatic properties and crystal structures of an aminopropyltransferase from P. calidifontis (Pc-SpeE). Pc-SpeE shared 75% amino acid identity with the thermospermine synthase from Pyrobaculum aerophilum, and recombinant Pc-SpeE could synthesize both thermospermine (334) and spermine (343) from spermidine and decarboxylated S-adenosyl methionine (dcSAM). Recombinant Pc-SpeE showed high enzymatic activity when aminopropylagmatine and norspermidine were used as substrates. By comparison, Pc-SpeE showed low affinity toward putrescine, and putrescine was not stably bound in its active site. Norspermidine was produced from thermospermine by oxidative degradation using a cell-free extract of P. calidifontis, whereas 1,3-diaminopropane (3) formation was not detected. These results suggest that thermospermine was mainly produced from arginine via agmatine, aminopropylagmatine, and spermidine. Norspermidine was produced from thermospermine by an unknown polyamine oxidase/dehydrogenase followed by norspermine formation by Pc-SpeE

Substrate Specificity of an Aminopropyltransferase and the Biosynthesis Pathway of Polyamines in the Hyperthermophilic Crenarchaeon <i>Pyrobaculum calidifontis</i>

The facultative anaerobic hyperthermophilic crenarchaeon Pyrobaculum calidifontis possesses norspermine (333), norspermidine (33), and spermidine (34) as intracellular polyamines (where the number in parentheses represents the number of methylene CH2 chain units between NH2, or NH). In this study, the polyamine biosynthesis pathway of P. calidifontis was predicted on the basis of the enzymatic properties and crystal structures of an aminopropyltransferase from P. calidifontis (Pc-SpeE). Pc-SpeE shared 75% amino acid identity with the thermospermine synthase from Pyrobaculum aerophilum, and recombinant Pc-SpeE could synthesize both thermospermine (334) and spermine (343) from spermidine and decarboxylated S-adenosyl methionine (dcSAM). Recombinant Pc-SpeE showed high enzymatic activity when aminopropylagmatine and norspermidine were used as substrates. By comparison, Pc-SpeE showed low affinity toward putrescine, and putrescine was not stably bound in its active site. Norspermidine was produced from thermospermine by oxidative degradation using a cell-free extract of P. calidifontis, whereas 1,3-diaminopropane (3) formation was not detected. These results suggest that thermospermine was mainly produced from arginine via agmatine, aminopropylagmatine, and spermidine. Norspermidine was produced from thermospermine by an unknown polyamine oxidase/dehydrogenase followed by norspermine formation by Pc-SpeE

C(sp³)–H Bond Hydroxylation Catalyzed by Myoglobin Reconstituted with Manganese Porphycene

Myoglobin reconstituted with manganese porphycene was prepared in an effort to generate a new biocatalyst and was characterized by spectroscopic techniques. The X-ray crystal structure of the reconstituted protein reveals that the artificial cofactor is located in the intrinsic heme-binding site with weak ligation by His93. Interestingly, the reconstituted protein catalyzes the H₂O₂-dependent hydroxylation of ethylbenzene to yield 1-phenylethanol as a single product with a turnover number of 13 at 25 °C and pH 8.5. Native myoglobin and other modified myoglobins do not catalyze C–H hydroxylation of alkanes. Isotope effect experiments yield KIE values of 2.4 and 6.1 for ethylbenzene and toluene, respectively. Kinetic data, log <i>k</i>_obs versus BDE­(C­(sp³)–H) for ethylbenzene, toluene, and cyclohexane, indicate a linear relationship with a negative slope. These findings clearly indicate that the reaction occurs via a rate-determining step that involves hydrogen-atom abstraction by a Mn­(O) species and a subsequent rebound hydroxylation process which is similar to the reaction mechanism of cytochrome P450