Doowon Lee

Characterization of a corrinoid protein involved in the C1 metabolism of strict anaerobic bacterium Moorella thermoacetica

The strict anaerobic, thermophilic bacterium Moorella thermoacetica metabolizes C1 compounds for example CO2/H2, CO, formate, and methanol into acetate via the Wood/Ljungdahl pathway. Some of the key steps in this pathway include the metabolism of the C1 compounds into the methyl group of methylenetetrahydrofolate (MTHF) and the transfer of the methyl group from MTHF to the methyl group of acetyl‐CoA catalyzed by methyltransferase, corrinoid protein and CO dehydrogenase/acetyl CoA synthase. Recently, we reported the crystallization of a 25 kDa methanol‐induced corrinoid protein from M. thermoacetica (Zhou et al., Acta Crystallogr F 2005; 61:537–540). In this study we analyzed the crystal structure of the 25 kDa protein and provide genetic and biochemical evidences supporting its role in the methanol metabolism of M. thermoacetia. The 25 kDa protein was encoded by orf1948 of contig 303 in the M. thermoacetica genome. It resembles similarity to MtaC the corrinoid protein of the methanol:CoM methyltransferase system of methane producing archaea. The latter enzyme system also contains two additional enzymes MtaA and MtaB. Homologs of MtaA and MtaB were found to be encoded by orf2632 of contig 303 and orf1949 of contig 309, respectively, in the M. thermoacetica genome. The orf1948 and orf1949 were co‐transcribed from a single polycistronic operon. Metal analysis and spectroscopic data confirmed the presence of cobalt and the corrinoid in the purified 25 kDa protein. High resolution X‐ray crystal structure of the purified 25 kDa protein revealed corrinoid as methylcobalamin with the imidazole of histidine as the α‐axial ligand replacing benziimidazole, suggesting base‐off configuration for the corrinoid. Methanol significantly activated the expression of the 25 kDa protein. Cyanide and nitrate inhibited methanol metabolism and suppressed the level of the 25 kDa protein. The results suggest a role of the 25 kDa protein in the methanol metabolism of M. thermoacetica. Proteins 2007.

Isolation, crystallization and preliminary X-ray analysis of a methanol-induced corrinoid protein from Moorella thermoacetica

A corrinoid protein was induced and overexpressed in methanol-grown cells of the thermophilic anaerobic bacterium Moorella thermoacetica. The protein was purified from cytosolic extracts. After screening for crystallization conditions and optimization, crystals were obtained that diffracted strongly on a rotating-anode X-ray source. A diffraction data set was collected and processed including reflections to 1.9 A resolution. Reflections were indexed in a primitive orthorhombic cell with unit-cell parameters a = 55.69, b = 62.74, c = 34.54 A. N-terminal amino-acid sequencing indicates that the crystals contain a C-terminal fragment of the protein.

Synthesis of enantiomerically pure cyclopropyl carbocyclic nucleosides

Abstract The enantiomeric synthesis of cyclopropyl carbocyclic nucleosides has been achieved and the key intermediate was characterized by X-ray crystallography.

The high-throughput protein-to-structure pipeline at SECSG.

Using a high degree of automation, the crystallography core at the Southeast Collaboratory for Structural Genomics (SECSG) has developed a high-throughput protein-to-structure pipeline. Various robots and automation procedures have been adopted and integrated into a pipeline that is capable of screening 40 proteins for crystallization and solving four protein structures per week. This pipeline is composed of three major units: crystallization, structure determination/validation and crystallomics. Coupled with the protein-production cores at SECSG, the protein-to-structure pipeline provides a two-tiered approach for protein production at SECSG. In tier 1, all protein samples supplied by the protein-production cores pass through the pipeline using standard crystallization screening and optimization procedures. The protein targets that failed to yield diffraction-quality crystals (resolution better than 3.0 A) become tier 2 or salvaging targets. The goal of tier 2 target salvaging, carried out by the crystallomics core, is to produce the target proteins with increased purity and homogeneity, which would render them more likely to yield well diffracting crystals. This is performed by alternative purification procedures and/or the introduction of chemical modifications to the proteins (such as tag removal, methylation, surface mutagenesis, selenomethionine labelling etc.). Details of the various procedures in the pipeline for protein crystallization, target salvaging, data collection/processing and high-throughput structure determination/validation, as well as some examples, are described.

1,3-Dioxolane C-Nucleosides: Asymmetric Synthesis of Four Stereoisomers of 2-[2-(Hydroxymethyl)-1,3-Dioxolan-S-yl)-1,3-Thiazole-4-Carboxamide

Abstract Asymmetric synthesis of four novel C-nucleosides, (2′R,5′R)-, (2′S,5′R)-, (2′S,5′S)- and (2′R,5′S)-2-[2-hydroxymelhy])-1,3-dioxolan-5-yl]-1 ,3-thiazole-4-carboxamide has been accomplished by the condensation of key intermediates, 2-(1R- and 1S-glycol-1-yl)-4-ethoxycarbonyl-1,3-thiazole with 2-benzoyloxy acetaldehyde dimethyl acetal.

Asymmetric methylene transfer reactions II: Asymmetric synthesis of oxiranes from carbonyl compounds by methylene transfer reaction using chiral S-neomenthyl and S-exo-2-bornyl sulfoximines☆

Abstract Three chiral sulfoximines, viz. , (1R,Ss)-(−)-S-methyl-S- exo -2-bornyl-N-tosyl sulfoximine, 9a , its epimer at sulfur, (1R,Rs)-(+)- 9b , and (1R,3S,4S,1′S,Rs)-(+)-S-methyl-S-neomenthyl-N-(camphor-10-sulfonyl) sulfoximine, 19 , were used as methylene transfer reagents in the preparation of nonracemic oxiranes (enantiomeric excess, 19–86%) from prochiral carbonyl compounds. Sulfoximines 9a and 9b were found to be less effective (ee, 19–68%) than the corresponding S-neomenthyl sulfoximines 2a and 2b (ee, 56–86%), and, sulfoximine 19 , despite having a chiral auxiliary on nitrogen in place of the tosyl group, was found to be only as effective as the corresponding N-tosyl sulfoximine 2b in influencing the steric course of these reactions. The absolute configuration at sulfur of all the synthesised sulfoxides and sulfoximines has been established by X-ray studies.

The structure of the π-molecular complex between 1,4-dihydro-9,10-anthrahydroquinone and 1,4-dihydro-9,10-anthraquinone

The structure of the π-molecular complex (10) was assigned on the basis of the solid state13C-nmr spectrum. The solid state13C-nmr spectrum of quinhydrone (12) has also been determined. Accurate1H and13C chemical shift assignments have been made for the compounds3,5,6,7,8, and10 on the basis of HMQC and HMBC spectral data. The π-molecular complex10 crystallizes in the space groupP21In with cell parameters:a=4.052 (1) Å,b=6.477 (1) Å,c=19.093 (2) Å, β=90.17 (1)o,z=1,Dc=1.400 g mc−32. Crystal and molecular structure of the title compound, C28H22O4, has been determined by an X-ray analysis of10 by direct methods from diffractometer data and refined by full-matrix least-squares

Heterolytic fragmentation of deltaline, a norditerpenoid alkaloid

Abstract The rearrangement product obtained by treatment of the norditerpenoid alkaloid deltaline ( 1 ) with SOCl 2 in moist benzene has been assigned structure 5 . This compound was also obtained by treatment of 10-chloro-10-deoxydeltaline ( 4 ) with aqueous MeOH. However, when ( 4 ) was treated with methanolic KOH, a novel rearranged pyrrolidine ( 8 ) was obtained. Structures 5 and 8 for these heterolytic fragmentation products were established by nmr spectroscopic data and an X-ray crystal structure determination of 8 .

Three‐dimesional structure of GlcNAcα1‐4Gal releasing Endo‐β‐Galactosidase from Clostridium perfringens

Introduction. Clostridium perfringens is a ubiquitous anaerobic bacterium that is commonly found not only in soil but also in the gastrointestinal tract of higher animals. Its pathogenicity has been reviewed extensively. As a major cause of gastrointestinal infections in humans and animals, C. perfringens is suspected to employ hydrolytic enzymes in the degradation of gastrointestinal mucous glycoproteins. A unique endo-galactosidase, Endo-GalGnGa, capable of releasing GlcNAc 1-4Gal from porcine gastric mucin was discovered serendipitously as a contaminant of a commercially available C. perfringens sialidase preparation. Further characterization revealed small but significant sequence similarities with known glycosidases, including several enzymes from Bacillus sp. Endo-GalGnGa has been cloned and overexpressed in Escherichia coli. Single-crystal X-ray diffraction studies of Endo-GalGnGa were undertaken in order to explain the distinctive behavior of these enzymes based on a structural model in atomic detail.

Salvaging Pyrococcus furiosus protein targets at SECSG.

Proteins derived from the coding regions of Pyrococcus furiosus are targets for three-dimensional X-ray and NMR structure determination by the Southeast Collaboratory for Structural Genomics (SECSG). Of the 2200 open reading frames (ORFs) in this organism, 220 protein targets were cloned and expressed in a high-throughput (HT) recombinant system for crystallographic studies. However, only 96 of the expressed proteins could be crystallized and, of these, only 15 have led to structures. To address this issue, SECSG has recently developed a two-tier approach to protein production and crystallization. In this approach, tier-1 efforts are focused on producing protein for new Pfu(italics?) targets using a high-throughput approach. Tier-2 protein production efforts support tier-1 activities by (1) producing additional protein for further crystallization trials, (2) producing modified protein (further purification, methylation, tag removal, selenium labeling, etc) as required and (3) serving as a salvaging pathway for failed tier-1 proteins. In a recent study using this two-tiered approach, nine structures were determined from a set of 50 Pfu proteins, which failed to produce crystals suitable for X-ray diffraction analysis. These results validate this approach and suggest that it has application to other HT crystal structure determination applications.