Woo Cheol Lee

Crystal Structure and Desulfurization Mechanism of 2′-Hydroxybiphenyl-2-sulfinic Acid Desulfinase

The desulfurization of dibenzothiophene in Rhodococcus erythropolis is catalyzed by two monooxygenases, DszA and DszC, and a desulfinase, DszB. In the last step of this pathway, DszB hydrolyzes 2′-hydroxybiphenyl-2-sulfinic acid into 2-hydroxybiphenyl and sulfite. We report on the crystal structures of DszB and an inactive mutant of DszB in complex with substrates at resolutions of 1.8Å or better. The overall fold of DszB is similar to those of periplasmic substrate-binding proteins. In the substrate complexes, biphenyl rings of substrates are recognized by extensive hydrophobic interactions with the active site residues. Binding of substrates accompanies structural changes of the active site loops and recruits His60 to the active site. The sulfinate group of bound substrates forms hydrogen bonds with side chains of Ser27, His60, and Arg70, each of which is shown by site-directed mutagenesis to be essential for the activity. In our proposed reaction mechanism, Cys27 functions as a nucleophile and seems to be activated by the sulfinate group of substrates, whereas His60 and Arg70 orient the syn orbital of sulfinate oxygen to the sulfhydryl hydrogen of Cys27 and stabilize the negatively charged reaction intermediate. Cys, His, and Arg residues are conserved in putative proteins homologous to DszB, which are presumed to constitute a new family of desulfinases.

Crystal structure of the PIN domain of human telomerase-associated protein EST1A

Saccharomyces cerevisiae Est1p is a telomerase‐associated protein essential for telomere length homeostasis. hEST1A is one of the three human Est1p homologues and is considered to be involved not only in regulation of telomere elongation or capping but also in nonsense‐mediated degradation of RNA. hEST1A is composed of two conserved regions, Est1p homology and PIN (PilT N‐terminus) domains. The present study shows the crystal structure of the PIN domain at 1.8 Å resolution. The overall structure is composed of an α/β fold or a core structure similar to the counterpart of 5′ nucleases and an extended structure absent from archaeal PIN‐domain proteins and 5′ nucleases. The structural properties of the PIN domain indicate its putative active center consisting of invariant acidic amino acid residues, which is geometrically similar to the active center of 5′ nucleases and an archaeal PAE2754 PIN‐domain protein associated with exonuclease activity. Proteins 2007.

Improvement of 2'-Hydroxybiphenyl-2-sulfinate Desulfinase, an Enzyme Involved in the Dibenzothiophene Desulfurization Pathway, from Rhodococcus erythropolis KA2-5-1 by Site-Directed Mutagenesis

In the microbial dibenzothiophene desulfurization pathway, 2′-hydroxybiphenyl-2-sulfinate is converted to 2-hydroxybiphenyl and sulfinate by desulfinase (DszB) at the last step, and this reaction is rate-limiting for the whole pathway. The catalytic activity and thermostability of DszB were enhanced by the two amino acid substitutions. Based on information on the 3-D structure of DszB and a comparison of amino acid sequences between DszB and reported thermophilic and thermostable homologs (TdsB and BdsB), two amino acid residues, Tyr63 and Gln65, were selected as targets to mutate and improve DszB. These two residues were replaced by several amino acids, and the promising mutant enzymes were purified and their properties were examined. Among the wild-type and mutant enzymes, Y63F had higher catalytic activity but similar thermostability, and Q65H showed higher thermostability but less catalytic activity and affinity for the substrate. To compensate for these drawbacks, the double mutant enzyme Y63F-Q65H was purified and its properties were investigated. This mutant enzyme showed higher thermostability without loss of catalytic activity or affinity for the substrate. These superior properties of the mutant enzyme have also been confirmed with resting cells harboring the mutant gene.

The structure of brazzein, a sweet-tasting protein from the wild African plant Pentadiplandra brazzeana

Brazzein is the smallest sweet-tasting protein and was isolated from the wild African plant Pentadiplandra brazzeana. The brazzein molecule consists of 54 amino-acid residues and four disulfide bonds. Here, the first crystal structure of brazzein is reported at 1.8 Å resolution and is compared with previously reported solution structures. Despite the overall structural similarity, there are several remarkable differences between the crystal and solution structures both in their backbone folds and side-chain conformations. Firstly, there is an additional α-helix in the crystal structure. Secondly, the atomic r.m.s.d.s between the corresponding C(α)-atom pairs are as large as 2.0-2.2 Å between the crystal and solution structures. Thirdly, the crystal structure exhibits a molecular shape that is similar but not identical to the solution structures. The crystal structure of brazzein reported here will provide additional information and further insights into the intermolecular interaction of brazzein with the sweet-taste receptor.

Crystal structure of the YjgF/YER057c/UK114 family protein from the hyperthermophilic archaeon Sulfolobus tokodaii strain 7

ammoniumacetate, and 20% (v/v) glycerol and were then flash-cooled ina nitrogen stream. Diffraction data were collected at 100 Kwith an R-AXIS VII image plate detector mounted on aRigaku FR-E rotating-anode X-ray generator (Rigaku, Ja-pan) using the operation software CrystalClear (Rigaku/MSC) and were processed with MOSFLM

Crystal structures of apo-DszC and FMN-bound DszC from Rhodococcus erythropolis D-1

The release of SO2 from petroleum products derived from crude oil, which contains sulfur compounds such as dibenzothiophene (DBT), leads to air pollution. The ‘4S’ metabolic pathway catalyzes the sequential conversion of DBT to 2‐hydroxybiphenyl via three enzymes encoded by the dsz operon in several bacterial species. DszC (DBT monooxygenase), from Rhodococcus erythropolis D‐1 is involved in the first two steps of the ‘4S’ pathway. Here, we determined the first crystal structure of FMN‐bound DszC, and found that two distinct conformations occur in the loop region (residues 131–142) adjacent to the active site. On the basis of the DszC–FMN structure and the previously reported apo structures of DszC homologs, the binding site for DBT and DBT sulfoxide is proposed.

The effect of amphiphilic additives on the growth and morphology of Aspergillus niger acid proteinase A crystals

Abstract The effect of amphiphilic additives, dimethyl sulfoxide (DMSO) and alkane diols (HO(CH 2 ) n OH , n=2 – 6) , on the crystallization of Aspergillus niger acid proteinase A was investigated by optical and confocal laser microscopy. As DMSO concentration was increased, while keeping ammonium sulfate concentration constant, the crystal clusters became larger and their number decreased. The crystal cluster morphology changed from thin needles to rod-like. The amphiphilic nature of DMSO molecules seemed to suppress unfavorable hydrophobic interaction between protein molecules in the solution and in the crystal. To control the amphiphilic nature of additives, an alkane diol series of n =2–6 was examined in addition to DMSO. Single crystals were obtained at n =4 and 5, while only crystal clusters were obtained at n =2 and 3 and no crystals appeared at n =6. These results suggest that the balance of hydrophobicity and hydrophilicity of additives is important in protein crystallization.

Crystallization and preliminary X-ray analysis of the PIN domain of human EST1A

Human EST1A (ever shorter telomeres 1A) is associated with most or all active telomerase in cell extracts and is involved either directly or indirectly in telomere elongation and telomere capping. The C-terminal region of EST1A contains the PIN (PilT N-terminus) domain, a putative nuclease domain. The PIN domain of human EST1A was expressed, purified and crystallized by the sitting-drop vapour-diffusion method. The crystals belonged to space group C2, with unit-cell parameters a = 107.3, b = 51.6, c = 100.5 angstroms, beta = 119.3 degrees, and diffracted X-rays to 1.8 angstroms resolution. The asymmetric unit contained two molecules of the PIN domain and the solvent content was 57%.

Crystallization and preliminary X-ray analysis of carboxypeptidase 1 from Thermus thermophilus.

Carboxypeptidase 1 from the thermophilic eubacterium Thermus thermophilus (TthCP1, 58 kDa), a member of the M32 family of metallocarboxypeptidases, was crystallized by the sitting-drop vapour-diffusion method using PEG 8000 as the precipitant. The crystals diffracted X-rays to beyond 2.6 A resolution using a synchrotron-radiation source. The crystals belonged to the orthorhombic space group C222(1), with unit-cell parameters a = 171.0, b = 231.6, c = 124.9 A. The crystal contains three molecules in an asymmetric unit (VM = 2.11 A3 Da(-1)) and has a solvent content of 61.5%.

Crystal structure of dibenzothiophene sulfone monooxygenase BdsA from Bacillus subtilis WU-S2B

The dibenzothiophene (DBT) sulfone monooxygenase BdsA from Bacillus subtilis WU‐S2B catalyzes the conversion of DBT sulfone to 2′‐hydroxybiphenyl 2‐sulfinate. We report the crystal structures of BdsA at a resolution of 2.80 Å. BdsA exists as a homotetramer with a dimer‐of‐dimers configuration in the crystal, and the interaction between E288 and R296 in BdsA is important for tetramer formation. A structural comparison with homologous proteins shows that the orientation and location of the α9‐α12 helices in BdsA are closer to those of the closed form than those of the open form in the EDTA monooxygenase EmoA. Proteins 2017; 85:1171–1177.