Hyung-Yeon Park

Furanone derivatives as quorum-sensing antagonists of Pseudomonas aeruginosa

The biofilm formation of Pseudomonas aeruginosa, an opportunistic human pathogen, is developed by cell-to-cell signaling, so-called quorum sensing (QS). To control the biofilm formation, we designed and synthesized new QS inhibitors of P. aeruginosa based on the structure of the previously known QS inhibitor, furanone. Newly synthesized compounds were a series of analogs of (5-oxo-2,5-dihydrofuran-3-yl)methyl alkanoate, and the structures of all six synthesized compounds was confirmed by NMR and GC/MS analyses. These new QS inhibitor candidates could remarkably inhibit both Pseudomonas QS signaling and biofilm formation, which were assayed by using the recombinant reporter system and flow cell confocal microscopy. The degree of QS inhibition by these new inhibitors varied from 20% to 90%. For the profound understanding about inhibition mechanism, we tried to estimate the binding energy between QS receptor, LasR, and our inhibitors from the in silico modeling system. The predicted binding pattern from the modeling system and our experimental data about QS inhibition were in good agreement. From these results, we suggest a new approach to develop the QS inhibitors and biofilm control agents based on structural modeling.

Expanding substrate specificity of GT‐B fold glycosyltransferase via domain swapping and high‐throughput screening

Glycosyltransferases (GTs) are crucial enzymes in the biosynthesis and diversification of therapeutically important natural products, and the majority of them belong to the GT‐B superfamily, which is composed of separate N‐ and C‐domains that are responsible for the recognition of the sugar acceptor and donor, respectively. In an effort to expand the substrate specificity of GT, a chimeric library with different crossover points was constructed between the N‐terminal fragments of kanamycin GT (kanF) and the C‐terminal fragments of vancomycin GT (gtfE) genes by incremental truncation method. A plate‐based pH color assay was newly developed for the selection of functional domain‐swapped GTs, and a mutant (HMT31) with a crossover point (N‐kanF‐669 bp and 753 bp‐gtfE‐C) for domain swapping was screened. The most active mutant HMT31 (50 kDa) efficiently catalyzed 2‐DOS (aglycone substrate for KanF) glucosylation using dTDP‐glucose (glycone substrate for GtfE) with kcat/Km of 162.8 ± 0.1 mM−1 min−1. Moreover, HMT31 showed improved substrate specificity toward seven more NDP‐sugars. This study presents a domain swapping method as a potential means to glycorandomization toward various syntheses of 2‐DOS‐based aminoglycoside derivatives. Biotechnol. Bioeng. 2009;102: 988–994.

Prediction of densities for solid energetic molecules with molecular surface electrostatic potentials

The densities of high energetic molecules in the solid state were calculated with a simplified scheme based on molecular surface electrostatic potentials (MSEP). The MSEP scheme for density estimation, originally developed by Politzer et al., was further modified to calculate electrostatic potential on a simpler van der Waals surface. Forty‐one energetic molecules containing at least one nitro group were selected from among a variety of molecular types and density values, and were used to test the suitability of the MSEP scheme for predicting the densities of solid energetic molecules. For comparison purposes, we utilized the group additivity method (GAM) incorporating the parameter sets developed by Stine (Stine‐81) and by Ammon (Ammon‐98 and ‐00). The absolute average error in densities from our MSEP scheme was 0.039 g/cc. The results based on our MSEP scheme were slightly better than the GAM results. In addition, the errors in densities generated by the MSEP scheme were almost the same for various molecule types, while those predicted by GAM were somewhat dependent upon the molecule types.

Structural understanding of quorum-sensing inhibitors by molecular modeling study in Pseudomonas aeruginosa

Inhibitors of 3OC12, an initial signal molecule of the quorum sensing (QS) signaling cascade in Pseudomonas aeruginosa have been developed. Eight inhibitor candidates were synthesized by substituting the head part of 3-oxododecanoyl-homoserine lactone (3OC12) with different aromatic rings, and their docking poses and scores (binding energies) were predicted by in silico modeling study. All compounds gave better docking scores than 3OC12 and good inhibition effects on LasR activity in the in vivo bioassay. Like the modifications in the tail part of 3OC12 in our previous study Kim et al. (2008), the head-part modifications also showed inhibition activity in a fairly good proportion to the docking scores from the modeling analysis. This implies that the head part of 3OC12 also contributes significantly to forming the active conformation of the LasR-3OC12 complex, and its modification could effectively induce the inactive conformation of the complex. We suggest that the head part of 3OC12 is also a good target moiety to develop the structure-based Pseudomonas QS inhibitors.

Engineering of daidzein 3'-hydroxylase P450 enzyme into catalytically self-sufficient cytochrome P450.

A cytochrome P450 (CYP) enzyme, 3’-daidzein hydroxylase, CYP105D7 (3’-DH), responsible for daidzein hydroxylation at the 3’-position, was recently reported. CYP105D7 (3’-DH) is a class I type of CYP that requires electrons provided through electron transfer proteins such as ferredoxin and ferredoxin reductase. Presently, we constructed an artificial CYP in order to develop a reaction host for the production of a hydroxylated product. Fusion-mediated construction with the reductase domain from self-sufficient CYP102D1 was done to increase electron transfer efficiency and coupling with the oxidative process. An artificial self-sufficient daidzein hydroxylase (3’-ASDH) displayed distinct spectral properties of both flavoprotein and CYP. The fusion enzyme catalyzed hydroxylation of daidzein more efficiently, with a kcat/Km value of 16.8 μM-1 min-1, which was about 24-fold higher than that of the 3’-DH-camA/B reconstituted enzyme. Finally, a recombinant Streptomyces avermitilis host for the expression of 3’-ASDH and production of the hydroxylated product was developed. The conversion that was attained (34.6%) was 5.2-fold higher than that of the wild-type.

Characterization of a new ScbR-like γ-butyrolactone binding regulator (SlbR) in Streptomyces coelicolor

Abstractγ-Butyrolactones in Streptomyces are well recognized as bacterial hormones, and they affect secondary metabolism of Streptomyces. γ-Butyrolactone receptors are considered important regulatory proteins, and various γ-butyrolactone synthases and receptors have been reported in Streptomyces. Here, we characterized a new regulator, SCO0608, that interacted with SCB1 (γ-butyrolactone of Streptomyces coelicolor) and bound to the scbR/A and adpA promoters. The SCO0608 protein sequences are not similar to those of any known γ-butyrolactone binding proteins in Streptomyces such as ScbR from S. coelicolor or ArpA from Streptomyces griseus. Interestingly, SCO0608 functions as a repressor of antibiotic biosynthesis and spore formation in R5 complex media. We showed the existence of another type of γ-butyrolactone receptor in Streptomyces, and this SCO0608 was named ScbR-like γ-butyrolactone binding regulator (SlbR) in S. coelicolor.

Development of inhibitors against TraR quorum-sensing system in Agrobacterium tumefaciens by molecular modeling of the ligand-receptor interaction

The quorum sensing (QS) inhibitors that antagonize TraR, a receptor protein for N-3-oxo-octanoyl-L-homoserine lactones (3-oxo-C8-HSL), a QS signal of Agrobacterium tumefaciens were developed. The structural analogues of 3-oxo-C8-HSL were designed by in silico molecular modeling using SYBYL packages, and synthesized by the solid phase organic synthesis (SPOS) method, where the carboxamide bond of 3-oxo-C8-HSL was replaced with a nicotinamide or a sulfonamide bond to make derivatives of N-nicotinyl-L-homoserine lactones or N-sulfonyl-L-homoserine lactones. The in vivo inhibitory activities of these compounds against QS signaling were assayed using reporter systems and compared with the estimated binding energies from the modeling study. This comparison showed fairly good correlation, suggesting that the in silico interpretation of ligand-receptor structures can be a valuable tool for the pre-design of better competitive inhibitors. In addition, these inhibitors also showed anti-biofilm activities against Pseudomonas aeruginosa.

Frequent false-positive reactions in pronase-treated T-cell flow cytometric cross-match tests.

OBJECTIVE Pretransplantation cross-match (XM) is essential in organ transplantation. The flow cytometric XM (FCXM) is the most sensitive cell-based XM technique. Pronase treatment is used to improve the sensitivity and specificity of the B-cell FCXM. Thus, pronase-treated (PT) T cells are tested in a single tube T-cell/B-cell technique. Observing discrepancies between PT and pronase-nontreated (PN) T- FCXM results, we investigated their incidence, clinical significance, and possible causes. METHODS We tested 226 serum samples from 167 kidney transplantation candidates or posttransplantation follow-up patients using PT and PN T-FCXM in parallel using 3-color and 2-color immunofluorescence staining, respectively. We reviewed panel-reactive antibody (PRA) and donor-specific antibody (DSA) status as well as HLA data and clinical outcomes. RESULTS The T-FCXM positive rate was significantly higher among PT versus PN tests (24.3% vs 11.1%; P < .001). Less than half of the PT-positive cases were positive in the PN test (45.5%; 25/55). Discrepancies were observed in 30 cases (13.3%), all of which gave PT(+)/PN(-) results. Our findings suggested that PT(+)/PN(-) results might arise from non-HLA antibodies. Class I DSA-positive rate (6.3% vs 2.2%; P = .45) and antibody-mediated rejection rate (0% vs 16.3%; P = .32) were not different between PT(+)/PN(-) and PT(-)/PN(-) groups. Moreover, 2 cases of PT(+)/PN(-) were observed among HLA-A, B, DR-identical donor-recipient pairs. CONCLUSION Pronase treatment is prone to give false-positive reactions in T-FCXM test probably due to the participation of non-HLA antibodies including autoantibodies. Patients might be inappropriately excluded from receiving organs. In laboratories using PT single tube T/B FCXM, caution is needed to avoid false-positive reporting of results.

Enzymatic reduction of levulinic acid by engineering the substrate specificity of 3-hydroxybutyrate dehydrogenase

Enzymatic reduction of levulinic acid (LA) was performed for the synthesis of 4-hydroxyvaleric acid (4HV)--a monomer of bio-polyester and a precursor of bio-fuels--using 3-hydroxybutyrate dehydrogenase (3HBDH) from Alcaligenes faecalis. Due to the catalytic inactivity of the wild-type enzyme toward LA, engineering of the substrate specificity of the enzyme was performed. A rational design approach with molecular docking simulation was applied, and a double mutant, His144Leu/Trp187Phe, which has catalytic activity (kcat/Km=578.0 min(-1) M(-1)) toward LA was generated. Approximately 57% conversion of LA to 4HV was achieved with this double mutant in 24 h, while no conversion was achieved with the wild-type enzyme.

Effect of His-tag location on the catalytic activity of 3-hydroxybutyrate dehydrogenase

The effect of hexahistidine-tag (His-tag) location at either the C or N-terminus on the catalytic activity of 3-hydroxybutyrate dehydrogenase (3HBDH) from Alcaligenes faecalis was studied. The kinetic parameters of 3HBDHs with C and N-terminal His-tags were investigated, and the enzyme with an N-terminal His-tag was found to have approximately 1,200-fold higher catalytic efficiency than its C-terminal counterpart. Furthermore, the effect of His-tag location on the catalytic activity of 3 engineered variants of 3HBDH that were previously developed for the conversion of levulinic acid to 4-hydroxyvaleric acid was also investigated. All of the N-terminal variants exhibited higher catalytic efficiency for levulinic acid than did the C-terminal counterparts. The structural basis of the His-tag effect was studied by investigating the structure of 3HBDH obtained from in silico His-tag modification, and the results revealed that the modification of the C-terminal structure could deform the hinge region of the active site entry loop, disrupting the catalytic motion of the enzyme. In contrast, due to the location of the N-terminus far from the active site of the enzyme, the catalytic activity of the enzyme was not severely affected by the N-terminal His-tag.