Li-Chu Tsai

DNA binding and cleavage by the periplasmic nuclease Vvn: a novel structure with a known active site

The Vibrio vulnificus nuclease, Vvn, is a non‐specific periplasmic nuclease capable of digesting DNA and RNA. The crystal structure of Vvn and that of Vvn mutant H80A in complex with DNA were resolved at 2.3 Å resolution. Vvn has a novel mixed α/β topology containing four disulfide bridges, suggesting that Vvn is not active under reducing conditions in the cytoplasm. The overall structure of Vvn shows no similarity to other endonucleases; however, a known ‘ββα–metal’ motif is identified in the central cleft region. The crystal structure of the mutant Vvn–DNA complex demonstrates that Vvn binds mainly at the minor groove of DNA, resulting in duplex bending towards the major groove by ∼20°. Only the DNA phosphate backbones make hydrogen bonds with Vvn, suggesting a structural basis for its sequence‐independent recognition of DNA and RNA. Based on the enzyme–substrate and enzyme–product structures observed in the mutant Vvn–DNA crystals, a catalytic mechanism is proposed. This structural study suggests that Vvn hydrolyzes DNA by a general single‐metal ion mechanism, and indicates how non‐specific DNA‐binding proteins may recognize DNA.

Crystal Structure of a Natural Circularly Permuted Jellyroll Protein: 1,3-1,4-β-d-Glucanase from Fibrobacter succinogenes

The 1,3-1,4-beta-D-glucanase from Fibrobacter succinogenes (Fsbeta-glucanase) is classified as one of the family 16 glycosyl hydrolases. It hydrolyzes the glycosidic bond in the mixed-linked glucans containing beta-1,3- and beta-1,4-glycosidic linkages. We constructed a truncated form of recombinant Fsbeta-glucanase containing the catalytic domain from amino acid residues 1-258, which exhibited a higher thermal stability and enzymatic activity than the full-length enzyme. The crystal structure of the truncated Fsbeta-glucanase was solved at a resolution of 1.7A by the multiple wavelength anomalous dispersion (MAD) method using the anomalous signals from the seleno-methionine-labeled protein. The overall topology of the truncated Fsbeta-glucanase consists mainly of two eight-stranded anti-parallel beta-sheets arranged in a jellyroll beta-sandwich, similar to the fold of many glycosyl hydrolases and carbohydrate-binding modules. Sequence comparison with other bacterial glucanases showed that Fsbeta-glucanase is the only naturally occurring circularly permuted beta-glucanase with reversed sequences. Structural comparison shows that the engineered circular-permuted Bacillus enzymes are more similar to their parent enzymes with which they share approximately 70% sequence identity, than to the naturally occurring Fsbeta-glucanase of similar topology with 30% identity. This result suggests that protein structure relies more on sequence identity than topology. The high-resolution structure of Fsbeta-glucanase provides a structural rationale for the different activities obtained from a series of mutant glucanases and a basis for the development of engineered enzymes with increased activity and structural stability.

Metal ions and phosphate binding in the H-N-H motif: Crystal structures of the nuclease domain of ColE7/Im7 in complex with a phosphate ion and different divalent metal ions

H‐N‐H is a motif found in the nuclease domain of a subfamily of bacteria toxins, including colicin E7, that are capable of cleaving DNA nonspecifically. This H‐N‐H motif has also been identified in a subfamily of homing endonucleases, which cleave DNA site specifically. To better understand the role of metal ions in the H‐N‐H motif during DNA hydrolysis, we crystallized the nuclease domain of colicin E7 (nuclease‐ColE7) in complex with its inhibitor Im7 in two different crystal forms, and we resolved the structures of EDTA‐treated, Zn2+‐bound and Mn2+‐bound complexes in the presence of phosphate ions at resolutions of 2.6 Å to 2.0 Å. This study offers the first determination of the structure of a metal‐free and substrate‐free enzyme in the H‐N‐H family. The H‐N‐H motif contains two antiparallel β‐strands linked to a C‐terminal α‐helix, with a divalent metal ion located in the center. Here we show that the metal‐binding sites in the center of the H‐N‐H motif, for the EDTA‐treated and Mg2+‐soaked complex crystals, were occupied by water molecules, indicating that an alkaline earth metal ion does not reside in the same position as a transition metal ion in the H‐N‐H motif. However, a Zn2+ or Mn2+ ions were observed in the center of the H‐N‐H motif in cases of Zn2+ or Mn2+‐soaked crystals, as confirmed in anomalous difference maps. A phosphate ion was found to bridge between the divalent transition metal ion and His545. Based on these structures and structural comparisons with other nucleases, we suggest a functional role for the divalent transition metal ion in the H‐N‐H motif in stabilizing the phosphoanion in the transition state during hydrolysis.

Structural Studies of the Pigeon Cytosolic Nadp+ -Dependent Malic Enzyme

Malic enzymes are widely distributed in nature, and have important biological functions. They catalyze the oxidative decarboxylation of malate to produce pyruvate and CO2 in the presence of divalent cations (Mg2+, Mn2+). Most malic enzymes have a clear selectivity for the dinucleotide cofactor, being able to use either NAD+ or NADP+, but not both. Structural studies of the human mitochondrial NAD+‐dependent malic enzyme established that malic enzymes belong to a new class of oxidative decarboxylases. Here we report the crystal structure of the pigeon cytosolic NADP+‐dependent malic enzyme, in a closed form, in a quaternary complex with NADP+, Mn2+, and oxalate. This represents the first structural information on an NADP+‐dependent malic enzyme. Despite the sequence conservation, there are large differences in several regions of the pigeon enzyme structure compared to the human enzyme. One region of such differences is at the binding site for the 2′‐phosphate group of the NADP+ cofactor, which helps define the cofactor selectivity of the enzymes. Specifically, the structural information suggests Lys362 may have an important role in the NADP+ selectivity of the pigeon enzyme, confirming our earlier kinetic observations on the K362A mutant. Our structural studies also revealed differences in the organization of the tetramer between the pigeon and the human enzymes, although the pigeon enzyme still obeys 222 symmetry.

Improving nanowire sensing capability by electrical field alignment of surface probing molecules.

We argue that the structure ordering of self-assembled probing molecular monolayers is essential for the reliability and sensitivity of nanowire-based field-effect sensors because it can promote the efficiency for molecular interactions as well as strengthen the molecular dipole field experienced by the nanowires. In the case of monolayers, we showed that structure ordering could be improved by means of electrical field alignment. This technique was then employed to align multilayer complexes for nanowire sensing applications. The sensitivity we achieved for detection of hybridization between 15-base single-strand DNA molecules is 0.1 fM and for alcohol sensors is 0.5 ppm. The reliability was confirmed by repeated tests on chips that contain multiple nanowire sensors.

Directed Mutagenesis of Specific Active Site Residues onFibrobacter succinogenes1,3–1,4-β-d-Glucanase Significantly Affects Catalysis and Enzyme Structural Stability

The functional and structural significance of amino acid residues Met39, Glu56, Asp58, Glu60, and Gly63 ofFibrobacter succinogenes1,3–1,4-β-d-glucanase was explored by the approach of site-directed mutagenesis, initial rate kinetics, fluorescence spectroscopy, and CD spectrometry. Glu56, Asp58, Glu60, and Gly63 residues are conserved among known primary sequences of the bacterial and fungal enzymes. Kinetic analyses revealed that 240-, 540-, 570-, and 880-fold decreases in k cat were observed for the E56D, E60D, D58N, and D58E mutant enzymes, respectively, with a similar substrate affinity relative to the wild type enzyme. In contrast, no detectable enzymatic activity was observed for the E56A, E56Q, D58A, E60A, and E60Q mutants. These results indicated that the carboxyl side chain at positions 56 and 60 is mandatory for enzyme catalysis. M39F, unlike the other mutants, exhibited a 5-fold increase inK m value. Lower thermostability was found with the G63A mutant when compared with wild type or other mutant forms ofF. succinogenes 1,3–1,4-β-d-glucanase. Denatured wild type and mutant enzymes were, however, recoverable as active enzymes when 8 m urea was employed as the denaturant. Structural modeling and kinetic studies suggest that Glu56, Asp58, and Glu60 residues apparently play important role(s) in the catalysis of F. succinogenes 1,3–1,4-β-d-glucanase.

EGCG/gelatin-doxorubicin gold nanoparticles enhance therapeutic efficacy of doxorubicin for prostate cancer treatment.

AIM Development of epigallocatechin gallate (EGCG) and gelatin-doxorubicin conjugate (GLT-DOX)-coated gold nanoparticles (DOX-GLT/EGCG AuNPs) for fluorescence imaging and inhibition of prostate cancer cell growth. MATERIALS & METHODS AuNPs alternatively coated with EGCG and DOX-GLT conjugates were prepared by a layer-by-layer assembly method. The physicochemical properties of the AuNPs and the effect of Laminin 67R receptor-mediated endocytosis on the anticancer efficacy of the AuNPs were examined. RESULTS The AuNPs significantly inhibit the proliferation of PC-3 cancer cell and the enzyme-responsive intracellular release of DOX could be tracked by monitoring the recovery of the fluorescence signal of DOX. CONCLUSION Laminin 67R receptor-mediated delivery of DOX using the AuNPs enhanced cellular uptake of DOX and improved apoptosis of PC-3 cells.

Structural modeling and further improvement in pH stability and activity of a highly-active xylanase from an uncultured rumen fungus

Rumen fungi are a rich source of enzymes degrading lignocelluloses. XynR8 is a glycosyl hydrolase family 11 xylanase previously cloned from unpurified rumen fungal cultures. Phylogenetic analysis suggested that xynR8 was obtained from a Neocallimastix species. Recombinant XynR8 expressed in Escherichia coli was highly active and stable between pH 3.0 and 11.0, and displayed a V(max) of 66,672μmolmin(-1)mg(-1), a k(cat) of 38,975s(-1), and a K(m) of 11.20mg/mL towards soluble oat spelt xylan. Based on molecular modeling, residues N41 and N58, important in stabilizing two loops and the structure of XynR8, were mutated to D. Both mutant enzymes showed higher tolerance to pH 2.0. The V(max), k(cat) and K(m) of the N41D and N58D mutant enzymes were 79,645μmolmin(-1)mg(-1), 46,493s(-1), 29.29mg/mL, and 96,689μmolmin(-1)mg(-1), 56,503s(-1), and 21.24mg/mL, respectively. Thus, they are good candidates for application, including biofuel production.

Structural modeling of glucanase–substrate complexes suggests a conserved tyrosine is involved in carbohydrate recognition in plant 1,3-1,4-β-d-glucanases

Glycosyl hydrolase family 16 (GHF16) truncated Fibrobacter succinogenes (TFs) and GHF17 barley 1,3-1,4-β-d-glucanases (β-glucanases) possess different structural folds, β-jellyroll and (β/α)8, although they both catalyze the specific hydrolysis of β-1,4 glycosidic bonds adjacent to β-1,3 linkages in mixed β-1,3 and β-1,4 β-d-glucans or lichenan. Differences in the active site region residues of TFs β-glucanase and barley β-glucanase create binding site topographies that require different substrate conformations. In contrast to barley β-glucanase, TFs β-glucanase possesses a unique and compact active site. The structural analysis results suggest that the tyrosine residue, which is conserved in all known 1,3-1,4-β-d-glucanases, is involved in the recognition of mixed β-1,3 and β-1,4 linked polysaccharide.

A highly active beta-glucanase from a new strain of rumen fungus Orpinomyces sp.Y102 exhibits cellobiohydrolase and cellotriohydrolase activities

A new strain of rumen fungus was isolated from Bos taurus, identified and designated Orpinomyces sp.Y102. A clone, celC7, isolated from the cDNA library of Orpinomyces sp.Y102, was predicted to encode a protein containing a signal peptide (Residues 1-17), an N-terminal dockerin-containing domain, and a C-terminal cellobiohydrolase catalytic domain of glycoside hydrolase family 6. CelC7 was insoluble when expressed in Escherichia coli. Deletion of 17 or 105 residues from the N-terminus significantly improved its solubility. The resulting enzymes, CelC7(-17) and CelC7(-105), were highly active to β-glucan substrates and were stable between pH 5.0 and 11.0. CelC7(-105) worked as an exocellulase releasing cellobiose and cellotriose from acid-swollen Avicel and cellooligosaccharides, and displayed a Vmax of 6321.64μmole/min/mg and a Km of 2.18mg/ml to barley β-glucan. Further, the crude extract of CelC7(-105) facilitated ethanol fermentation from cellulose. Thus, CelC7(-105) is a good candidate for industrial applications such as biofuel production.