Importance of Arg-599 of β-galactosidase (Escherichia coli) as an anchor for the open conformations of Phe-601 and the active-site loop

Structural and kinetic data show that Arg-599 of β-galactosidase plays an important role in anchoring the "open" conformations of both Phe-601 and an active-site loop (residues 794-803). When alanine was substituted for Arg-599, the conformations of Phe-601 and the loop shifted towards the "closed" positions because interactions with the guanidinium side chain were lost. Also, Phe-601, the loop, and Na+, which is ligated by the backbone carbonyl of Phe-601, lost structural order, as indicated by large B-factors. IPTG, a substrate analog, restored the conformations of Phe-601 and the loop of R599A-β-galactosidase to the open state found with IPTG-complexed native enzyme and partially reinstated order. ?-Galactonolactone, a transition state analog, restored the closed conformations of R599A-β-galactosidase to those found with ?-galactonolactone-complexed native enzyme and completely re-established the order. Substrates and substrate analogs bound R599A-β-galactosidase with less affinity because the closed conformation does not allow substrate binding and extra energy is required for Phe-601 and the loop to open. In contrast, transition state analog binding, which occurs best when the loop is closed, was several-fold better. The higher energy level of the enzyme?substrate complex and the lower energy level of the first transition state means that less activation energy is needed to form the first transition state and thus the rate of the first catalytic step (k2) increased substantially. The rate of the second catalytic step (k3) decreased, likely because the covalent form is more stabilized than the second transition state when Phe-601 and the loop are closed. The importance of the guanidinium group of Arg-599 was confirmed by restoration of conformation, order, and activity by guanidinium ions.     

RPA physically interacts with the human DNA glycosylase NEIL1 to regulate excision of oxidative DNA base damage in primer-template structures

The human DNA glycosylase NEIL1, activated during the S-phase, has been shown to excise oxidized base lesions in single-strand DNA substrates. Furthermore, our previous work demonstrating functional interaction of NEIL1 with PCNA and flap endonuclease 1 (FEN1) suggested its involvement in replication-associated repair. Here we show interaction of NEIL1 with replication protein A (RPA), the heterotrimeric single-strand DNA binding protein that is essential for replication and other DNA transactions. The NEIL1 immunocomplex isolated from human cells contains RPA, and its abundance in the complex increases after exposure to oxidative stress. NEIL1 directly interacts with the large subunit of RPA (K_d ～20 nM) via the common interacting interface (residues 312–349) in NEIL1's disordered C-terminal region. RPA inhibits the base excision activity of both wild-type NEIL1 (389 residues) and its C-terminal deletion CΔ78 mutant (lacking the interaction domain) for repairing 5-hydroxyuracil (5-OHU) in a primer-template structure mimicking the DNA replication fork. This inhibition is reduced when the damage is located near the primer-template junction. Contrarily, RPA moderately stimulates wild-type NEIL1 but not the CΔ78 mutant when 5-OHU is located within the duplex region. While NEIL1 is inhibited by both RPA and Escherichia coli single-strand DNA binding protein, only inhibition by RPA is relieved by PCNA. These results showing modulation of NEIL1's activity on single-stranded DNA substrate by RPA and PCNA support NEIL1's involvement in repairing the replicating genome.     

Probing protein-surfactant interaction by steady state and time-resolved fluorescence spectroscopy

The microenvironment of the probe coumarin 153 (C-153) in 1% bovine serum albumin (BSA) is more hydrophobic in nature compared to that in pure micelles or protein-surfactant complexes. In the native state of protein, we have not observed any solvation using C-153 as a probe but we have observed a slow dynamics on protein surface using 8-anilino-1-naphthalenesulfonic acid (ANS) as a probe. This may be due to the location of the probe (C-153) in the hydrophobic, solvent-inaccessible pocket of the BSA. Solvation dynamics in the BSA-surfactant (SDS) complexes in the solution phase is markedly different from that in pure micelles. This is may be due to the formation of 'necklace and bead' structure in the complexes. The rotational motion is also severely hindered in the surface of the protein.     

Stimulation of DNA glycosylase activity of OGG1 by NEIL1: functional collaboration between two human DNA glycosylases

The eukaryotic 8-oxoguanine-DNA glycosylase 1 (OGG1) provides the major activity for repairing mutagenic 7,8-dihydro-8-oxoguanine (8-oxoG) induced in the genome due to oxidative stress. Earlier in vitro studies showed that, after excising the base lesion, the human OGG1 remains bound to the resulting abasic (AP) site in DNA and does not turn over efficiently. The human AP-endonuclease (APE1), which cleaves the phosphodiester bond 5' to the AP site, in the next step of repair, displaces the bound OGG1 and thus increases its turnover. Here we show that NEIL1, a DNA glycosylase/AP lyase specific for many oxidized bases but with weak 8-oxoG excision activity, stimulates turnover of OGG1 in a fashion similar to that of APE1 and carries out betadelta-elimination at the AP site. This novel collaboration of two DNA glycosylases, which do not stably interact with each other, in stimulating 8-oxoguanine repair is possible because of higher AP site affinity and stronger AP lyase activity of NEIL1 relative to OGG1. Comparable levels of NEIL1 and OGG1 in some human cells raise the possibility that NEIL1 serves as a backup enzyme to APE1 in stimulating 8-oxoG repair in vivo.     

Conserved nuclear export sequences in Schizosaccharomyces pombe Mex67 and human TAP function in mRNA export by direct nuclear pore interactions

Mex67, the homolog of human TAP, is not an essential mRNA export factor in Schizosaccharomyces pombe. Here we show that S. pombe encodes a homolog of the TAP cofactor that we have also named p15, whose function in mRNA export is not essential. We have identified and characterized two distinct nuclear export activities, nuclear export signal (NES) I and NES II, within the region of amino acids 434-509 of Mex67. These residues map within the known NTF2-like fold of TAP (amino acids 371-551). We show that the homologs of these two NESs are present and are functionally conserved in TAP. The NES I, NES II, and NES I + II of TAP and Mex67 directly bind with -phenylalanine-glycine (-FG)-containing sequences of S. pombe Nup159 and Nup98 but not with human p62. Mutants of NES I or NES II of Mex67/TAP that do not bind -FG Nup159 and Nup98 in vitro are unable to mediate nuclear export of a heterologous protein in S. pombe and in HeLa cells. Fused with the RNA recognition motifs (RRMs) of Crp79 and green fluorescent protein (GFP) (RRM-NES-GFP), the NES I and NES II of Mex67 or TAP can suppress the mRNA export defect of the Deltap15 rae1-167 synthetic lethal S. pombe strain, suggesting that the NESs can function in the absence of p15. These novel nuclear export sequences may provide additional routes for delivering Mex67/TAP to the nuclear pore complex.     

Protein-protein interaction conferring stability to an extracellular acetyl (xylan) esterase produced by Termitomyces clypeatus

Acetyl esterase (AE) activity present in the culture filtrate of Termitomyces clypeatus was separated into lower molar mass (LMM) and higher molar mass (HMM) protein fractions during BioGel P-200 gel chromatography. AE was purified as a 30 kDa nonglycosylated protein from LMM fractions by CM-Sepharose ion exchange chromatography and HPGPLC. Although the HMM fraction had a number of enzyme activities (sucrase, beta-xylosidase, beta-glucosidase, and alpha-L-arabinofuranosidase) other than AE, protein present in the fraction was eluted as a single protein peak in HPGPLC and gave a single band in native PAGE. The fraction, subsequently purified by DEAE-Sephadex chromatography, was a SDS-PAGE homogeneous 80 kDa glycoprotein, but with both AE and cellobiase activities. The aggregate dissociated during ConA-Sepharose chromatography and 30 kDa AE and 56 kDa glycosylated cellobiase were purified separately. The dissociation caused significant loss of cellobiase activity but not that of AE. AE purified from both HMM and LMM fractions was characterized to be the same enzyme in terms of molar masses, pI (7.3), and other physicochemical properties. AE as an aggregate with cellobiase showed higher thermostability, temperature optimum, and resistance toward chemical denaturants than those of purified AE. Compared to cellobiase purified earlier from the same fungus, the enzyme present with AE in the aggregate also showed higher catalytic activity, thermostability, and temperature optimum. The study indicated that the formation of such SDS-resistant enzyme aggregate was associated with significant changes in the physicochemical properties of the enzymes, mainly toward improvement of rigidity of enzymes, and sometimes with the improvement of catalytic activity.     

Multilamellar Vesicular Clusters of Phosphatidylcholine and Their Sensitivity to Spectrin: A Study by Fractal Analysis

The cluster patterns of multilamellar vesicles (MLV) of dimyristoylphosphatidylcholine (DMPC) were analyzed using a combination of fractal analysis and lattice simulation. Self-assembly of DMPC MLVs resulted in two types of microscopically observable clusters. The clusters were classified on the basis of their mass fractal dimension, two-dimensional porosity, and the light scattering properties. Spectrin, a cytoskeletal protein, well known for its role in determining the cellular morphology, was used to perturb such spontaneously formed clusters. The fragmentation of the clusters by hydrodynamic perturbation followed a power law, implying again a fractal behavior. A lattice-based simulation was performed generating different class of cluster patterns. The observed correspondence between the cluster patterns and their stability was discussed in the framework of the proposed lattice simulation.     

Genotypic control of peanut somatic embryogenesis

The protocol for obtaining a high frequency of plant development via somatic embryogenesis from mature zygotic embryo-derived leaflets of peanut (Arachis hypogaea L.) involves multiple stages; these include the induction of embryogenic masses, development of embryos, radicle emergence/conversion of embryos and the development of plants from rooted abnormal embryos. Sixteen genotypes were subjected to this protocol by exposing mature zygotic embryo-derived leaflets to the common media sequence and comparing responses. Although the protocol was effective for all the genotypes, variation in frequency of response at each stage of development indicated that, with the exception of root meristem differentiation and subsequent radicle emergence, the whole process of somatic embryogenesis depended on the genotypic constitution of the original plant. The failure of somatic embryos to undergo conversion to plantlets could be a genotype-dependent characteristic. Received: 5 June 1997 / Revision received: 2 December 1997 / Accepted: 12 December 1997     

Appearance of storage lipid (triglycerides) in somatic embryos of peanut (arachis hypogaea L.)

Summary We estimated the level of triglycerides (triacylglycerol) at intervals of 2 wk during somatic embryogenesis in peanut. The initial triglyceride content in the leaflet explants was depleted during the formation of embryogenic tissue. It increased with the onset of somatic embryogenesis. Concentration of triglyceride in a fully developed embryo increased further if incubated in the same dehydrated medium for a longer period of time. Transferring these embryos to fresh medium led to germination of somatic embryos with a depletion of storage lipids.     

Action of human endonucleases III and VIII upon DNA-containing tandem dihydrouracil

We have shown previously that endonuclease III from Escherichia coli, its yeast homolog Ntg1p and E. coli endonuclease VIII recognize single dihydrouracil (DHU) lesions efficiently. However, these enzymes have limited capacities for completely removing DHU, when the lesion is present on duplex DNA as a tandem lesion. A duplex 30-mer (duplex1920) containing tandem DHU lesions at positions 19 and 20 from the 5' terminus was used as a substrate for human endonuclease III (hNTH) and endonuclease VIII (NEIL1). Two cleavage products, 18beta and 19beta were formed, when duplex1920 was treated with hNTH. The 18beta corresponded to the expected beta-elimination product generated from duplex1920, when the 5'-DHU of the tandem DHU was processed by hNTH. Similarly, 19beta is the beta-elimination product generated, when the 3'-DHU of the tandem DHU was processed by hNTH; 19beta thus still contained a DHU lesion at the 3' terminus. When these hNTH reaction products were further treated with human APE1, a single new product that corresponded to an 18mer was observed. These data suggested that human APE1 can help to process the 3' terminals following the action of hNTH on DHU lesions. Similarly, when duplex1920 was treated with NEIL1, two cleavage products, 18p and 19p were observed. The 18p and 19p corresponded to the expected beta,delta-elimination products derived from NEIL1 induced cleavage at the 5'-DHU and 3'-DHU of the tandem DHU, respectively. The 3'-phosphoryl group present in 18p can be readily removed by T4 polynucleotide kinase (PNK) to yield an 18mer that is suitable for repair synthesis. However, 19p required the participation of both PNK and APE1 to generate the 18mer. Together, we suggest that the processing of DNA-containing tandem DHU lesions, initiated by hNTH and NEIL1 can be channeled into two sub-pathways, the PNK-independent, APE1-dependent and the PNK, APE1-dependent pathways, respectively.