The regulation of growth hormone gene expression by thyroid hormone in cultured GH1 cells is mediated by a chromatin-associated receptor. We have previously described a photoaffinity label derivative of 3,5,3'-triiodo-L-thyronine (L-T3) in which the alanine side chain was modified to form N-2-diazo-3,3,3-trifluoropropionyl-L-T3 (L-[125I]T3-PAL). On exposure to 254 nm UV light, L-[125I]T3-PAL generates a carbene which covalently modifies two thyroid hormone receptor forms in intact GH1 cells; an abundant 47,000 Mr species and a less abundant 57,000 Mr form. We have now synthesized similar photoaffinity label derivatives of 3,5,3',5'-tetraiodo-L-thyronine (L-T4) and 3,3',5'-triiodo-L-thyronine (L-rT3). Both compounds identify the same receptor forms in intact cells and in nuclear extracts in vitro as L-[125I]T3-PAL. Labeling by L-[125I]rT3-PAL was low and consistent with the very low occupancy of receptor by L-rT3. Underivatized L-[125I]T3 and L-[125I]T4 labeled the same receptor forms at 254 nm but at a markedly lower efficiency than their PAL derivatives. In contrast, N-bromoacetyl-L-[125I]T3, a chemical affinity labeling agent, did not derivatize either receptor form in vitro. The relative efficiency of coupling to receptor at 254 nm was L-[125I]T4-PAL greater than L-[125I]T3-PAL greater than L-[125I]T4 greater than L-[125I]T3. Although L-[125I]T4-PAL has a lower affinity for receptor than L-[125I]T3-PAL, its coupling efficiency was 5-10-fold higher. This suggests that the alanine side chain of L-[125I]T4-PAL is positioned in the ligand binding region near a residue which is efficiently modified by photoactivation. With L-[125I]T4-PAL we were able to identify three different molecular weight receptor species in human fibroblast nuclei.

Geobacillus stearothermophilus T1 is a Gram-positive thermophilic soil bacterium that contains an extensive system for the utilization of plant cell-wall polysaccharides, including xylan, arabinan and galactan. The bacterium uses a number of extracellular enzymes that break down the high-molecular-weight polysaccharides into short oligosaccharides, which enter the cell and are further hydrolyzed into sugar monomers by dedicated intracellular glycoside hydrolases. The interest in the biochemical characterization and structural analysis of these proteins originates mainly from the wide range of their potential biotechnological applications. Studying the different hemicellulolytic utilization systems in G. stearothermophilus T1, a new galactan-utilization gene cluster was recently identified, which encodes a number of proteins, one of which is a GH1 putative 6-phospho-β-galactosidase (Gan1D). Gan1D has recently been cloned, overexpressed, purified and crystallized as part of its comprehensive structure-function study. The best crystals obtained for this enzyme belonged to the triclinic space group P1, with average crystallographic unit-cell parameters of a = 67.0, b = 78.1, c = 92.1 Å, α = 102.4, β = 93.5, γ = 91.7°. A full diffraction data set to 1.33 Å resolution has been collected for the wild-type enzyme, as measured from flash-cooled crystals at 100 K, using synchrotron radiation. These data are currently being used for the detailed three-dimensional crystal structure analysis of Gan1D.

In order to identify a rice gibberellin ester β-D-glucosidase, gibberellin A4 β-D-glucosyl ester (GA4-GE) was synthesized and used to screen rice β-glucosidases. Os3BGlu6 was found to have the highest hydrolysis activity to GA4-GE among five recombinantly expressed rice glycoside hydrolase family GH1 enzymes from different phylogenic clusters. The kinetic parameters of Os3BGlu6 and its mutants E178Q, E178A, E394D, E394Q and M251N for hydrolysis of p-nitrophenyl β-D-glucopyranoside (pNPGlc) and GA4-GE confirmed the roles of the catalytic acid/base and nucleophile for hydrolysis of both substrates and suggested M251 contributes to binding hydrophobic aglycones. The activities of the Os3BGlu6 E178Q and E178A acid/base mutants were rescued by azide, which they transglucosylate to produce β-D-glucopyranosyl azide, in a pH-dependent manner, while acetate also rescued Os3BGlu6 E178A at low pH. High concentrations of sodium azide (200-400 mM) inhibited Os3BGlu6 E178Q but not Os3BGlu6 E178A. The structures of Os3BGlu6 E178Q crystallized with either GA4-GE or pNPGlc had a native α-D-glucosyl moiety covalently linked to the catalytic nucleophile, E394, which showed the hydrogen bonding to the 2-hydroxyl in the covalent intermediate. These data suggest that a GH1 β-glucosidase uses the same retaining catalytic mechanism to hydrolyze 1-O-acyl glucose ester and glucoside.

Monolignol glucosides and their β-glucosidases are found in monocots, but their biological roles are unclear. Phylogenetic analysis of rice (Oryza sativa L.) glycoside hydrolase family GH1 β-glucosidases indicated that Os4BGlu14, Os4BGlu16, and Os4BGlu18 are closely related to known monolignol β-glucosidases. An optimized Os4BGlu16 cDNA and cloned Os4BGlu18 cDNA were used to express fusion proteins with His6 tags in Pichia pastoris and Escherichia coli, respectively. The secreted Os4BGlu16 fusion protein was purified from media by immobilized metal affinity chromatography (IMAC), while Os4BGlu18 was extracted from E. coli cells and purified by anion exchange chromatography, hydrophobic interaction chromatography and IMAC. Os4BGlu16 and Os4BGlu18 hydrolyzed the monolignol glucosides coniferin (kcat/KM, 21.6mM(-1)s(-1) for Os4BGlu16 and for Os4BGlu18) and syringin (kcat/KM, 22.8mM(-1)s(-1) for Os4BGlu16 and 24.0mM(-1)s(-1) for Os4BGlu18) with much higher catalytic efficiencies than other substrates. In quantitative RT-PCR, highest Os4BGlu14 mRNA levels were detected in endosperm, embryo, lemma, panicle and pollen. Os4BGlu16 was detected highest in leaf from 4 to 10 weeks, endosperm and lemma, while Os4BGlu18 mRNA was most abundant in vegetative stage from 1 week to 4 weeks, pollen and lemma. These data suggest a role for Os4BGlu16 and Os4BGlu18 monolignol β-glucosidases in both vegetative and reproductive rice tissues.

Most patients with hereditary isolated growth hormone deficiency (IGHD) are either heterozygous or homozygous for a growth hormone (GH) gene abnormality. GH1 gene deletions (6.7 and 7.6 kb) from eight Japanese families with IGHD type IA has been detected by Southern blot analysis or polymerase chain reaction and Smal digestion. Heterozygous point mutations at the donor splice site of intron 3 in the GH1 gene have been identified among autosomal dominant IGHD type II patients. Recently, we have identified two kinds of splicing mutations in intron 3 in four Japanese families with IGHD type II. We believe a newly diagnosed G to A mutation at the fifth base of intron 3 in a Japanese family is responsible for the IGHD type II phenotype.

Triiodothyronine (T3) dependent growth of GH1 rat pituitary tumor cells in serum-free defined culture requires apotransferrin (apoTf) (Sirbasku et al.: Mol. Cell. Endocrinol., 77:C47-C55, 1991). Diferric transferrin (2Fe.Tf) also is necessary as an iron source (Eby et al.: Anal. Biochem., 203:317-325, 1992). Further, T3 dependence is prevented by soluble Fe(III) addition to the medium (Sato et al.: In Vitro Cell. Dev. Biol., 27A:599-602, 1991). While our data suggested that apoTf caused growth by chelation of Fe(III), direct evidence was required. We used urea polyacrylamide gel electrophoresis along with autoradiography and Western immunoblotting to measure the Fe(III) content of growing GH1 cell cultures and identify the apoTf, mono-metal transferrins and 2Fe.Tf present. We found that apoTf per se did not cause growth but instead chelated inhibitory levels of Fe(III). In fact, apoTf need not be present at all provided that Fe(III) is reduced to < or = 0.6 microM. In addition, other protein and non-protein Fe(III) chelators were shown to be as effective as apoTf. Here, we report that pituitary cells are completely inhibited by>> or = 1.2 microM Fe(III), which are concentrations which might be expected in many culture media and usually are not thought to influence growth. The high sensitivity of pituitary cells to Fe(III) suggests further study to determine what cellular functions are affected and how they interfere with thyroid hormone dependence.

Dominantly inherited isolated GH deficiency is mainly caused by a heterozygous donor site mutation of intron 3 in the GH1 gene. An exon 3 deletion in GH (del32-71 GH) is produced from a mutant allele, whereas wild-type GH is produced from the other allele. Several studies have demonstrated a dominant negative effect of del32-71 GH on wild-type GH secretion, but the precise molecular mechanisms remain unclear. We hypothesized that unfolded del32-71 GH accumulates in the endoplasmic reticulum (ER) and causes ER stress and apoptosis in somatotrophs, promoting GH deficiency. To evaluate del32-71 GH-mediated ER stress, we established GH4C1 cell lines with doxycycline (dox)-controlled del32-71 GH expression. In 20 of 23 dox-controlled cell lines, the concentration of wild-type GH in the culture medium significantly decreased with del32-71 GH induction, demonstrating the dominant negative effect of this mutant. Cell viability, mRNA abundance of ER stress-response genes, caspase activation, and DNA fragmentation were evaluated in 5 dox-controlled cell lines selected as cellular models. In 4 of the 5 cell lines, del32-71 GH induction decreased cell viability, increased expression of 3 major ER stress response pathways (PRKR-like endoplasmic reticulum kinase [PERK], activating transcription factor-6 [ATF6], and inositol requirement 1 [IRE1]), and induced caspase-3 and caspase-7 activation. In 1 of the 4 cell lines, DNA fragmentation was demonstrated. Finally, overexpression of XBP1(S), a nuclear transcription factor downstream of IRE1, completely reversed the observed caspase activation. These data suggested that del32-71 GH-mediated ER stress and apoptosis contributed to the decrease in wild-type GH secretion observed in GH deficiency due to the GH1 gene slice-site mutations.

Growth hormone (GH) transgenic Amago (Oncorhynchus masou ishikawae), containing the sockeye GH1 gene fused with metallothionein-B promoter from the same species, were generated and the physiological condition through lipid metabolism compared among homozygous (Tg/Tg) and heterozygous GH transgenic (Tg/+) Amago and the wild type control (+/+). Previously, we have reported that the adipose tissue was generally smaller in GH transgenic fish compared to the control, and that the Δ-6 fatty acyl desaturase gene was down-regulated in the Tg/+ fish. However, fatty acid (FA) compositions have not been measured previously in these fish. In this study we compared the FAs composition and content in the liver using gas chromatography. Eleven kinds of FA were detected. The composition of saturated and monounsaturated fatty acids (SFA and MUFA) such as myristic acid (14:0), palmitoleic acid (16:1n-7), and cis-vaccenic acid (cis-18:1n-7) was significantly (P<0.05) decreased in GH transgenic Amago. On the other hand, the composition of polyunsaturated fatty acids (PUFAs) such as linoleic acid (18:2n-6), arachidonic acid (20:4n-6), and docosapentaenoic acid (22:5n-3) was significantly (P<0.05) increased. Levels of serum glucose and triacylglycerol were significantly (P<0.05) decreased in the GH transgenics compared with +/+ fish. Furthermore, 3'-tag digital gene expression profiling was performed using liver tissues from Tg/Tg and +/+ fish, and showed that Mid1 interacting protein 1 (Mid1ip1), which is an important factor to activate Acetyl-CoA carboxylase (ACC), was down-regulated in Tg/Tg fish, while genes involved in FA catabolism were up-regulated, including long-chain-fatty-acid-CoA ligase 1 (ACSL1) and acyl-coenzyme A oxidase 3 (ACOX3). These data suggest that liver tissue from GH transgenic Amago showed starvation by alteration in glucose and lipid metabolism due to GH overexpression. The decrease of serum glucose suppressed Mid1ip1, and caused a decrease of de novo FA synthesis, resulting in a decrease of SFA and MUFA. This induced expression of ACSL1 and ACOX3 to produce energy through β-oxidation in the GH transgenic Amago.

BACKGROUND

The pituitary-specific transcription factor 1 plays a key role in the development and differentiation of three pituitary cell types: somatotrophs, lactotrophs, and thyrotrophs. Several mutations of the human gene (called POU1F1) have been shown to be responsible for a phenotype of combined pituitary hormone deficiency involving GH, prolactin (PRL), and TSH.

OBJECTIVE

We have identified a novel homozygous C to G mutation in exon 4 of the POU1F1 gene (S179R) in a patient with this rare phenotype. We analyzed the functional consequences of this S179R mutation associated with a single-amino acid change in the POU-specific domain.

METHODS

Consequences of this mutation on transcriptional activities by transfection studies in alphaT3 cells, DNA binding ability by EMSA, structural properties, and nuclear accumulation of POU1F1 were investigated.

RESULTS

The transactivation capacity of this mutant was markedly decreased on the GH1, PRL, TSHbeta, and POU1F1 genes. Interestingly, this mutation abolished the functional interaction of POU1F1 on the PRL promoter with the coactivator cAMP response element-binding protein-binding protein but not with the transcription factor LIM homeodomain transcription factor 3. The S179R mutant displayed normal nuclear accumulation but a markedly decreased binding to a DNA response element in keeping with crystallographic data, suggesting that the S179R mutation might interfere with DNA binding.

CONCLUSIONS

Together with previous data, our study indicates that both DNA binding and interaction with cofactors like cAMP response element-binding protein-binding protein are critical for POU1F1 function and that functional and structural properties of abnormal POU1F1 proteins are variously influenced by the type of mutations.

Pituitary development depends on a complex cascade of interacting transcription factors and signaling molecules. Lesions in this cascade lead to isolated or combined pituitary hormone deficiency (CPHD). The aim of this study was to identify copy number variants (CNVs) in genes known to cause CPHD and to determine their structure. We analyzed 70 CPHD patients from 64 families. Deletions were found in three Turkish families and one family from northern Iraq. In one family we identified a 4.96 kb deletion that comprises the first two exons of POU1F1. In three families a homozygous 15.9 kb deletion including complete PROP1 was discovered. Breakpoints map within highly homologous AluY sequences. Haplotype analysis revealed a shared haplotype of 350 kb among PROP1 deletion carriers. For the first time we were able to assign the boundaries of a previously reported PROP1 deletion. This gross deletion shows strong evidence to originate from a common ancestor in patients with Kurdish descent. No CNVs within LHX3, LHX4, HESX1, GH1 and GHRHR were found. Our data prove multiplex ligation-dependent probe amplification to be a valuable tool for the detection of CNVs as cause of pituitary insufficiencies and should be considered as an analytical method particularly in Kurdish patients.

OBJECTIVE

Short stature caused by biologically inactive GH is clinically characterized by lack of GH action despite normal-high secretion of GH, pathologically low IGF1 concentrations and marked catch-up growth on GH replacement therapy.

METHODS

Adopted siblings (girl and a boy) of unknown family history were referred for assessment of short stature (-4.5 and -5.6 SDS) at the age of 10 and 8.1 years respectively. They had delayed bone ages (6.8 and 4.5 years), normal GH peaks at stimulation tests, and severely reduced IGF1 concentrations (-3.5 and -4.0 SDS). Genetic analysis of the GH1 gene showed a heterozygous P59S mutation at position involved in binding to GH receptor (GHR).

RESULTS

Isoelectric focusing analysis of secreted GH in patient serum revealed the presence of higher GH-P59S peak compared with that of wt-GH. Furthermore, computational simulation of GH-P59S binding to GHR suggested problems in correct binding of the mutant to the GHR. In vitro GHR binding studies revealed reduced binding affinity of GH-P59S for GHR (IC₅₀, 30 ng/ml) when compared with the wt-GH (IC₅₀, 11.8 ng/ml) while a significantly decreased ability of the mutant to activate the Jak2/Stat5 signaling pathway was observed at physiological concentrations of 25-100 ng/ml.

CONCLUSIONS

The clinical and biochemical data of our patients support the diagnosis of partial bioinactive GH syndrome. The higher amount of GH-P59S secreted in their circulation combined with its impact on the wt-GH function on GHR binding and signaling may alter GHR responsiveness to wt-GH and could ultimately explain severe short stature found in our patients.

This study investigated human cytomegalovirus (HCMV) glycoprotein genotypes in the genital tract tissue of 125 tubal pregnancy patients. The HCMV glycoprotein-B N-terminus (gBn, 54 - 485 NT), gB endoprotease cleavage site (gBclv, 1284 - 1600 NT) and glycoproteinH (gH, -58 - 213 NT) gene fragments were amplified by nested polymerase chain reaction and sequenced to identify gB and gH genotypes. Of 16 gBn-positive samples, four were gBn1, one was gBn2 and 11 were the gBn3 genotype. Of 13 positive gBclv samples, seven were gBclv1, two were gBclv2 and four were the gBclv3 genotype. Of 20 positive gH samples, 10 were gH1, six were gH2 and four were a combined gH1/gH2 genotype. In 10 of the samples that were positive for the gBn and gBclv genotypes, the gBn and gBclv genotypes were not consistent (four were gBclv1-gBn3). This study showed that: (i) HCMV infection with the gB1 - gB3 glycoprotein genotypes is present in tubal pregnancy; (ii) the gBclv and gBn genotypes are not strictly consistent; and (iii) intragenetic variability within the gB gene due to homologous recombination occurs frequently.

This study was performed to investigate human cytomegalovirus (HCMV) infection and genetic variations within glycoprotein B (gB) and H (gH) genes in Chinese transplant recipients. A total of 245 ethylene-diamine tetraacetic acid (EDTA)-treated blood samples were obtained from 79 transplant recipients in southeast China. Based on the sequences of highly variable regions of the gB endoprotease cleavage site (gBclv), N-terminus of gp116 (gBn), and the gH N-terminus (gH), nested polymerase chain reaction assays for the detection of HCMV were established. Nucleotide sequencing was employed to differentiate gB and gH genotypes. Twenty-six of 79 (32.9%) transplant recipients were proved HCMV positive. The distribution of genotypes was gBclv1, 12/25; gBclv2, 3/25; gBclv3, 4/25; gBn1, 6/23; gBn2, 2/23; gBn3, 11/23; and no gBclv/n 4-related genotypes were presented. The distribution of gH genotypes was gH1, 11/26; gH2, 9/26; and co-infected with both gH1/2 in 6/26. These data show that genetic variability within the gB genes occurs frequently. Mixtures of gB and gH genotypes infection were common in Chinese solid organ transplant recipients.

β-Glucosidases (EC 3.2.1.21) can be found in all domains of living organisms, where they play essential roles in the removal of terminal glucosyl residues from nonreducing ends of saccharides and glycosides. Two active site amino acid residues, a nucleophile and a proton donor, play key roles in the hydrolytic mechanism. Besides these two highly conserved catalytic residues, there are other conserved amino acids in the active site of β-glucosidases that make direct hydrogen bonds to the glycosyl moiety at the -1 subsite. In this study, the catalytic mechanism of a GH1 β-glucosidase (BGlu1) is systematically studied. On the basis of the quantum mechanical studies, the side chain of Tyr315 in an interaction with both O5 of the glucose ring and the nucleophilic glutamate contributes significantly to the energy profile. Glu440 and the conserved Asn175 are the other residues in the polar interaction with -1 glucose with considerable influence on the free energy of the reaction. Gln29, His130, and Trp441, which also form hydrogen bonds to the glycosyl moiety, are found to have relatively a minor effect on the reaction. Different arrangements of active site residues in the high-level [quantum mechanics (QM)] and low-level [molecular mechanics (MM)] regions during the hybrid QM/MM calculations indicate that Tyr315 lowers the energy barrier in the deglycosylation step (by 11.95 kcal/mol) while Glu440 mainly reduces the energy barrier of the glycosylation step. Exclusion of either of these two residues from the QM region results in deviation of the geometric parameters of the enzyme-substrate complex from those expected for the preactivated distorted structure of the substrate.

β-glucosidases (EC 3.2.1.21) have been described as essential to second-generation biofuel production. They act in the last step of the lignocellulosic saccharification, cleaving the β-1,4 glycosidic bonds in cellobiose to produce two molecules of glucose. However, β-glucosidases have been described as strongly inhibited by glucose, causing an increment of cellobiose concentration. Also, cellobiose is an inhibitor of other enzymes used in this process, such as exoglucanases and endoglucanases. Hence, the engineering of thermostable and glucose-tolerant β-glucosidases has been targeted by many studies. In this study, we performed high sampling accelerated molecular dynamics for a wild glucose-tolerant GH1 β-glucosidase (Bgl1A), a wild non-tolerant (Bgl1B), and a set of glucose-tolerant Bgl1B's mutants: V302F, N301Q/V302F, F172I, V227M, G246S, T299S, and H228T. Our results suggest that point mutations promissory to induce glucose tolerance trend to enhance the mobility of the flexible loops around the active site. Mutations affected B and C loops regions, and an αβ-hairpin motif between them. Conformational clusters and free energy landscape profiles suggest that the mobility acquired by mutants allows a higher closure of the substrate channel. This closure is compatible with a higher impedance for glucose entrance and stimulus of its withdrawal. Based on mutants' structural analyses, we inferred that both the direct stereochemical effect on the glucose path and the changes in the mobility affect glucose tolerance. We hope these results be useful for the rational design of glucose-tolerant and industrially promising enzymes.

β-glucosidases play a critical role among the enzymes in enzymatic cocktails designed for plant biomass deconstruction. By catalysing the breakdown of β-1, 4-glycosidic linkages, β-glucosidases produce free fermentable glucose and alleviate the inhibition of other cellulases by cellobiose during saccharification. Despite this benefit, most characterised fungal β-glucosidases show weak activity at high glucose concentrations, limiting enzymatic hydrolysis of plant biomass in industrial settings. In this study, structural analyses combined with site-directed mutagenesis efficiently improved the functional properties of a GH1 β-glucosidase highly expressed by Trichoderma harzianum (ThBgl) under biomass degradation conditions. The tailored enzyme displayed high glucose tolerance levels, confirming that glucose tolerance can be achieved by the substitution of two amino acids that act as gatekeepers, changing active-site accessibility and preventing product inhibition. Furthermore, the enhanced efficiency of the engineered enzyme in terms of the amount of glucose released and ethanol yield was confirmed by saccharification and simultaneous saccharification and fermentation experiments using a wide range of plant biomass feedstocks. Our results not only experimentally confirm the structural basis of glucose tolerance in GH1 β-glucosidases but also demonstrate a strategy to improve technologies for bioethanol production based on enzymatic hydrolysis.

UNASSIGNED

Compost habitats sustain a vast ensemble of microbes that engender the degradation of cellulose, which is an important part of global carbon cycle. β-Glucosidase is the rate-limiting enzyme of degradation of cellulose. Thus, analysis of regulation of β-glucosidase gene expression in composting is beneficial to a better understanding of cellulose degradation mechanism. Genetic diversity and expression of β-glucosidase-producing microbial communities, and relationships of cellulose degradation, metabolic products and the relative enzyme activity during natural composting and inoculated composting were evaluated.

UNASSIGNED

Compared with natural composting, adding inoculation agent effectively improved the degradation of cellulose, and maintained high level of the carboxymethyl cellulose (CMCase) and β-glucosidase activities in thermophilic phase. Gene expression analysis showed that glycoside hydrolase family 1 (GH1) family of β-glucosidase genes contributed more to β-glucosidase activity in the later thermophilic phase in inoculated compost. In the cooling phase of natural compost, glycoside hydrolase family 3 (GH3) family of β-glucosidase genes contributed more to β-glucosidase activity. Intracellular β-glucosidase activity played a crucial role in the regulation of β-glucosidase gene expression, and upregulation or downregulation was also determined by extracellular concentration of glucose. At sufficiently high glucose concentrations, the functional microbial community in compost was altered, which may contribute to maintaining β-glucosidase activity despite the high glucose content.

UNASSIGNED

This research provides an ecological functional map of microorganisms involved in carbon metabolism in cattle manure-rice straw composting. The performance of the functional microbial groups in the two composting treatments is different, which is related to the cellulase activity and cellulose degradation, respectively.

OBJECTIVE

Soils are rich, diversified environments where β-glucosidases abound because of their importance in organic matter degradation. The aim of this work was to discover new β-glucosidases by constructing two metagenomic DNA libraries from soil samples collected in winter and spring from a field of winter wheat.

RESULTS

Both libraries were screened on esculin-supplemented medium so as to isolate candidates showing β-glucosidase activity. Candidate analysis revealed seven putative β-glycosidases and two putative glycosyltransferases, displaying 25 to 82% identity to known enzymes. The putative β-glycosidases belong to families GH1, GH3 and GH20 and the two putative glycosyltransferases, probably, to new families. In characterization tests performed on bacteria in suspension or spread on agar plates, some candidates appeared to hydrolyse several natural and synthetic substrates. These tests also highlighted interesting industrial characteristics, such as the activity of four β-glycosidases under alkaline conditions and the esculin-hydrolysing activity of a β-glucosidase candidate in the presence of glucose.

CONCLUSIONS

Seven putative β-glycosidases and two putative glycosyltransferases were found by functional screening of two metagenomic DNA libraries derived from agricultural soil.

CONCLUSIONS

This study has identified β-glycosidases and putative glycosyltransferases that have or may have interesting industrial characteristics.

Plants produce an array of natural products with important ecological functions. These compounds are often decorated with oligosaccharide groups that influence bioactivity, but the biosynthesis of such sugar chains is not well understood. Triterpene glycosides (saponins) are a large family of plant natural products that determine important agronomic traits, as exemplified by avenacins, antimicrobial defense compounds produced by oats. Avenacins have a branched trisaccharide moiety consisting of l-arabinose linked to 2 d-glucose molecules that is critical for antifungal activity. Plant natural product glycosylation is usually performed by uridine diphosphate-dependent glycosyltransferases (UGTs). We previously characterized the arabinosyltransferase that initiates the avenacin sugar chain; however, the enzymes that add the 2 remaining d-glucose molecules have remained elusive. Here we characterize the enzymes that catalyze these last 2 glucosylation steps. AsUGT91G16 is a classical cytosolic UGT that adds a 1,2-linked d-glucose molecule to l-arabinose. Unexpectedly, the enzyme that adds the final 1,4-linked d-glucose (AsTG1) is not a UGT, but rather a sugar transferase belonging to Glycosyl Hydrolase family 1 (GH1). Unlike classical UGTs, AsTG1 is vacuolar. Analysis of oat mutants reveals that AsTG1 corresponds to Sad3, a previously uncharacterized locus shown by mutation to be required for avenacin biosynthesis. AsTG1 and AsUGT91G16 form part of the avenacin biosynthetic gene cluster. Our demonstration that a vacuolar transglucosidase family member plays a critical role in triterpene biosynthesis highlights the importance of considering other classes of carbohydrate-active enzymes in addition to UGTs as candidates when elucidating pathways for the biosynthesis of glycosylated natural products in plants.

An Amazon soil microbial community metagenomic fosmid library was functionally screened for β-glucosidase activity. Contig analysis of positive clones revealed the presence of two ORFs encoding novel β-glucosidases, AmBGL17 and AmBGL18, from the GH3 and GH1 families, respectively. Both AmBGL17 and AmBGL18 were functionally identified as β-glucosidases. The enzymatic activity of AmBGL17 was further characterized. AmBGL17 was tested with different substrates and showed highest activity on pNPβG substrate with an optimum temperature of 45 °C and an optimum pH of 6. AmBGL17 showed a Vmax of 116 mM s(-1) and Km of 0.30 ± 0.017 mM. This is the first report of β-glucosidases from an Amazon soil microbial community using a metagenomic approach.

β-glucosidase is a crucial element of the microbial cellulose multienzyme complex since it is responsible for the regulation of the entire cellulose hydrolysis process. Therefore, the aim of the present work was to explore the diversity and distribution of glycosyl hydrolase family 1 β-glucosidase genes in three different environmental niches including, Himalayan soil, cow dung and compost by metagenomic approach. Preliminary evaluation through metabolic profiling using BIOLOG based utilization patterns of carbon, nitrogen, phosphorus and sulfur revealed the environment and substrate specific nature of the indigenous microbial population. Furthermore, clonal library selection, screening and sequence analysis revealed that most of the GH1 β-glucosidase proteins had low identities with the available database. Analysis of the distribution of GH1 β-glucosidase gene fragments and β-glucosidase producing microbial community revealed the environment specific nature. The OTUs obtained from Himalayan soil and compost metagenomic libraries were grouped into 19 different genera comprising 6 groups. The cow dung sample displayed the least diversity of GH1 β-glucosidase sequences, with only 14 genera, distributed among three groups- Bacteroidetes, Firmicutes, and Actinobacteria. The metagenomic study coupled with metabolic profiling of GH1 β-glucosidase illustrated the existence of intricate relationship between the geochemical environmental factors and inherent microbial community.

A novel technique of chimeric somatic cell cloning was applied to produce a transgenic rabbit (NT20). Karyoplasts of transgenic adult skin fibroblasts with Tg(Wap-GH1) gene construct as a marker were microsurgically transferred into one, previously enucleated, blastomere of 2-cell non-transgenic embryos, while the second one remained intact. The reconstructed embryos either were cultured in vitro up to the blastocyst stage (Experiment I) or were transferred into recipient-females immediately after the cloning procedure (Experiment II). In Experiment I, 25/102 (24.5%) embryos formed blastocysts from whole embryos and 46/102 (44.12%) embryos developed to the blastocyst stage from single non-operated blastomeres, while the reconstructed blastomeres were damaged and degenerated. Thirteen (12.7%) embryos did not exceed 3- to 4-cell stages and 18 (17.7%) embryos were inhibited at the initial 2-cell stage. Out of 14 blastocysts which were subjected to molecular analysis, the transgene was detected in the cells of 4 blastocysts. In Experiment II, 163/217 (75.0%) embryos were transferred into 9 pseudopregnant recipient-rabbits (an average of 18 embryos per recipient). Four recipient-females (44.4%) became pregnant and delivered a total of 24 (14.7%) pups. Molecular analysis confirmed that two pups (1.2%), one live and one stillborn, showed a positive transgene signal. Live transgenic rabbit NT20 appeared healthy and anatomically as well as physiologically normal. The results of our experiments showed that transgenic adult skin fibroblast cell nuclei, which have been introduced into the cytoplasmic microenvironment of single enucleated blastomeres from 2-cell stage rabbit embryos, are able to direct the development of chimeric embryos not only to the blastocyst stage but also up to term.

Tannin acyl hydrolases, or tannases (EC 3.1.1.20), are enzymes with potential biotechnological applications. In this work, we describe the gene and amino acid sequences of the tannase from Aspergillus niger GH1. In addition, we engineered Pichia pastoris strains to produce and secrete the enzyme, and the produced tannase was characterized biochemically. The nucleotide sequence of mature tannase had a length of 1,686 bp, and encodes a protein of 562 amino acids. A molecular model of mature A. niger GH1 tannase showed the presence of two structural domains, one with an α/β-hydrolase fold and one lid domain that covers the catalytic site, likely being residues Ser-196, Asp-448, and His-494 the putative catalytic triad, which are connected by a disulfide bond between the neighboring cysteines, Cys-195 and Cys-495. A 120-ml shake flask culture with a constructed recombinant P. pastoris strain showed extracellular tannase activity at 48 h induction of 0.57 U/ml. The produced tannase was N-glycosylated, consisted of two subunits, likely linked by a disulfide bond, and had an optimum pH of 5.0 and optimum temperature of 20 °C. These biochemical properties differed from those of native A. niger GH1 tannase. The recombinant tannase could be suitable for food and beverage applications.