Reaction mechanisms of Trp120.fwdarw.Phe and wild-type glucoamylases from Aspergillus niger. Interactions with maltooligodextrins and acarbose

Background:Little is known about BYDV protein synthesis initiation mechanisms. Results: eIF4F binding correlates with 3Ј BTE translation efficiency and is enthalpically and entropically favorable. Conclusion: Binding of eIF4F to the 3Ј BTE is important for efficient BYDV translation. Significance: Binding of eIF4F to the 3Ј-UTR to initiate assembly of the protein synthesis initiation complex is a novel mechanism that may be used by other viruses containing a 3Ј BTE.

Section: An Increase Inmentioning

confidence: 99%

Eukaryotic Initiation Factor (eIF) 4F Binding to Barley Yellow Dwarf Virus (BYDV) 3′-Untranslated Region Correlates with Translation Efficiency

Banerjee

Goss

2014

“…The data were fitted to the single-and double-exponential functions. Fitted curves correspond to the following single-exponential equation (40),…”

Section: Construction Of Expression Vector For Wild Type Vpg and N-tementioning

confidence: 99%

Potyvirus Genome-linked Protein, VPg, Directly Affects Wheat Germ in Vitro Translation

Khan

Mizumoto

Gallie

et al. 2008

Potyvirus genome linked protein, VPg, interacts with translation initiation factors eIF4E and eIFiso4E, but its role in protein synthesis has not been elucidated. We show that addition of VPg to wheat germ extract leads to enhancement of uncapped viral mRNA translation and inhibition of capped viral mRNA translation. This provides a significant competitive advantage to the uncapped viral mRNA. To understand the molecular basis of these effects, we have characterized the interaction of VPg with eIF4F, eIFiso4F, and a structured RNA derived from tobacco etch virus (TEV RNA). When VPg formed a complex with eIF4F, the affinity for TEV RNA increased more than 4-fold compared with eIF4F alone (19.4 and 79.0 nM, respectively). The binding affinity of eIF4F to TEV RNA correlates with translation efficiency. VPg enhanced eIFiso4F binding to TEV RNA 1.6-fold (178 nM compared with 108 nM). Kinetic studies of eIF4F and eIFiso4F with VPg show ϳ2.6-fold faster association for eIFiso4F⅐VPg as compared with eIF4F⅐VPg. The dissociation rate was ϳ2.9-fold slower for eIFiso4F than eIF4F with VPg. These data demonstrate that eIFiso4F can kinetically compete with eIF4F for VPg binding. The quantitative data presented here suggest a model where eIF4F⅐VPg interaction enhances cap-independent translation by increasing the affinity of eIF4F for TEV RNA. This is the first evidence of direct participation of VPg in translation initiation.Most eukaryotic mRNAs contain a cap structure m 7 GpppX 2 (where X is any nucleotide) at the 5Ј-end that is required for efficient initiation of translation. The cap serves as the binding site for initiation factors eIF4F or eIFiso4F, an isozyme form of eIF4F present in higher plants, either of which is involved in the assembly of the initiation complex. Alternative translation mechanisms have been shown to take place under a unique set of circumstances. One such example is that of viral infection. Viruses employ different strategies for preferential translation of their mRNAs often using internal ribosome entry sites (IRES).Recently attention has focused on the possible role in translation initiation of the viral protein linked to the genome (VPg) of potyvirus (1, 2). It has been suggested that VPg may serve as an analog of the m 7 G cap of the mRNAs and plays a role in mRNA translation because it interacts with the cap-binding proteins eIF4E and eIFiso4E (3, 4), subunits of eIF4F and eIFiso4F. Potyviruses have a messenger-polarity single-strand RNA genome, a poly(A) tail, and VPg covalently attached to the 5Ј terminus (1, 5), which upon infection of a suitable host is cleaved from the genome RNA by an unknown cellular enzyme. The 5Ј-pUpU-processed RNA serves as a template for viral protein synthesis as well as early rounds of RNA synthesis (6). Potyviruses have been shown to be capable of cap-independent translation (7) making the role of VPg in translation uncertain. Studies support a biological role for the VPg linked to the viral RNA in virions. The covalent tyrosine residue-mediated linkage between ...

“…AT and CG represent A. tumefaciens and C. gilvus, respectively. mutation could be attributed to modification in hydrogen bonding (32,34,35). The relative rates of hydrolysis of cello-oligosaccharides by parental and chimeric enzymes are shown in Table II.…”

mentioning

confidence: 99%

Construction of Chimeric β-Glucosidases with Improved Enzymatic Properties

Singh

Hayashi

1995

The amino acid sequences of ␤-glucosidases from Cellvibrio gilvus and Agrobacterium tumefaciens show about 40% similarity. The pH/temperature optima and stabilities and substrate specificities of the two enzymes are quite different. C. gilvus ␤-glucosidase exhibits an optimum pH of 6.2-6.4 and temperature of 35°C, whereas the corresponding values for A. tumefaciens are 7.2-7.4 and 60°C, respectively. The substrate specificity of A. tumefaciens enzyme toward different aryl glycosides is broader than C. gilvus enzyme. To analyze these properties further, three chimeric ␤-glucosidases were constructed by substituting segments from the Cterminal homologous region of C. gilvus ␤-glucosidase gene with that of A. tumefaciens. The chimeric enzymes were characterized with respect to pH/temperature activity and stability and substrate specificity. Chimeric enzymes exhibited chromatographic behavior similar to that of C. gilvus enzyme. However, enzymatic properties of chimeras were admixtures of those of the two parents. The chimeric enzymes were optimally active at 45-50°C and pH 6.6 -7.0. K m values of chimeric enzymes for the various saccharides were admixtures of both parental enzymes. These results suggest that the two domains of C. gilvus and A. tumefaciens enzymes probably can fold independently. The homologous C-terminal region in ␤-glucosidase appears to play an important role in determining enzyme characteristics. Changes in the properties on substitution of segments in this region might be related to the enzyme specificity, and ␤-glucosidases with improved properties can be prepared by manipulating this region.The enzyme ␤-glucosidase (EC 3.2.1.21) catalyzes the hydrolysis of alkyl-and aryl-␤-D-glucosides (methyl-␤-D-glucoside and p-nitrophenyl-␤-D-glucoside) as well as glycosides containing only carbohydrate residues (Cellobiose). On the basis of substrate specificity, ␤-glucosidases can be classified as aryl-␤-glucosidases, cellobiases, and those hydrolyzing both aryl-␤-glucosides and oligosaccharides. The last group is often found in cellulolytic microorganisms (1, 2). On the basis of sequence homology, ␤-glucosidases have been divided into two subfamilies (2): BGA (␤-glucosidases and phospho-␤-glucosidases from bacteria to mammals) and BGB (␤-glucosidases from yeasts, molds, and rumen bacteria). It is one of the components of the cellulase enzyme complex required for the hydrolysis of cellulose to glucose by catalyzing the final step which converts cellobiose to glucose (3, 4).The study of these enzymes has been facilitated by the use of recombinant DNA technology (1,5,6). Although a number of cellulase genes including several ␤-glucosidases have been cloned and expressed in both Escherichia coli and Saccharomyces cerevisiae (7-10), their enzymological properties, especially structure-function relationships, have not been well understood, partially because most of the cellulases show little sequence homology. Analysis of structure-function relationships may be facilitated by the formation of chimeric genes/e...