No abstract

No abstract

The middle base (U35) of the anticodon of tRNAGII is a major element ensuring the accuracy of aminoacylation by Escherichia coUl glutaminyl-tRNA synthetase (GlnRS). An opal suppressor of tRNAGI, (su+2UGA) containing C35 (anticodon UCA) was isolated by genetic selection and mutagenesis. Suppression of a UGA mutation in the E. colifol gene followed by N-terminal sequence analysis of purified dihydrofolate reductase showed that this tRNA was an efficient suppressor that inserted predominantly tryptophan. Mutations of the 3-70 base pair (U70 and A3U70) were made.

Direct electron transfer (DET)-type bioelectrocatalysis, which couples the electrode reactions and catalytic functions of redox enzymes without any redox mediator, is one of the most intriguing subjects that has been studied over the past few decades in the field of bioelectrochemistry. In order to realize the DET-type bioelectrocatalysis and improve the performance, nanostructures of the electrode surface have to be carefully tuned for each enzyme. In addition, enzymes can also be tuned by the protein engineering approach for the DET-type reaction. This review summarizes the recent progresses in this field of the research while considering the importance of nanostructure of electrodes as well as redox enzymes. This review also describes the basic concepts and theoretical aspects of DET-type bioelectrocatalysis, the significance of nanostructures as scaffolds for DET-type reactions, protein engineering approaches for DET-type reactions, and concepts and facts of bidirectional DET-type reactions from a cross-disciplinary viewpoint.

No abstract

Wild-type Escherichia coli glutaminyl-tRNA synthetase (GlnRS; EC 6.1.1.18) poorly aminoacylates opal suppressors (GLN) derived from tRNAGIII. Mutations in ginS (the gene encoding GlnRS) that compensate for impaired aminoacylation were isolated by genetic selection. Two ginS mutants were obtained by using opal suppressors differing in the nucleotides composing the base pair at 3 70: glnS113 with an Asp-235 Asn change selected with GLNA3U70 (GLN carrying G3 A and C70 --U changes), and glnS114 with a Gln-318 --Arg change selected with GLNU70 (GLN carrying a C70 --U change). The Asp-235 --Asn change was identified previously by genetic selection. Additional mutants were isolated by site-directed mutagenesis followed by genetic selection; the mutant enzymes have single amino acid changes (Lys-317 -+ Arg and Gln-318 -* Lys). A number of mutants with no phenotype also were obtained randomly. In vitro aminoacylation of a tRNAGIn transcript by GlnRS enzymes with Lys-317 --Arg, Gln-318 --Lys, or Gln-318 -+ Arg changes shows that the enzyme's kinetic parameters are not greatly affected by the mutations. However, aminoacylation of a tRNAGIn transcript with an opal (UCA) anticodon shows that the specificity constants (kc,t/Kin) for the mutant enzymes were 5-10 times above that of the wild-type GhnRS. Interactions between Lys-317 and Gln-318 with the inside of the L-shaped tRNA and with the side chain of Gln-234 provide a connection between the acceptor end-binding and anticodon-binding domains of GlnRS. The GlnRS mutants isolated suggest that perturbation of the interactions with the inside of the tRNA L shape results in relaxed anticodon recognition.

No abstract

Bioelectrocatalysis has become one of the most important research fields in electrochemistry and provided a firm base for the application of important technology in various bioelectrochemical devices, such as biosensors, biofuel cells, and biosupercapacitors. The understanding and technology of bioelectrocatalysis have greatly improved with the introduction of nanostructured electrode materials and protein-engineering methods over the last few decades. Recently, the electroenzymatic production of renewable energy resources and useful organic compounds (bioelectrosynthesis) has attracted worldwide attention. In this review, we summarize recent progress in the applications of enzymatic bioelectrocatalysis.