Periplasmic chitooligosaccharide-binding protein requires a three-domain organization for substrate translocation.

Periplasmic solute-binding proteins (SBPs) specific for chitooligosaccharides, (GlcNAc)n (n = 2, 3, 4, 5 and 6), are involved in the uptake of chitinous nutrients and the negative control of chitin signal transduction in Vibrios. Most translocation processes by SBPs across the inner membrane have been explained thus far by two-domain open/closed mechanism. Here we propose three-domain mechanism of the (GlcNAc)n translocation based on experiments using a recombinant VcCBP, SBP specific for (GlcNAc)n from Vibrio cholerae. X-ray crystal structures of unliganded or (GlcNAc)3-liganded VcCBP solved at 1.2-1.6 Å revealed three distinct domains, the Upper1, Upper2 and Lower domains for this protein. Molecular dynamics simulation indicated that the motions of the three domains are independent and that in the (GlcNAc)3-liganded state the Upper2/Lower interface fluctuated more intensively, compared to the Upper1/Lower interface. The Upper1/Lower interface bound two GlcNAc residues tightly, while the Upper2/Lower interface appeared to loosen and release the bound sugar molecule. The three-domain mechanism proposed here was fully supported by binding data obtained by thermal unfolding experiments and ITC, and may be applicable to other translocation systems involving SBPs belonging to the same cluster.

Photoclick reaction for rapid and simple fluorescence detection of itaconic acid and its derivatives in fungal cultures.

Itaconic acid (IA) and its derivatives produced by fungi have significant potential as industrial feedstocks. We recently developed a method for the detection of these compounds based on their terminal C-C double bonds. However, the presence of reducing agents, such as glucose and other fungal metabolites, leads to undesirable side reactions, and consequently, deteriorates the detection specificity. Therefore, we developed a fluorescence detection method for IA and its derivatives underpinned by a photoclick reaction. The photoclick reaction between conjugated IA and 5-(4-methoxyphenyl)-2-phenyl-2H-tetrazole under UV irradiation affords a fluorescent product. No fluorescence was detected when succinic acid was subjected to the reaction, indicating that a terminal C-C double bond is required to induce fluorescence. Optimal reaction conditions were determined to be a combination of 80% final dimethyl sulfoxide concentration, 30-s UV irradiation, and a pH of 2. Two weeks after the reaction at 4 °C, 89.0% of the initial intensity was retained, indicating that the reaction product was relatively stable. Glucose and kojic acid did not induce fluorescence after the reaction, indicating that these reducing agents did not affect fluorescence. IA was detected in a culture of Aspergillus terreus, and its quantification using the photoclick reaction was in agreement with the results obtained using high-performance liquid chromatography analysis. Interestingly, the IA derivative avenaciolide present in submillimolar quantities was also detectable in a culture of Aspergillus avenaceus using this method. The established method will enable the development of high-throughput screening methods to identify fungi that produce IA and its derivatives.