Mechanism of transcription regulation by Acinetobacter baumannii HpaR in the catabolism of p-hydroxyphenylacetate.

HpaR is a transcription regulator in the MarR family that controls the expression of the gene cluster responsible for conversion of p-hydroxyphenylacetate to pyruvate and succinate for cellular metabolism. Here, we report the biochemical and structural characterization of Acinetobacter baumannii HpaR (AbHpaR) and its complex with cognate DNA. Our study revealed that AbHpaR binds upstream of the divergently transcribed hpaA gene and the meta-cleavage operon, as well as the hpaR gene, thereby repressing their transcription by blocking access of RNA polymerase. Structural analysis of AbHpaR-DNA complex revealed that the DNA binding specificity can be achieved via a combination of both direct and indirect DNA sequence readouts. DNA binding of AbHpaR is weakened by 3,4-dihydroxyphenylacetate (DHPA), which is the substrate of the meta-cleavage reactions; this likely leads to expression of the target genes. Based on our findings, we propose a model for how A. baumannii controls transcription of HPA-metabolizing genes, which highlights the independence of global catabolite repression and could be beneficial for metabolic engineering toward bioremediation applications. DATABASES: The structural data that support these findings are openly available in the wwPDB at https://doi.org/10.2210/pdb7EL2/pdb and https://doi.org/10.2210/pdb7EL3/pdb for AbHpaR and AbHpaR-DNA complex, respectively.

Crystallization, structural characterization and kinetic analysis of a GH26 ß-mannanase from Klebsiella oxytoca KUB-CW2-3.

ß-Mannanase (EC 3.2.1.78) is an enzyme that cleaves within the backbone of mannan-based polysaccharides at ß-1,4-linked D-mannose residues, resulting in the formation of mannooligosaccharides (MOS), which are potential prebiotics. The GH26 ß-mannanase KMAN from Klebsiella oxytoca KUB-CW2-3 shares 49-72% amino-acid sequence similarity with ß-mannanases from other sources. The crystal structure of KMAN at a resolution of 2.57 Šrevealed an open cleft-shaped active site. The enzyme structure is based on a (ß/α)8-barrel architecture, which is a typical characteristic of clan A glycoside hydrolase enzymes. The putative catalytic residues Glu183 and Glu282 are located on the loop connected to ß-strand 4 and at the end of ß-strand 7, respectively. KMAN digests linear MOS with a degree of polymerization (DP) of between 4 and 6, with high catalytic efficiency (kcat/Km) towards DP6 (2571.26 min-1 mM-1). The predominant end products from the hydrolysis of locust bean gum, konjac glucomannan and linear MOS are mannobiose and mannotriose. It was observed that KMAN requires at least four binding sites for the binding of substrate molecules and hydrolysis. Molecular docking of mannotriose and galactosyl-mannotetraose to KMAN confirmed its mode of action, which prefers linear substrates to branched substrates.

Development of mini-spectrophotometer for determination of plasma glucose.

This work demonstrates a novel compact spectrophotometer, "Mini-spectrophotometer", designed for plasma glucose detection. Unlike conventional spectrophotometer, a light source of the mini spectrophotometer is replaced by a light-emitting diode (LED), and a fabricated polymer-based microwell is used as a cuvette. To validate the downsizing spectrophotometer prototype, the efficiency and reliability for glucose determination are investigated. Using a certain light intensified from LED, the within-run precision of mini-spectrophotometer is found to be 3.9-8.4% while the between-run precision is 6.7-10.8%. The linearity for the quantification of glucose was up to 500 mg dL-1 and the recovery 99.1 ±â€¯3.4% is obtained. The sensitive and selective detection of glucose has been observed; with limit of detection (LOD) of 13.5 mg dL-1 and limit of quantification (LOQ) of 46.2 mg dL-1, respectively. Hemoglobin and triglyceride at high concentration slightly interferes with the proposed instrument. From comparative studies, there are no significant differences between the glucose concentration measured by mini-spectrophotometer and Shimadzu (r2 = 0.9862) or CECIL spectrophotometer (r2 = 0.9853). Using Passing-Bablok regression analysis, the results obtained from mini-spectrophotometer are in close agreement with the two conventional spectrophotometers. Furthermore, using microwell, the sample volume and reagent used in the process can be reduced. The in-house developed mini-spectrophotometer is capable of detecting plasma glucose while maintaining a compact system, demonstrating the potential of high performance, cost-effective, and portable spectrophotometer for clinical chemistry analysis in small routine, research, and teaching medical laboratory technologist.