Journal of Microbiology

Research Support, Non-U.S. Gov't

The crystal structure of the D-alanine-D-alanine ligase from Acinetobacter baumannii suggests a flexible conformational change in the central domain before nucleotide binding: Kim-Hung Huynh , Myoung-ki Hong , Clarice Lee , Huyen-Thi Tran , Sang Hee Lee , Yeh-Jin Ahn , Sun-Shin Cha , Lin-Woo Kang; J. Microbiol. 2015;53(11):776-782. Published online October 28, 2015; DOI: https://doi.org/10.1007/s12275-015-5475-8

Abstract: Acinetobacter baumannii, which is emerging as a multidrugresistant nosocomial pathogen, causes a number of diseases, including pneumonia, bacteremia, meningitis, and skin infections. With ATP hydrolysis, the D-alanine-D-alanine ligase (DDL) catalyzes the synthesis of D-alanyl-D-alanine, which is an essential component of bacterial peptidoglycan. In this study, we determined the crystal structure of DDL from A. baumannii (AbDDL) at a resolution of 2.2 Å. The asymmetric unit contained six protomers of AbDDL. Five protomers had a closed conformation in the central domain, while one protomer had an open conformation in the central domain. The central domain with an open conformation did not interact with crystallographic symmetry-related protomers and the conformational change of the central domain was not due to crystal packing. The central domain of AbDDL can have an ensemble of the open and closed conformations before the binding of substrate ATP. The conformational change of the central domain is important for the catalytic activity and the detail information will be useful for the development of inhibitors against AbDDL and putative antibacterial agents against A. baumannii. The AbDDL structure was compared with that of other DDLs that were in complex with potent inhibitors and the catalytic activity of AbDDL was confirmed using enzyme kinetics assays.

Kinetic Study on the Enzymatic Production of D-Alanine from D-Aspartic Acid: Jae-Heung Lee , Moon-Hee Sung , Yeong-Joong Jeon; J. Microbiol. 2002;40(1):33-37.

Abstract: An enzymatic reaction for the production of D-alanine from D-aspartic acid and pyruvate as substrates by a thermostable D-amino acid aminotransferase (D-AAT) was investigated at various conditions in the temperature range of 40-70 C and pH range of 6.0-9.5. The D-AAT was produced with recombinant E. coli BL21, which hosted the chimeric plasmid pTLK2 harboring the D-AAT from the novel thermophilic Bacillus sp. LK-2. The enzyme reaction was shown to follow the Ping Pong Bi Bi mechanism. The K m values for D-aspartic acid and pyruvate were 4.38 mM and 0.72 mM, respectively. It was observed that competitive inhibition by D-alanine, the product of this reaction, was evident with the inhibition constant K i value of 0.1 mM. A unique feature of this reaction scheme is that the decarboxylation of oxaloacetic acid, one of the products, spontaneously produces pyruvate. Therefore, only a catalytic amount of pyruvate is necessary for the enzyme conversion reaction to proceed. A typical time-course kinetic study showed that D-alanine up to 88 mM could be produced from 100 mM of D-aspartic acid with a molar yield of 1.0.

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