Complex Structure Research Of Alanine Dehydrogenase From Bacillus Megaterium

Alanine dehydrogenase (Ald, EC 1.4.1.1) which is an amino acid dehydrogenase with a nicotinamide adenine dinucleotide (NAD+) dependent enzyme, catalyzes a reversible oxidative deamination of L-alanine to pyruvate and ammonia, with NADH as cofactor. In vivo, Ald can regulate steady equilibrium state of ketonic acid and amino acid, and participate in amino acid metabolism and glucose metabolism. Pyruvate, a promising reaction product, has a widely application in medicine, food and scientific research. The recent data was summarized about this enzyme, including its expression, purification, crystallization and enzyme structure in the research.The research studied mainly alanine dehydrogenase from Bacillus megaterium, the recombinant plasmid was gained through constructing pET-28a (+) expression system, and it expressed successfully a large number of recombinant proteins. A pure protein was obtained by Ni2+-NTA affinity chromatography and anion exchange column successively, which provided favorable conditions for protein crystal to crystallization.Initial screening was performed by using Crystal Screen â…  & â…¡, Index â…  & â…¡ and PEG/Ion â…  & â…¡ for crystallization of single crystal and complex eutectic. The nubby single crystal and tebrahedron complex eutectic were obtained through optimization of initial crystal conditions unceasingly. X-ray diffraction data were collected to a resolution of 2.97 A and a resolution of 2.88 A fronisingle crystal and binary complex belonging to the trigonal space group R32, and ternary complex diffracted to 2.67 A resolution belonging to the orthorhombic space group P2. We find that single crystal and binary complex contain one protein molecule per asymmetric unit in crystal structure by SFCHECK software analysis.The crystal structure of single crystal and binary complex have been parsed, the crystal structure of ternary complex is parsing.In order to study further the catalytic mechanism of alanine dehydrogenase, we also did the crystallization experiment of the quaternary complex, but we did not get crystals with good resolution and the crystals need optimization deeply. It is meaningful that we obtained three-dimensional structure to utilize enzyme protein for the industrial production, or develop suitable bacteriostatic agent and treatment.