Characterisation of the first enzymes committed to lysine biosynthesis in Arabidopsis thaliana.

In plants, the lysine biosynthetic pathway is an attractive target for both the development of herbicides and increasing the nutritional value of crops given that lysine is a limiting amino acid in cereals. Dihydrodipicolinate synthase (DHDPS) and dihydrodipicolinate reductase (DHDPR) catalyse the first two committed steps of lysine biosynthesis. Here, we carry out for the first time a comprehensive characterisation of the structure and activity of both DHDPS and DHDPR from Arabidopsis thaliana. The A. thaliana DHDPS enzyme (At-DHDPS2) has similar activity to the bacterial form of the enzyme, but is more strongly allosterically inhibited by (S)-lysine. Structural studies of At-DHDPS2 show (S)-lysine bound at a cleft between two monomers, highlighting the allosteric site; however, unlike previous studies, binding is not accompanied by conformational changes, suggesting that binding may cause changes in protein dynamics rather than large conformation changes. DHDPR from A. thaliana (At-DHDPR2) has similar specificity for both NADH and NADPH during catalysis, and has tighter binding of substrate than has previously been reported. While all known bacterial DHDPR enzymes have a tetrameric structure, analytical ultracentrifugation, and scattering data unequivocally show that At-DHDPR2 exists as a dimer in solution. The exact arrangement of the dimeric protein is as yet unknown, but ab initio modelling of x-ray scattering data is consistent with an elongated structure in solution, which does not correspond to any of the possible dimeric pairings observed in the X-ray crystal structure of DHDPR from other organisms. This increased knowledge of the structure and function of plant lysine biosynthetic enzymes will aid future work aimed at improving primary production.

Crystallization and preliminary X-ray diffraction analysis of dihydrodipicolinate synthase 2 from Arabidopsis thaliana.

Dihydrodipicolinate synthase (DHDPS; EC 4.2.1.52) catalyzes the first committed step of the lysine-biosynthetic pathway in plants and bacteria. Since (S)-lysine biosynthesis does not occur in animals, DHDPS is an attractive target for rational antibiotic and herbicide design. Here, the cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of DHDPS2 from Arabidopsis thaliana are reported. Diffraction-quality protein crystals belonged to space group P2(1)2(1)2.

Characterization of cryogel monoliths for extraction of minor proteins from milk by cation exchange.

Extraction and purification of high-value minor proteins directly from milk without pre-treatment is a challenge for the dairy industry. Pre-treatment of milk before extraction of proteins by conventional packed-bed chromatography is usually necessary to prevent column blockage but it requires several steps that result in significant loss of yield and activity for many minor proteins. In this paper, we demonstrate that it is possible to pass 40-50 column volumes of various milk samples (raw whole milk, homogenized milk, skim milk and acid whey) through a 5 mL cryogel chromatographic column at 550 cm/h without exceeding its pressure limits if the processing temperature is maintained above 35 degrees C. The dynamic binding capacity obtained for the cryogel matrix (2.1 mg/mL) was similar to that of the binding capacity (2.01 mg/mL) at equilibrium with 0.1 mg/mL of lactoferrin in the feed samples. The cryogel column selectively binds lactoferrin and lactoperoxidase with only minor leakage in flowthrough fractions. Lactoferrin was recovered from elution fractions with a yield of over 85% and a purity of more than 90%. These results, together with the ease of manufacture, low cost and versatile surface chemistry of cryogels suggest that they may be a good alternative to packed-bed chromatography for direct capture of proteins from milk.