Characterization of recombinant dihydrodipicolinate synthase from the bread wheat Triticum aestivum.

MAIN CONCLUSION: Recombinant wheat DHDPS was produced for the first time in milligram quantities and shown to be an enzymatically active tetramer in solution using analytical ultracentrifugation and small angle X-ray scattering. Wheat is an important cereal crop with an extensive role in global food supply. Given our rapidly growing population, strategies to increase the nutritional value and production of bread wheat are of major significance in agricultural science to satisfy our dietary requirements. Lysine is one of the most limiting essential amino acids in wheat, thus, a thorough understanding of lysine biosynthesis is of upmost importance to improve its nutritional value. Dihydrodipicolinate synthase (DHDPS; EC 4.3.3.7) catalyzes the first committed step in the lysine biosynthesis pathway of plants. Here, we report for the first time the expression and purification of recombinant DHDPS from the bread wheat Triticum aestivum (Ta-DHDPS). The optimized protocol yielded 36 mg of > 98% pure recombinant Ta-DHDPS per liter of culture. Enzyme kinetic studies demonstrate that the recombinant Ta-DHDPS has a KM (pyruvate) of 0.45 mM, KM (l-aspartate-4-semialdehyde) of 0.07 mM, kcat of 56 s-1, and is inhibited by lysine (IC 50 LYS of 0.033 mM), which agree well with previous studies using labor-intensive purification from wheat suspension cultures. We subsequently employed circular dichroism spectroscopy, analytical ultracentrifugation and small angle X-ray scattering to show that the recombinant enzyme is folded with 60% α/ß structure and exists as a 7.5 S tetrameric species with a Rg of 33 Å and Dmax of 118 Å. This study is the first to report the biophysical properties of the recombinant Ta-DHDPS in aqueous solution and offers an excellent platform for future studies aimed at improving nutritional value and primary production of bread wheat.

Comparison of untagged and his-tagged dihydrodipicolinate synthase from the enteric pathogen Vibrio cholerae.

Given the emergence of multi drug resistant Vibrio cholerae strains, there is an urgent need to characterize new anti-cholera targets. One such target is the enzyme dihydrodipicolinate synthase (DHDPS; EC 4.3.3.7), which catalyzes the first committed step in the diaminopimelate pathway. This pathway is responsible for the production of two key metabolites in bacteria and plants, namely meso-2,6-diaminopimelate and L-lysine. Here, we report the cloning, expression and purification of untagged and His-tagged recombinant DHDPS from V. cholerae (Vc-DHDPS) and provide comparative structural and kinetic analyses. Structural studies employing circular dichroism spectroscopy and analytical ultracentrifugation demonstrate that the recombinant enzymes are folded and exist as dimers in solution. Kinetic analyses of untagged and His-tagged Vc-DHDPS show that the enzymes are functional with specific activities of 75.6 U/mg and 112 U/mg, KM (pyruvate) of 0.14 mM and 0.15 mM, KM (L-aspartate-4-semialdehyde) of 0.08 mM and 0.09 mM, and kcat of 34 and 46 s-1, respectively. These results demonstrate there are no significant changes in the structure and function of Vc-DHDPS upon the addition of an N-terminal His tag and, hence, the tagged recombinant product is suitable for future studies, including screening for new inhibitors as potential anti-cholera agents. Additionally, a polyclonal antibody raised against untagged Vc-DHDPS is validated for specifically detecting recombinant and native forms of the enzyme.

Identification of a dimeric KDG aldolase from Agrobacterium tumefaciens.

Agrobacterium tumefaciens is a Gram-negative bacterium and causative agent of Crown Gall disease that infects a variety of economically important plants. The annotated A. tumefaciens genome contains 10 putative dapA genes, which code for dihydrodipicolinate synthase (DHDPS). However, we have recently demonstrated that only one of these genes (dapA7) encodes a functional DHDPS. The function of the other nine putative dapA genes is yet to be determined. Here, we demonstrate using bioinformatics that the product of the dapA5 gene (DapA5) possesses all the catalytic residues canonical to 2-keto-3-deoxygluconate (KDG) aldolase, which is a class I aldolase involved in glucose metabolism. We therefore expressed, purified, and characterized recombinant DapA5 using mass spectrometry, circular dichroism spectroscopy, analytical ultracentrifugation, and enzyme kinetics. The results show that DapA5 (1) adopts an α/ß structure consistent with the TIM-barrel fold of KDG aldolases, (2) possesses KDG aldolase enzyme activity, and (3) exists as a tight dimer in solution. This study shows for the first time that dapA5 from A. tumefaciens encodes a functional dimeric KDG aldolase.

Dihydrodipicolinate synthase is absent in fungi.

The class I aldolase dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step of the diaminopimelate (DAP) lysine biosynthesis pathway in bacteria, archaea and plants. Despite the existence, in databases, of numerous fungal sequences annotated as DHDPS, its presence in fungi has been the subject of contradictory claims. We report the characterization of DHDPS candidates from fungi. Firstly, the putative DHDPS from Coccidioides immitis (PDB ID: 3QFE) was shown to have negligible enzyme activity. Sequence analysis of 3QFE showed that three out of the seven amino acid residues critical for DHDPS activity are absent; however, exact matches to catalytic residues from two other class I aldolases, 2-keto-3-deoxygluconate aldolase (KDGA), and 4-hydroxy-2-oxoglutarate aldolase (HOGA), were identified. The presence of both KDGA and HOGA activity in 3QFE was confirmed in vitro using enzyme assays, the first report of such dual activity. Subsequent analyses of all publically available fungal sequences revealed that no entry contains all seven residues important for DHDPS function. The candidate with the highest number of identities (6 of 7), KIW77228 from Fonsecaea pedrosoi, was shown to have trace DHDPS activity in vitro, partially restored by substitution of the seventh critical residue, and to be incapable of complementing DHDPS-deficient E. coli cells. Combined with the presence of all seven sequences for the alternative α-aminoadipate (AAA) lysine biosynthesis pathway in C. immitis and F. pedrosoi, we believe that DHDPS and the DAP pathway are absent in fungi, and further, that robust informed methods for annotating genes need to be implemented.

Structural insight for substrate tolerance to 2-deoxyribose-5-phosphate aldolase from the pathogen Streptococcus suis.

2-deoxyribose-5-phosphate aldolase (DERA) is a class I aldolase that catalyzes aldol condensation of two aldehydes in the active site, which is particularly germane in drug manufacture. Structural and biochemical studies have shown that the active site of DERA is typically loosely packed and displays broader substrate specificity despite sharing conserved folding architecture with other aldolases. The most distinctive structural feature of DERA compared to other aldolases is short and flexible C-terminal region. This region is also responsible for substrate recognition. Therefore, substrate tolerance may be related to the C-terminal structural features of DERA. Here, we determined the crystal structures of full length and C-terminal truncated DERA from Streptococcus suis (SsDERA). In common, both contained the typical (α/ß)8 TIM-barrel fold of class I aldolases. Surprisingly, C-terminal truncation resulting in missing the last α9 and ß8 secondary elements, allowed DERA to maintain activity comparable to the fulllength enzyme. Specifically, Arg186 and Ser205 residues at the C-terminus appeared mutually supplemental or less indispensible for substrate phosphate moiety recognition. Our results suggest that DERA might adopt a shorter C-terminal region than conventional aldolases during evolution pathway, resulting in a broader range of substrate tolerance through active site flexibility.