Identification and biochemical characterization of a novel NAD+-dependent homoserine dehydrogenase from the symbiotic Polynucleobacter necessarius

Homoserine dehydrogenase is an important enzyme in the aspartate pathway. This enzyme catalyzes the conversion of l-aspartate-β-semialdehyde to l-homoserine along with NAD(P)H as a reducing agent for the forward reaction and NAD(P)⁺ as an oxidizing agent for the backward reaction. The product of this reaction, l-homoserine, serves as a precursor for the synthesis of three amino acids – l-threonine, l-leucine, and l-methionine. Some of these enzymes consist of an ACT regulatory domain (aspartokinase, TyrA, and chorismate mutase). So far, Neisseria gonorrhea homoserine dehydrogenase is the only monofunctional enzyme that has NAD⁺-dependent activity and has an ACT domain. Polynucleobacter necessarius has two endosymbiotic and free-living species, with both species having more than 99% similarity concerning the 16S RNA sequence.

In a new study by Professor Wanggang Tang, Mengqing Guo, Xu Jiang, Haonan Xu from the Bengbu Medical College assessed the coenzyme specificity of homoserine dehydrogenase enzymes and the P. necessarius aspartate pathway characteristics. The study also reported the cloning, overexpression, and biochemical characterization of an NAD⁺-dependent monofunctional homoserine dehydrogenase with ACT domain on the C-terminal of the enzyme in symbiotic P. subsp. necessarius. The authors used a variety of advanced molecular and cellular biology techniques in their experiments such as sequence analysis, western blotting, chemical cross-linking analysis, and size-exclusion chromatography to identify the biochemical characteristics of symbiotic P. subsp. necessarius homoserine dehydrogenase (PnHSD).

The study yielded several key findings. The sequence analysis revealed that PnHSD, encoded by the hom gene, comprises 436 amino acid residues and is of monofunctional type with an ACT domain. The other two domains that were identified include the catalytic and NAD⁺ binding domain. Western blotting showed that PnHSD can be overexpressed in E. coli BL21 (DE3) cells using the SUMO fusion technique. Another technique – size-exclusion chromatography – demonstrated that purified and homogenized PnHSD has a homotetrameric structure with a molecular mass of approximately 160 kDa. Kinetic analysis studies demonstrate that the enzyme PnHSD has an up to 1460-fold preference for NAD⁺ over NADP⁺. The study also revealed that the enzyme exhibits maximal activity given that the optimal temperature at 35 ºC and pH 11. Moreover, the enzyme activity is neither stimulated by NaCl and KCl nor inhibited by l-threonine as studied using the oxidation reaction. The original research article is now published in the peer-reviewed journal Protein Expression and Purification.

The research team were indeed successful in achieving overexpression of PnHSD in E. coli as a soluble form, using the SUMO fusion technique as compared to fusion with other tags. The success of the SUMO fusion technique in overexpression of the enzyme in E. coli is contributed by the insertion of a favor mRNA structure coding for the SUMO tag. The reverse reaction of the enzyme takes place at an alkaline pH of 11 and a temperature of 35 ºC. The activity of this enzyme is insensitive to KCl, NaCl, and l-threonine. The unique feature of this enzyme is its relatively reduced affinity for NADP⁺ with the coenzyme specificity attributed to alteration of the NADPH 2′-phosphate group. In summary, the study by Professor Wanggang Tang and colleagues provides a better and more comprehensive understanding of the biochemical characteristics and coenzyme specificity of PnHSD.