Crystal Structure of the Hexachlorocyclohexane Dehydrochlorinase (LinA-Type2): Mutational Analysis, Thermostability and Enantioselectivity

Abstract

Hexachlorocyclohexane dehydrochlorinase (LinA) mediates dehydrochlorination of γ-HCH to 1, 3, 4, 6-tetrachloro-1,4-cyclohexadiene that constitutes first step of the aerobic degradation pathway. We report the 3.5 Å crystal structure of a thermostable LinA-type2 protein, obtained from a soil metagenome, in the hexagonal space group P6322 with unit cell parameters a = b = 162.5, c = 186.3 Å, respectively. The structure was solved by molecular replacement using the co-ordinates of LinA-type1 that exhibits mesophile-like properties. Structural comparison of LinA-type2 and -type1 proteins suggests that thermostability of LinA-type2 might partly arise due to presence of higher number of ionic interactions, along with 4% increase in the intersubunit buried surface area. Mutational analysis involving the differing residues between the -type1 and -type2 proteins, circular dichroism experiments and functional assays suggest that Q20 and G23 are determinants of stability for LinA-type2. It was earlier reported that LinA-type1 exhibits enantioselectivity for the (−) enantiomer of α-HCH. Contrastingly, we identified that -type2 protein prefers the (+) enantiomer of α-HCH. Structural analysis and molecular docking experiments suggest that changed residues K20Q, L96C and A131G, vicinal to the active site are probably responsible for the altered enantioselectivity of LinA-type2. Overall the study has identified features responsible for the thermostability and enantioselectivity of LinA-type2 that can be exploited for the design of variants for specific biotechnological applications.

Novel LinA Type 3 δ-Hexachlorocyclohexane Dehydrochlorinase

“…Inserts in the clones that were carrying the desired linA genes, i.e., identical to linA type 1 (GenBank accession number D90355), linA type 2 (accession number EU863871), and the newly identified linA type 3, were reamplified by using primers that contained NdeI and XhoI sites in the forward primer described above and a reverse primer that consisted of nucleotides 448 to 471 of linA type 1, respectively. These inserts were ligated with the pET-28a(ϩ) vector (Novagen, Darmstadt, Germany) and cloned into E. coli BL21(DE3) cells, as described previously (9). After induction with 0.1 mM isopropyl-␤-D-thiogalactopyranoside (IPTG) and cell lysis, the expressed proteins present in the clear supernatant were purified by using a Ni-nitrilotriacetic acid (NTA) Superflow column (Qiagen, Hilden, Germany) at 4°C (9).…”

“…Two variants of LinA, LinA type 1 and LinA type 2, have been characterized (9). While LinA type 1, also referred to as LinA-UT26 or LinA2 in some studies (8,10), was first described for Sphingobium japonicum UT26 (11) and subsequently described for several other organisms (3,4), the gene for LinA type 2 was discovered in a soil metagenome (9).…”

“…LinA type 2 is distinct from another variant, LinA1, which was characterized in Sphingobium indicum B90 and consists of 154 amino acids (4,8,10). LinA1 is 99% identical to LinA type 2 in the region from amino acids 1 to 148 but differs completely from it in the C-terminal region (residues 149 to 156) due to a replacement of the amino acids IHFAPSGA by ALLQKS (9). LinA type 1 and LinA type 2 exhibit substantial differences in enantioselectivity and thermostability (9).…”

“…LinA1 is 99% identical to LinA type 2 in the region from amino acids 1 to 148 but differs completely from it in the C-terminal region (residues 149 to 156) due to a replacement of the amino acids IHFAPSGA by ALLQKS (9). LinA type 1 and LinA type 2 exhibit substantial differences in enantioselectivity and thermostability (9). Thus, while LinA type 1 exhibits enantioselectivity for the transformation of (Ϫ)-␣-HCH, LinA type 2 shows a preference for (ϩ)-␣-HCH (9).…”

“…LinA type 1 and LinA type 2 exhibit substantial differences in enantioselectivity and thermostability (9). Thus, while LinA type 1 exhibits enantioselectivity for the transformation of (Ϫ)-␣-HCH, LinA type 2 shows a preference for (ϩ)-␣-HCH (9). Also, while LinA type 1 undergoes rapid denaturation after it is preincubated at 45°C, LinA type 2 remains stable for up to 8 h (9, 12).…”