Isolation of a novel thermostable dehydrochlorinase (LinA) from a soil metagenome

Macwan, Ankit S.; Javed, Saleem; Kumar, Ashwani

doi:10.1007/s13205-011-0012-x

Cited by 8 publications

(16 citation statements)

References 11 publications

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“…Thus, while LinA-type2 is thermostable and remains active even after 8 hours of incubation at 45°C, >50% activity of LinA-type1 is lost after incubation for 60 min at the same temperature [6]. Their Tm, analyzed by circular dichroism studies, are 65 and 45°C, respectively.…”

Section: Introductionmentioning

confidence: 98%

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

et al. 2012

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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.

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Section: Introductionmentioning

confidence: 98%

“…It consists of 156 amino acids, and is referred to here as LinA-type1. One of our groups recently described isolation of another variant, LinA-type2, that differs from LinA-type1 by 10 residues [6]. Characterization of yet another variant, LinA1-B90, from Sphingobium indicum Β90 has also been described [7].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2012

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“…Metagenomic DNA was extracted from the soil samples, and PCR amplification of linA genes from this DNA was done as described previously (12), except that the Platinum Taq thermostable high-fidelity DNA polymerase enzyme (Invitrogen, Carlsbad, CA, USA) was used. The forward primer (5=-CATATGAGTGATCTAGACAGACTTGCAA-3=) consisted of 1 to 25 nucleotides from the 5= end of linA type 1 with an NdeI site at its 5= end (underlined).…”

Section: Methodsmentioning

confidence: 99%

“…The reverse primer (5=-CTCGAGCGATTTTTGCAACAG AGC-3=) consisted of nucleotides 1 to 21 from the 5= end of IS6100, which was shown previously to be present downstream of the termination codon of several linA genes (3), and an overhang of the XhoI site at its 5= end (underlined). The selected amplified DNA product was eluted from the agarose gel and ligated with the TA cloning vector pGEM-T Easy (Promega, Madison, WI, USA), and Escherichia coli DH5␣ cells were transformed as described previously (12). Inserts from the clones were sequenced on an ABI Prism-3100 genetic analyzer (Applied Biosystems, Foster City, CA, USA) by using universal forward and reverse M13 primers.…”

Section: Methodsmentioning

confidence: 99%

“…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). The activities of both variants are high with ␥-HCH, moderate with ␣-HCH, and low with ␦-HCH, and no activity was observed with ␤-HCH (12). In this report, we describe the identification and characterization of a new variant, LinA type 3, whose gene was obtained from the metagenome of an HCH-contaminated soil sample.…”

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Novel LinA Type 3 δ-Hexachlorocyclohexane Dehydrochlorinase

Shrivastava

Prokop

Kumar

2015

Appl Environ Microbiol

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cLinA is the first enzyme of the microbial degradation pathway of a chlorinated insecticide, hexachlorocyclohexane (HCH), and mediates the dehydrochlorination of ␣-, ␥-, and ␦-HCH. Its two variants, LinA type 1 and LinA type 2, which differ at 10 out of 156 amino acid residues, have been described. Their activities for the metabolism of different HCH isomers differ considerably but overall are high for ␥-HCH, moderate for ␣-HCH, low for ␦-HCH, and lacking for ␤-HCH. Here, we describe the characterization of a new variant of this enzyme, LinA type 3, whose gene was identified from the metagenome of an HCH-contaminated soil sample. Its deduced primary structure in the region spanning amino acid residues 1 to 147 of the protein exhibits 17 and 12 differences from LinA type 1 and LinA type 2, respectively. In addition, the residues GIHFAPS, present at the region spanning residues 148 to 154 in both LinA type 1 and LinA type 2, are deleted in LinA type 3.The activity of LinA type 3 for the metabolism of ␦-HCH is several orders of magnitude higher than that of LinA type 1 or LinA type 2 and can be useful for improvement of the metabolism of ␦-HCH.

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Important amino acid residues of hexachlorocyclohexane dehydrochlorinases (LinA) for enantioselective transformation of hexachlorocyclohexane isomers

et al. 2017

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LinA-type1 and LinA-type2 are two well-characterized variants of the enzyme ‘hexachlorocyclohexane (HCH)-dehydrochlorinase’. They differ from each other at ten amino acid positions and exhibit differing enantioselectivity for the transformation of the (–) and (+) enantiomers of α-HCH. Amino acids responsible for this enantioselectivity, however, are not known. An in silico docking analysis identified four amino acids (K20, L96, A131, and T133) in LinA-type1 that could be involved in selective binding of the substrates. Experimental studies with constructed mutant enzymes revealed that a combined presence of three amino acid changes in LinA-type1, i.e. K20Q, L96C, and A131G, caused a reversal in its preference from the (–) to the (+) enantiomer of α-HCH. This preference was enhanced by the additional amino acid change T133 M. Presence of these four changes also caused the reversal of enantioselectivity of LinA-type1 for δ-HCH, and β-, γ-, and δ-pentachlorocyclohexens. Thus, the residues K20, L96, A131, and T133 in LinA-type1 and the residues Q20, C96, G131, and M133 in LinA-type 2 appear to be important determinants for the enantioselectivity of LinA enzymes.Electronic supplementary materialThe online version of this article (doi:10.1007/s10532-017-9786-9) contains supplementary material, which is available to authorized users.

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Isolation of a novel thermostable dehydrochlorinase (LinA) from a soil metagenome

Cited by 8 publications

References 11 publications

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

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

Novel LinA Type 3 δ-Hexachlorocyclohexane Dehydrochlorinase

Important amino acid residues of hexachlorocyclohexane dehydrochlorinases (LinA) for enantioselective transformation of hexachlorocyclohexane isomers

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