Biochemistry of microbial degradation of hexachlorocyclohexane and prospects for bioremediation

Abstract

Lindane, the gamma-isomer of hexachlorocyclohexane (HCH), is a potent insecticide. Purified lindane or unpurified mixtures of this and alpha-, beta-, and delta-isomers of HCH were widely used as commercial insecticides in the last half of the 20th century. Large dumps of unused HCH isomers now constitute a major hazard because of their long residence times in soil and high nontarget toxicities. The major pathway for the aerobic degradation of HCH isomers in soil is the Lin pathway, and variants of this pathway will degrade all four of the HCH isomers although only slowly. Sequence differences in the primary LinA and LinB enzymes in the pathway play a key role in determining their ability to degrade the different isomers. LinA is a dehydrochlorinase, but little is known of its biochemistry. LinB is a hydrolytic dechlorinase that has been heterologously expressed and crystallized, and there is some understanding of the sequence-structure-function relationships underlying its substrate specificity and kinetics, although there are also some significant anomalies. The kinetics of some LinB variants are reported to be slow even for their preferred isomers. It is important to develop a better understanding of the biochemistries of the LinA and LinB variants and to use that knowledge to build better variants, because field trials of some bioremediation strategies based on the Lin pathway have yielded promising results but would not yet achieve economic levels of remediation.

FIG. 1.

Axial versus equatorial arrangements of chlorine atoms in the five major isomers of HCH plus the less common η- and θ-isomers. Note that α-HCH also exists in two enantiomeric (+ and −) forms.

FIG. 2.

Known locations of HCH dumps in excess of 50,000 tons in Brazil (38), Canada (135), Germany (159, 183), Spain (26, 29, 181), India (30, 139), and the United States (135). HCH-degrading sphingomonads have been recovered in six countries in Europe and Asia, although precise locations are often not reported (see text).

FIG. 3.

Consensus anaerobic degradation pathway of γ- and β-HCH. Note that two intermediates that have been proposed but not yet observed empirically are shown in square brackets. The structures of TCCH and DCCH are shown in the planar format because their stereochemistries have not been established.

FIG. 4.

Upstream pathway for the aerobic degradation of α-, γ-, and δ-HCH initiated by two successive dehydrochlorination reactions. 1,3,4,6-TCDN and 2,4,5-DNOL are shown in square brackets because they have not been demonstrated empirically. A further intermediate in δ-HCH degradation that has not been demonstrated empirically in the BHC-A strain is also shown in square brackets. This intermediate may be 3,4,5,6-TCOL, as was demonstrated for B90A. Note that the LinA, LinB, and LinC enzymes believed to catalyze several of the reactions are indicated, although direct evidence for the role of these enzymes in the strains indicated is not always available (see text).

FIG. 5.

Upstream pathway for the aerobic degradation of β- and δ-HCH involving two successive hydrolytic dechlorination reactions. LinB has been directly implicated in these reactions in some although not all of the strains shown.

FIG. 6.

Downstream pathway for the aerobic degradation of the γ-HCH isomer in strain UT26 together with the Lin enzymes catalyzing various steps. The intermediate shown in the square brackets (acylchloride) is hypothetical, and the reaction shown by a question mark has not been demonstrated empirically. The enzyme catalyzing the conversion of γ-hydroxymuconic-6-semialdehyde to maleylacetate is not known.

FIG. 7.

Sequence differences among LinA variants from known HCH-degrading bacteria. Residues D25 and H73 that form the catalytic dyad are shown in boldface type. The four groups of sequences described in the text are separated by solid lines.

FIG. 8.

Sequence differences among LinB variants from known HCH-degrading bacteria. Residues D108, E132, and H272 that form the catalytic triad are shown in boldface type. The three groups of sequences described in the text are separated by solid lines. Note that the C terminus of the Xanthomonas sp. ICH12 LinB enzyme has not been determined.

FIG. 9.

Variant positions in the structure of LinB. The active-site residues D108, E132, and H272 are marked with red text. The coordinates used were those reported under Protein Data Bank accession number 1MJ5 (166).

FIG. 10.

Reactions of α-HCH and β- and γ-PCCH catalyzed by heterologously expressed LinB from B90A observed by Raina et al. (143). Product nomenclature was described previously by Raina et al. (143).