The evolution of new enzyme function: lessons from xenobiotic metabolizing bacteria versus insecticide-resistant insects

Here, we compare the evolutionary routes by which bacteria and insects have evolved enzymatic processes for the degradation of four classes of synthetic chemical insecticide. For insects, the selective advantage of such degradative activities is survival on exposure to the insecticide, whereas for the bacteria the advantage is simply a matter of access to additional sources of nutrients. Nevertheless, bacteria have evolved highly efficient enzymes from a wide variety of enzyme families, whereas insects have relied upon generalist esterase-, cytochrome P450- and glutathione-S-transferase-dependent detoxification systems. Moreover, the mutant insect enzymes are less efficient kinetically and less diverged in sequence from their putative ancestors than their bacterial counterparts. This presumably reflects several advantages that bacteria have over insects in the acquisition of new enzymatic functions, such as a broad biochemical repertoire from which new functions can be evolved, large population sizes, high effective mutation rates, very short generation times and access to genetic diversity through horizontal gene transfer. Both the insect and bacterial systems support recent theory proposing that new biochemical functions often evolve from ‘promiscuous’ activities in existing enzymes, with subsequent mutations then enhancing those activities. Study of the insect enzymes will help in resistance management, while the bacterial enzymes are potential bioremediants of insecticide residues in a range of contaminated environments

Kinetics and stereochemistry of LinB-catalyzed δ-HBCD transformation: Comparison of in vitro and in silico results

LinB is a haloalkane dehalogenase found in Sphingobium indicum B90A, an aerobic bacterium isolated from contaminated soils of hexachlorocyclohexane (HCH) dumpsites. We showed that this enzyme also converts hexabromocyclododecanes (HBCDs). Here we give new insights in the kinetics and stereochemistry of the enzymatic transformation of δ-HBCD, which resulted in the formation of two pentabromocyclododecanols (PBCDols) as first- (P1δ, P2δ) and two tetrabromocyclododecadiols (TBCDdiols) as second-generation products (T1δ, T2δ). Enzymatic transformations of δ-HBCD, α1-PBCDol, one of the transformation products, and α2-PBCDol, its enantiomer, were studied and modeled with Michaelis-Menten (MM) kinetics. Respective MM-parameters KM, vmax, kcat/KM indicated that δ-HBCD is the best LinB substrate followed by α2- and α1-PBCDol. The stereochemistry of these transformations was modeled in silico, investigating respective enzyme-substrate (ES) and enzyme-product (EP) complexes. One of the four predicted ES-complexes led to the PBCDol product P1δ, identical to α2-PBCDol with the 1R,2R,5S,6R,9R,10S-configuration. An SN2-like substitution of bromine at C6 of δ-HBCD by Asp-108 of LinB and subsequent hydrolysis of the alkyl-enzyme led to α2-PBCDol. Modeling results further indicate that backside attacks at C1, C9 and C10 are reasonable too, selectively binding leaving bromide ions in a halide pocket found in LinB. Docking with α2-PBCDol, also allowed productive enzyme binding. A TBCD-1,5-diol with the 1S,2S,5R,6R,9S,10R-configuration is the predicted second-generation product T1δ. In conclusion, in vitro- and in silico findings now allow a detailed description of step-wise enzymatic dehalohydroxylation reactions of δ-HBCD to specific PBCDols and TBCDdiols at Å-resolution and predictions of their stereochemistry

In Memoriam of Angel R. Ortiz (1966-2008)

Peer reviewe