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In recent weeks, two independent teams have reported long-sought X-ray crystal structures of two enzymes that remove halides from molecules with help from vitamin B12. These so-called reductive dehalogenase enzymes enable some bacteria to breathe organohalides like other organisms breathe oxygen. A better understanding of these enzymes could lead to improved bioremediation of pollutants such as halogenated solvents.
In one study, structural biologist Holger Dobbek of Humboldt University of Berlin; microbiologist Gabriele Diekert of Friedrich Schiller University, in Jena, Germany; and coworkers report crystal structures of a reductive dehalogenase from the bacterium Sulfurospirillum multivorans alone and in the presence of its substrate trichloroethylene (Science 2014, DOI: 10.1126/science.1258118). In the other study, structural biologist David Leys and coworkers at the University of Manchester, in England, report a crystal structure and propose two possible catalytic mechanisms of a reductive dehalogenase from the bacterium Nitratireductor pacificus (Nature 2014, DOI: 10.1038/nature13901).
These structures represent the first detailed pictures of a specific class of B12-dependent enzymes. Vitamin B12, also known as cobalamin, is a cofactor that consists of cobalt coordinated to a tetrapyrrole ring. In other classes of B12-dependent enzymes, the cobalt bonds to carbon on the substrate molecule. But in this class of enzymes, cobalt bonds with a halide atom on the substrate, the new structures reveal.
The S. multivorans enzyme uses a cofactor called norpseudo-B12, which differs slightly from B12. But Dobbek and coworkers got a surprise when they solved the structure. “The structure of this reductive dehalogenase is similar to a protein that humans use for taking up vitamin B12,” Dobbek says. That similarity was not obvious from the sequences of the two enzymes.
Another surprise was the size of the substrate-binding pocket. “We only realized once we had the substrate inside how tight this active site is,” Dobbek says. The trichloroethylene substrate fit in the space “like a hand in a glove.”
Leys and coworkers weren’t able to get a structure of the N. pacificus enzyme with its substrate. Instead, they performed electron paramagnetic resonance (EPR) experiments with the enzyme and 2,6-dibromophenol as a substrate to narrow down potential reaction mechanisms. They propose two possibilities that are consistent with their data.
In both mechanisms, formation of a cobalt-halogen bond drives catalysis. In one, the reaction includes nucleophilic attack of Co(I) on the halogen and transient formation of a halogen-Co(III) species with subsequent reduction to Co(II). The other possibility involves cleavage of the carbon-halogen bond following formation of an aryl radical. The combination of the crystal structure and EPR data is not sufficient to definitively rule either mechanism in or out.
“Guided by structural and elegant EPR spectroscopic data, Leys and colleagues propose a provocative mechanism for cobalamin-dependent reductive dehalogenases, which have been notoriously difficult to study,” says Ruma V. Banerjee, a professor of biological chemistry at the University of Michigan Medical School who is an expert on B12-dependent enzymes.
The researchers hope that organisms that use enzymes like the ones reported could be used in bioremediation. According to Leys, “Clearly, some of these bacteria have made it their business to survive entirely on mopping up whatever chlorinated entities are in the environment.”
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