Gene jumbling yields improved enzymes.

Gene jumbling yields improved enzymes

Guided by chemical and physical rules, strings of linked amino acids twist, bend and fold into functional three-dimensional proteins. For about a decade, researchers have used a technique called site-specific mutagenesis to interchange amino acids at individual locations in a protein and study the functional consequences. But since scientists have only partial knowledge of the rules involved and so cannot reliably predict outcomes of particular mutations, some have likened the technique to shooting in the dark.

Harvard University biochemist Jeremy R. Knowles and his co-workers have now developed a technique that produces almost all possible single-site mutations in a protein and thus can help provide the basic data for such predictions.

The researchers begin by inserting a gene into a strain of bacteria. This specific gene codes for a "sluggish" version of an enzyme called triose phosphate isomerase (TIM), crucial to cellular digestion of glucose. After generating random mutations throughout the gene, the scientists extract the mutant genes from the bacterial and insert them into a bacterial strain that lacks it own copy of an isomerage gene. Without either their own or an inserted TIM gene, these microbes die off in a growth medium that lacks the chemical lactate.

The medium thus serves as a laboratory version of natural selection, "choosing" only improved enzymes from among nearly 2 million mutations, Knowles says. In the January PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (Vol. 87, No. 2), the scientists report identifying six mutants that showed up to 20 times as much catalytic activity as the starting TIM. Knowles says he expects that running the most active mutants through more mutation-and-selection cycles should lead to enzymes that show even more activity.

"It's a classic piece of work," says biochemist Charles Craik of the University of California, San Francisco, adding that the technique might even empower researchers to change an enzyme's function. "Then you would be taking evolution in your own hands," he says.