SCIENCE & TECHNOLOGY
ENZYME BY DESIGN Computational design and directed evolution convert receptor protein into enzyme
fully placed amino acids with reactive side chains, such as glutamate, histidine, and lysine, within the active site. Seven of 14 designs showed increases in GAP production compared with background levels, and one design, which they dubbed NovoTiml.O, was significandy more active. NovoTiml.O, which was thermally unstable, was then used as a starting point to design proteins that were more stable.
The two main challenges in designing HE TRUE TEST OF UNDERSTANDthe enzyme were describing the problem ing a reaction is creating an enand solving the resulting combinatorial zyme from scratch to catalyze search problem. The "description probit. Biochemists at Duke Univerlem" involves "capturing the essential SOME DIFFICULTIES with the reaction sity Medical Center now have elements of the reaction mechanism," required Hellinga to improve the enzymes shown that they can turn a protein having Hellinga says. Such descriptions include using directed evolution. The most comno catalytic activity into an enzyme that the type of reaction, the transition state, mon carbon sources for gluconeogenesis speeds up a reaction that is unrelated to the geometry of the reaction, and ways to are lactate and glycerol; glycerol requires the protein's original function. stabilize the transition state. more enzyme activity Through a combination of computational design and directed evolution, bio"Our designed proteins grew on lactate The Duke researchers converted that chemistry professor Homme W. Hellinga, but not on glycerol," Hellinga says. "We description into algorithms that introduce working with graduate students Mary A. did a directed evolution experiment using mutations in the three-dimensional proDwyer and Loren L. Looger, turned the our selection method to ask for variants tein structure. They needed to specify the receptor ribose-binding protein (RBP) inthat were obtained randomly and that amino acid residues involved in the catalto an enzyme that mimics the natural enwould grow on glycerol. It turned out that ysis and to identify mutations that would zyme triose phosphate isomerase (TIM) a tiny tweak in activity was needed." facilitate interaction with the substrate and [Science, 304,1967 (2004)]. stabilize the protein. In an accompanying commentary [Science, 304,1916 (2004)}, German researchers "When you do these kinds of calcula"This is one of the very first times that we Reinhard Sterner from the Institute for Biohave been able to design, essentially from tions, you're faced with many different physics & Physical Biochemistry at the Unifirst principles, an enzymatic reaction using choices," Hellinga says. "In the end, we versity of Regensburg and Franz X. Schmid structure-based design," Hellinga says. "%u had 21 or 22 mutations that we played of the Laboratory for Biochemistry at the can dial in a particular reaction mechanism with, which is a huge computational University of Bayreuth write: "The new and turn a protein that normally doesn't do search problem." < work by the Hellinga lab exemanything into a little enzyme." plifies the enormous power of The team chose their example computational biology and illuscarefully selecting a well-undertrates how this approach can be stood reaction. T I M catalyzes the combined with directed evoluinterconversion of dihydroxytion. The latter is well suited to acetone phosphate (DHAP) and identify beneficial mutations far glyceraldehyde-3-phosphate from the active site. Such muta(GAP) through an isomerization tions are difficult to find by comreaction. T I M participates in glyputation but important for the colysis (the breakdown of glufine-tuning of catalysis." cose) and ingluconeogenesis (the production ofglucose from nonHellinga hopes to be able carbohydrate sources). eventually to design any enzyme at will. "I'm sure everybody's reIn a starting protein, the soaction to that is going to be called scaffold protein must be 'Good luck,'" he says. First, he able to accommodate the reacneeds to find out if his group tion in terms of molecular volcan do this again with another ume and geometry. "The only MIMIC Through a combination of computational reaction or if the first success thing you could say is a great simdesign and directed evolution, ribose-binding protein was just a fluke. His group memilarity [between RBP andTIM] was converted into an enzyme, called NovoTim1.2 bers have picked other reactions is that ribose-binding protein (right), that catalyzes the same reaction as the natural and have started to see if they has a cavity that would normalenzyme triose phosphate isomerase (left). can build enzymes for them. ly bind ribose that's large enough "We're quietly optimistic that this is a to hold the substrate of triose phosphate T h e researchers started by testing very nice way to go," Hellinga says. "My guess isomerase," he says. "That's the only sort whether they could redesign RBP to bind is that computational design in the near of decision that you make when you choose TIM's substrates, regardless of catalytic acterm is not going to get you all the way, a particular scaffold." tivity They changed the layer of amino acids meaning you'll get pretty good enzymes but that directly contact ribose in the normal Some protein-to-enzyme transformanot very good enzymes. Ifyou can then marprotein and found four designs that bind tions simply won't be possible. For examry that with clever directed evolution methboth DHAP and GAP The proteins were ple, Hellinga notes, "there's no way you're ods, it's like a marriage made in heaven. It constructed by mutagenesis in bacteria. going to turn ribose-binding protein into will go along, long way."—C ELI A HENRY a D N A polymerase." To introduce catalytic activity they care-
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