SCIENCE & TECHNOLOGY STYMIED Antifreeze proteins found in Arctic fish bind to forming ice crystals, preventing their growth. University of California, Riverside, the symposium highlighted the many ways in which scientists are trying to beef up en zymes' natural abilities, from degrading pesticides to producing vitamin C pre cursors. Attendees also heard about new technologies for screening huge numbers of mutants, as well as a systematic way to create libraries of protein fragments. In Arnold's lab, the latest focus is on cy tochrome P450, that ubiquitous family of enzymes found in virtually all living things. The enzymes perform some extremely dif ficult oxidation chemistry, with impres sive regio- and stereoselectivity and sub strate specificity
PROSPECTING FOR PROTEINS Chemical engineers' meeting highlights the big business of directed evolution ELIZABETH K. WILSON, C&EN WEST COAST NEWS BUREAU
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S FRANCES H . ARNOLD R E -
cently mused out loud to a gathering of chemical engi neers, just 10 years ago di rected evolution was consid ered to be on the lunatic fringe. The idea that enzymes, which had evolved precise functions over millions of years, could be made to do unnatural things, such as work in organic solvents, was heretical. That notion fell by the wayside quick ly, she said. But then critics came up with more complaints, charging that the com binatorial approach of screening thou sands of randomly mutated enzymes for improved traits wasn't real science. But that didn't dissuade Arnold, a cheinical engineering professor at California Insti tute of Technology, from putting her re search muscle behind it. But she and her peers are having the last laugh. Directed evolution is now flour HTTP://PUBS.ACS.ORG/CEN
ishing worldwide in both academia and industry, noted Huimin Zhao, chemical engineering professor at the University of Illinois, Urbana-Champaign. Σ A proliferation of biotech | companies working with di- | rected evolution, including Ϊ Maxygen, Diversa, Enchira £ Biotechnology, and others, £ have sprung up within the past five years. Zhao was speaking at the American Institute of Chem ical Engineers' meeting last month in Reno, Nev. A sym posium on biocatalysis and protein engineering featured A r n o l d a number of presentations on directed evolution; Arnold gave the meet ing's Professional Progress Award Lecture. Organized by Zhao and chemical engi neering professor Wilfred Chen at the
THE ENZYMES traverse a complicated re action pathway, requiring not only a cofactor but also at least one electron-trans fer protein to perform; to harness it for industrial use would be expensive and complicated. But scientists have discovered that cy tochrome P450s are also capable of by passing all those steps by using a "shunt" pathway that requires no cofactor and that employs hydrogen peroxide as a source of both electrons and oxygen. Unfortunate ly, this shunt pathway has some limita tions: It's extremely inefficient, and it requires H 2 0 2 in concentrations that in activate the enzyme after a short time. So Arnold and graduate student Patrick Cirino are evolving a cytochrome P450 to use this shunt pathway more efficient ly If such a feat were managed, they rea soned, it could set the stage for a wealth of interesting industrial and pharmaceu tical chemistry They selected a variety of cytochrome P450 known as BM-3 (forBacillusmegaterium, in which it's found), which cat alyzes fatty acid hydroxylation. A known mutant of the enzyme, F87A, uses H 2 0 2 more efficiendy than the wildtype enzyme. Cirino and Arnold first cre ated a library of randomly mu tated, enzyme-coding D N A strands, then expressed them in bacteria. To search for promising mutants, they used a fatty acid surrogate substrate in which/?nitrophenolate is incorporated at the acid's fatty end. If a mutant was effective, it re leased thejf>-nitrophenol, producing a sigC & E N / D E C E M B E R 17, 2001
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SCIENCE & TECHNOLOGY MUTATED Crystal structure of the P450 BM-3 heme domain. Blue residues show positions where mutations have occurred through directed evolution to make an active peroxide-utilizing mutant. L. Iverson at the University oflëxas, Austin, have addressed that problem with several technologies that make it possible to handle enormous numbers of enzymes. T h e i r test subject was the peptide-cleaving enzyme OmpTprotease, which normally breaks peptides at a spot between two arginines. Oc casionally, the enzyme cleaves a peptide between arginine and valine; the group attempted to increase the enzyme's affinity
nature yellow color. After five rounds of evolution, Cirino and Arnold obtained a mutant that uses the shunt pathway 10 times more effectively than F87A and roughly 100 times more effectively than the wild-type BM-3. THIS INITIAL result has already attracted the interest of one company, which hopes to use the mutated enzyme to change the solubility of surfactants. "In spite of what remarkable catalysts P450s are naturally, directed evolution is a necessary tool for figuring out where these enzymes can be used practically," Cirino said. It goes without saying that the larger
X, = Arg, X2 = Gly—Arg—CNH2 or
0
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X, = Val, X2 = Gly—Arg—Gty—Arg—CNH2
}—N—
C y s — Ala — Arg — X,—
Gly — Lys —
BROKEN UP To signal that an enzyme has cleaved a peptide substrate, researchers attach a fluorescent dye (Fl) and a quenching fluorophore (Q) to the substrate. When the enzyme breaks the substrate in two, the dye molecule is separated from its quenching partner and can then fluoresce. t h e library of m u t a t e d enzymes, the greater chance of finding one that behaves in the desired way Most libraries for directed evolution contain up to 100,000 elements. But a very large library—on the order of 107, up to even 10 9 elements— requires an assay that will quickly search through millions of enzymes for the favorable mutants. Chemical engineering professor George Georgiou and chemistry professor Brent
for that spot. Georgiou and Iverson expressed millions of randomly mutated D N A strands that coded for O m p T in Escherichia coll To screen the enzymes, they attached two fluorophore molecules to a peptide, one a green fluorescing dye, the other a quencher, in close proximity to each other. As long as the molecules sit side by side, the quencher will absorb energy from the green dye molecule, preventing
it from fluorescing. But if the enzyme does its job and cleaves the peptide, the quencher is released and the green dye can fluoresce. T h e y screened millions of enzyme-containing cells for green fluorescence using the so-called fluorescence-activated cell-sorting technique: Cells pass, single file, in front of a laser beam. If the cells fluoresce, then they're selected and isolated. With this method, the researchers have isolated a mutant OmpT that's up to 60 times better at cleaving peptides at ArgVal. The fluorophore/cell-sorting strategy should be quite general, Georgiou said. ORGANOPHOSPHATES, which make up a large percentage ofpesticides used worldwide, are a serious environmental problem, contaminating soil, water, and food supplies. Scientists have zeroed in on an enzyme known as organophosphorous hydrolase (ΟΡΗ)—produced by a naturally occurring bacteria—which degrades organophosphates. But the enzyme has a downside, Chen noted. "It doesn't hydrolyze all the substrates at high rates," he said. For example, while the enzyme ea gerly chews up paraoxon, a toxic degrada tion product of parathion, it only slowly degrades the ubiquitous methyl parathion, chlorpyrifos, and diazinon. It's a problem tailor-made for direct ed evolution: Chen and his colleagues are evolving Ο Ρ Η to expand its hydrolyzing capabilities to a broader spec trum of pesticides. T h e group randomly mutated the D N A strands that code for the enzyme. They inserted the D N A into E. colt bac teria, which then expressed mutated vari eties of the enzyme inside the cell. How ever, organophosphates are largely insoluble and therefore can't efficiently enter the bacteria where the enzyme can do its work. So, along with the D N A for the enzyme, Chen's group included ge-
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netic code that would direct the enzyme to be expressed on the surface of the bacteria instead. If one of the many mutant enzymes should be better at degrading methyl parathion, it would signal its presence with ayellow color, produced by/>-nitrophenol, one of the degradation products of organophosphates. The better the enzyme, the more intense the yellow color. Chen and coworker's first round of experiments generated a mutant that degraded methyl parathion six times more quickly than the naturally occurring, or wild-type, enzyme. After two rounds, they produced an enzyme that works 30 times faster. Not only that, but that mutant also degraded its more traditional targets— paraoxon, parathion, and coumaphos— more than three times more quickly than the wild-type enzyme does.
P O C K E T E D Structure of the binding pocket for the cofactor NADPH in 2,5diketo-D-gluconic acid reductase.
Curiously, they discovered that several mutations responsible for the effect are quite far away from the enzyme's active site—about 20 —a result they're still trying to understand. They're also trying to interest industry in their newly bred enzyme. Enzymes can also be evolved by handselecting the site and the amino acids to be changed, a technique known as site-directed mutagenesis. While it's much more work, its proponents say they can glean insights about the system. The bacterial enzyme 2,5-diketo-D-gluconic acid reductase catalyzes a reaction that produces a precursor to vitamin C. The enzymatic reaction requires the biological cofactor NADPH. This cofactor is quite similar to another common biological cofactor, NADH. But NADPH is more expensive, less stable, and less common in the cell. The biotech company Genencor International called on molecular biology and biochemistry professor Stephen Anderson at Rutgers University to engineer the enzyme to use NADH. Anderson and chemical engineering graduate student
Scott Banta substituted different amino acids infivedifferent sites in the enzyme. The best mutant, a combination of several different mutants, was better than the wild-type enzyme at using NADH by two orders of magnitude, Banta said.
O R G A N I Z E R S Zhao (left) and Chen orchestrated a biocatalysis and protein bioengineering symposium for AlChE.
Protein engineering isn't limited to directed evolution. For example, it's known that ifyou chop a protein's peptide backbone to create two protein fragments, some of those fragments will reassemble. Most of them won't, however. And find-
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E V O L V E D Structure of the pesticide-degrading enzyme organophosphorus hydrolase in the presence of a nonhydrolyzable inhibitor, diethyl 4-methylbenzylphosphonate (green). The mutations after the first and second round of evolution are shown in brown.
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SCIENCE & TECHNOLOGY ing exactly where you can split up a protein yet still have it reassemble is a difficult but fundamental question, the answer to which might aid understanding of protein folding or yield new technologies. Marc A. Ostermeier, assistant professor of chemical engineering atJohns Hopkins University, and his colleagues have developed a method for creating libraries of protein fragments: They take two long overlapping fragments of the same gene and successively chop single base pairs off the fragment ends. They place each new fragment—each time shorter by one base pair— into a library. They repeat the process, cutting a new DNA strand in two, this time such that part of one of the fragments overlaps with the previous fragment. Researchers can also sometimes force protein fragments to assemble by attaching them to dimerizing molecules—a strategy that could be used to develop sensors. For example, Ostermeier's group identified fragments of the aminoglycoside phosphotransferase protein that would assemble when leucine zippers were attached to the fragments.
"By doing this comprehensively, we can look at all possible places andfindall solutions," Ostermeier said.
TRUNCATED Structure of £ coli GAR transformylase. DNA strands coding for this enzyme are chopped at different places to create a library of fragments.
Thirty years ago, scientists discovered thatfishswimming in icy polar seas pro-
duced some remarkable proteins that act as natural antifreeze. The proteins, they eventually determined, bind to forming ice crystals, inhibiting their growth. The antifreeze proteins have obvious industrial potential, including in the development of frost-resistant economic crops as well as for use as nontoxic cryopreservation agents. But while nature is ingenious, it's not perfect. To be useful for industry, the antifreeze proteins, or AFPs, need to better protect against ice formation. That's where Zhao and his colleagues come in, with directed evolution. Early this year, they began an effort to enhance AFP performance. Their lab has cloned several AFP genes from Antarctic fish and expressed them in E. coli. Their highthroughput screening method monitors the survival of cells as temperatures drop. Sofer,the group members haven't identified improved mutants, largely because they're still trying to improve temperature control in their system, Zhao said. The applications for directed evolution should continue to grow, Chen said. "It's a technique that has no limits." •
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