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Profile of Michael A. Marletta

or more than three decades, largely in water, largely at a pH of 7.5, Michael Marletta has traveled and mostly at 37 degrees Celsius. Chem- a serpentine road, working to elu- ists would consider those conditions F ” cidate a molecule whose impor- about the worst to work with, he says. tance to human physiology is as well Marletta’s research during the next three recognized as his own contributions to his decades focused on the interface between field. Named “Molecule of the Year” in chemistry and biology. 1992 by Science, , a noxious Because of this work, he refuses to be atmospheric gas, plays a pivotal role in typecast. “At one time in my career, I biological functions as diverse as forming called myself a biochemist. Now, I call long-term memory and maintaining penile myself a chemical biologist, but I continue erections (1). Marletta, who was elected to do what I’ve always done—enzymol- to the National Academy of Sciences in ogy,” he says. 2006, has shed light on the biochemistry of When it came time to pick a Ph.D. nitric oxide and the cellular machinery program, Marletta was faced with a dearth through which the molecule performs of choices because there were not many its functions. chemistry departments during the early Marletta’s work on nitric oxide not only 1970s that were serious about biology. led to a fundamental understanding of However, a fortuitous tip from a professor reaction mechanisms, but it has at Fredonia, who had heard about a pro- informed the development of treatments. gram in pharmaceutical chemistry at For example, he showed that substrate UCSF, pointed the way to Marletta’s lookalikes, called analogs, block graduate studies. the activity of the enzyme nitric oxide His doctoral project in the laboratory of synthase. The analogs help curb the over- UCSF biochemist George Kenyon, now production of nitric oxide, preventing va- at the University of Michigan at Ann sodilation and the precipitous drop in Michael A. Marletta. Arbor, was to investigate the mechanism blood pressure experienced by patients of action of creatine kinase, an enzyme suffering from septic shock (2). His dis- would come home at night, smelling like found abundantly in muscle and brain covery helped other researchers develop a spice mill. I can remember reading books tissues, where it maintains constant levels treatments for septic shock. of ATP, the cellular energy currency, by in the library about the constituents of Marletta’s scientific foray began in reversibly catalyzing the transfer of California, where he obtained his doctor- , and I realized that smelled and a phosphoryl group from ATP to the or- ate in 1978 from the University of tasted the way they did because of the ganic acid creatine. “It was a perfect California, San Francisco (UCSF). That molecules in them,” Marletta says. project for me because it was my first journey, with stops in Massachusetts and He spent the next few years tinkering experience in mechanistic enzymology,” Michigan, has led him back to California, with chemicals in his basement laboratory. Marletta says. where he is now the Aldo DeBenedictis “In those days you could buy chemicals Marletta fashioned a substrate look- Distinguished Professor of Chemistry from drugstores. I had a paper route; I alike for rabbit skeletal muscle creatine and a professor of biochemistry and had money,” he chuckles. “And there was kinase, called epoxycreatine, to probe molecular biology at the University of a library with books to tell you what to the enzyme’s active site (3). Until then, California, Berkeley. mix.” By age 10, Marletta knew he wanted little structural information about the His love of science is deep-seated. Born to be a chemist. enzyme had been reported, and the en- ’ in Rochester, NY, to parents of Italian “ ” zyme s catalytic site had remained a black ancestry, Marletta says the space race of Biology Is for Wusses. box. By binding specifically to the en- the late 1950s spurred his scientific tem- Neither of Marletta’s parents attended zyme’s active site and blocking its action, perament. As a 6-year-old space buff, college, but both supported his interest in epoxycreatine helped explain the mecha- Marletta recalls watching Sputnik 1 fly science. “My mother was a strong, en- nism and kinetics of creatine kinase and overhead on a cold October night in 1957 couraging force. I can still hear her telling the bioenergetics of ATP use in muscles. in Rochester. “I was completely fascinated me that anything was possible,” he says. Additional studies using epoxycreatine by by the idea that a satellite launched by During the fall of 1969, he enrolled at the members of Kenyon’s laboratory showed humans was orbiting the earth,” he says. State University of New York College that a cysteine residue at position 282 of That Christmas, Marletta asked his father at Fredonia to study chemistry. Between the enzyme’s amino acid sequence was for a telescope and spent many a winter’s his sophomore and junior years, Marletta likely crucial to catalysis (4). Other re- night stargazing. Over the next few years, took a general biology course at the searchers have since solved the enzyme’s his interest turned from astronomical to University of Rochester while working crystal structure (5). biological; he peered at living organisms a summer job on campus. “Frankly, I Marletta’s doctoral work on procured from the neighborhood pond remember thinking biology is for wusses. was not the only factor that cemented his through a microscope. I figured if I got biology out of the way, long-term research interest. For his post- His Italian heritage largely cultivated his I could have more time to take advanced doctoral training between 1978 and 1980, interest in chemistry. “The Italians’ love chemistry courses,” he recalls. However, for food was certainly prevalent in my the course helped him realize that na- house. My father worked at the R.T. ture’s ability to do chemistry surpassed that of chemists. “It’s almost unbelievable This is a Profile of a recently elected member of the Na- French Company [a manufacturer of con- tional Academy of Sciences to accompany the member’s diments, such as French’s mustard]. He how nature does intracellular chemistry Inaugural Article on page 19753 in issue 47 of volume 106.

www.pnas.org/cgi/doi/10.1073/pnas.1007505107 PNAS Early Edition | 1of3 Downloaded by guest on October 1, 2021 Marletta worked with enzymologist ability to kill invaders,” Marletta recalls. P450 and requires oxygen, NADPH, and Christopher Walsh at the Massachusetts “I decided to take my chances and con- as cofactors for ca- Institute of Technology (MIT) on de- tinue to pursue that line of research rather talysis (14). “The enzyme catalyzes one veloping fluorinated analogs to study the than something more predictable that of the most complicated redox trans- mechanism of enzyme catalysis. The might have gotten me tenure at MIT.” formations known, and that’s what drew training turned into an assistant profes- At the University of Michigan at Ann me to it. I’ve always been interested in sorship for Marletta at MIT that lasted Arbor, where he was a faculty member for enzyme catalysis,” he says. 6 years. “During my first year in Walsh’s 14 years, Marletta pursued that high- This fact is finely illustrated by the slew lab, I managed to get a couple of papers stakes research, which paid off in a big of studies on that published, so I decided to test the job way. In 1980, pharmacologist Robert Marletta published while at the University market,” he says. Fortunately, MIT’s nu- Furchgott found that endothelial cells of Michigan, including reports on the trition department was being restructured lining blood vessels produced a chemical enzyme’s structure, catalytic mechanism, into the now-defunct applied biological compound, called endothelium-derived regulation, and inhibition (2, 15–18). sciences department into which Marletta relaxing factor (EDRF), which made was hired as an enzymologist for the the smooth muscles of vessel walls relax, A New Direction toxicology program. thereby causing vasodilation (10). Ex- “I’ve always picked projects by looking for tending Furchgott’s findings, biochemist fundamental science with implications for The Nitric Oxide Narrative Salvador Moncada at University human health and disease,” Marletta At MIT, he met toxicologist Steven Tan- says. Thus, he turned his attention in 1995 nenbaum, whose work led to Marletta’s to the biochemistry of malaria, a disease research on nitric oxide. Tannenbaum Nature walks a tricky that kills nearly 2 million people each studied how carcinogenic nitrosamines year. “My wife and I had our only child are made by the human body from ni- tightrope between two months before I won the fellowship. I trates found in drinking water and vege- asked myself what new project I should tables. Bacteria in saliva were known to toxicity and function. work on. Seventy percent of those dying convert into , which were from malaria are under the age of 10, so I then transformed into nitrosamines by decided to study the biochemistry of the intestinal bacteria. Tannenbaum’s dis- College London and researchers else- parasite,” he says. covery that there were surprisingly high where showed that EDRF was nitric oxide, The malarial parasite Plasmodium amounts of nitrates in the urine of ex- a discovery with implications for the devel- falciparum degrades the hemoglobin in perimental subjects fed a low- diet opment of blood pressure drugs (11, 12). host erythrocytes for its nutrition. The led him to hypothesize about and show “Moncada’s Nature paper showing parasite feeds on the amino acids released the existence of a novel metabolic path- that EDRF was NO tipped me off,” from the globin; however, free ’s way for nitrate biosynthesis in humans Marletta says. In a 1998 landmark Bio- intracellular reactivity is toxic to the par- (6). “Steve convinced me that mammals chemistry paper, Marletta showed that asite. To counter heme’s toxicity, a para- were making nitrate, and because I was macrophages produce nitric oxide from site called HRP-2 tucks heme into a biochemist, I had to figure out where it arginine through a biochemical pathway a complex called hemozoin. Marletta used was coming from,” Marletta says. similar to the one used by endothelial spectroscopy to study the heme-binding A serendipitous discovery by Tannen- cells for vasodilation (13). The find- site of HRP-2 and the molecular nature baum’s group pointed to the immune ing suggested that nitric oxide helped of the heme–HRP2 interaction (19). In system as a source of nitrates: One of the macrophages kill pathogens. “We found another study with UCSF molecular experimental subjects, who had intestinal that nitric oxide was the intermediate biologist Joseph DeRisi and others, flu, was excreting whopping amounts of between arginine and nitrate during Marletta showed that a highly conserved, nitrate in her urine, suggesting that the nitrate biosynthesis in macrophages.” five-amino acid signal sequence in the source of the nitrate was either the path- That discovery helped establish the role N terminus of HRP2, called PEXEL— ogen or the immune system. Tannenbaum of nitric oxide in immunity, in addition Plasmodium export element—undergoes ruled out the pathogen and showed that to its known roles in vasodilation and enzymatic cleavage and acetylation in the rats injected with bacterial lipopolysac- neurotransmission. parasite’s endoplasmic reticulum before charide to stimulate the immune system “Here’s an extremely toxic molecule that the parasite exports the protein to human excreted large amounts of nitrates in their the immune system uses to kill, but which erythrocytes (20). urine (7, 8). “That was a beautiful set of the body also uses to dilate blood vessels experiments that led me to ask what cells and to help neurons talk to each other. Chasing Fresh Challenges in the immune system were making the It’s stunning, and that’s why it captured Unwilling to be tethered to the present nitrate,” Marletta says. a lot of attention,” Marletta says. Nature and eager for fresh challenges, Marletta Using biochemical methods, Marletta walks a tricky tightrope between toxicity moved to the University of California, demonstrated that activated macrophages and function by deploying no more than Berkeley in 2001. “I wanted a change and produced nitrates and were capable of nanomolar to picomolar concentrations of a new environment. I also longed to be forming nitrosamines under physiological nitric oxide, which usually decomposes back on the West Coast where I was once conditions (9). “We figured out later that once its work is done and before it can a graduate student. All of that aligned macrophages were converting arginine in- wreak havoc. perfectly with my new position at Berke- to citrulline, , and nitrate,” he says. In 1995, Marletta won a MacArthur ley,” he says. Despite those important findings, Foundation fellowship for his contri- At Berkeley, Marletta’s research has Marletta failed to obtain tenure at MIT. butions to our understanding of the bio- focused on understanding how cells dis- “MIT is a tough place, and the nitric ox- chemistry of nitric oxide. Marletta was tinguish nitric oxide from chemically sim- ide story wasn’t fully developed until we among the first to show that nitric oxide ilar oxygen, among other aspects of nitric moved to Michigan. At the time, we synthase, the enzyme that makes nitric oxide biology. Given that cells contain didn’t know what tied the ability of mac- oxide in macrophages, is a heme-con- more oxygen than nitric oxide, it was long rophages to produce nitrate with their taining protein that resembles cytochrome unknown how trace amounts of nitric

2of3 | www.pnas.org/cgi/doi/10.1073/pnas.1007505107 Nair Downloaded by guest on October 1, 2021 oxide elicit specific cellular responses ing, heme would bind oxygen, as it does based biotechnology company that Mar- amid a sea of oxygen molecules. Nitric with the oxygen-toting blood protein letta cofounded in 2006 with his former oxide performs its physiological role hemoglobin. graduate students Stephen Cary and through an enzyme called soluble guany- Marletta has studied the H-NOX do- Jonathan Winger. The company devises late cyclase (sGC), which makes the sig- main in bacteria, worms, and flies, where novel ways based on the H-NOX domain naling second messenger cGMP (21). containing the domain selec- to selectively deliver oxygen or nitric cGMP, in turn, switches on a cascade of tively bind oxygen or nitric oxide for oxide to tissues, an application with signals that leads to smooth muscle re- specific cellular functions. “It turns out clinical implications for , surgery, laxation and vasodilation. Although nitric that the domain can be a freestanding and cardiovascular diseases. oxide toggles sGC between its active and signaling protein in prokaryotes or fused Marletta’s contribution to nitric oxide inactive states, its mechanism is largely to a larger protein, such as sGC,” he biology earned him membership in the a mystery. says. Pathogenic bacteria such as Vibrio Institute of Medicine in 1999 and in the Marletta found that the part of the cholera use the domain to bind small National Academy of Sciences in 2006. “I enzyme that binds nitric oxide contains amounts of nitric oxide, an ability that was excited when the IOM recognized a heme-binding region called heme-NO likely evolved as an evasive strategy to our accomplishments and their impact on and oxygen-binding (H-NOX) domain. neutralize the nitric oxide produced by medicine. But, as a scientist, I must say Together with Berkeley structural bi- macrophages (23). In collaboration with that being elected to the NAS is what I ologist John Kuriyan, Marletta solved Rockefeller University neuroscientist am still excited about,” he says. the crystal structure of a similar H-NOX Cornelia Bargmann, Marletta found that “We’re going to continue to probe domain from a signaling protein in an- the nematode Caenorhabditis elegans the expanding landscape of nitric oxide aerobic bacteria. The structure, pub- harbors a homolog of sGC in specialized biology in eukaryotes and chart nitric lished in a well-cited PNAS article in oxygen-sensing cells that the worm uses oxide signaling pathways in pathogens,” 2004, provided the clue to how cells to detect oxygen levels in its environment Marletta says. That effort, he believes, distinguish oxygen and nitric oxide (22). and to regulate its feeding behavior. Un- fi could lead to treatments for an array of Marletta showed that speci caminoacid like the H-NOX domain of sGC, the ailments, including rheumatoid arthritis, residuesintheH-NOXdomaininsGC heme-binding domain of the worm ho- inflammatory bowel disease, and in- prevent the enzyme’s heme from binding molog preferentially binds oxygen (24). fectious diseases. oxygen while allowing it to bind nitric Years of work on the H-NOX domain oxide. Without this structural rejigger- resulted in Omniox, Inc., a California- Prashant Nair, Science Writer

1. Koshland DE, Jr (1992) The molecule of the year. Sci- 9. Miwa M, Stuehr DJ, Marletta MA, Wishnok JS, 17. Stevens-Truss R, Marletta MA (1995) Interaction of ence 258:1861. Tannenbaum SR (1987) N-nitrosamine formation by with the inducible murine macrophage 2. Olken NM, Marletta MA (1992) NG-allyl- and NG-cy- macrophages. IARC Sci Publ 84:340–344. nitric oxide synthase. Biochemistry 34:15638–15645. clopropyl-L-arginine: Two novel inhibitors of macro- 10. Furchgott RF, Zawadzki JV (1980) The obligatory role 18. Perry JM, Marletta MA (1998) Effects of transition phage nitric oxide synthase. J Med Chem 35: of endothelial cells in the relaxation of arterial smooth metals on nitric oxide synthase catalysis. Proc Natl 1137–1144. muscle by acetylcholine. Nature 288:373–376. Acad Sci USA 95:11101–11106. 3. Marletta MA, Kenyon GL (1979) Affinity labeling of 11. Palmer RM, Ferrige AG, Moncada S (1987) Nitric oxide 19. Choi CY, Cerda JF, Chu HA, Babcock GT, Marletta MA creatine kinase by N-(2,3-epoxypropyl)-N-amidinogly- release accounts for the biological activity of endothe- (1999) Spectroscopic characterization of the heme- cine. J Biol Chem 254:1879–1886. lium-derived relaxing factor. Nature 327:524–526. binding sites in Plasmodium falciparum histidine-rich 4. Buechter DD, Medzihradszky KF, Burlingame AL, 12. Feelisch M, te Poel M, Zamora R, Deussen A, protein 2. Biochemistry 38:16916–16924. Kenyon GL (1992) The active site of creatine kinase. Moncada S (1994) Understanding the controversy over 20. Chang HH, et al. (2008) N-terminal processing of pro- Affinity labeling of cysteine 282 with N-(2,3-epox- the identity of EDRF. Nature 368:62–65. teins exported by malaria parasites. Mol Biochem ypropyl)-N-amidinoglycine. JBiolChem 267:2173–2178. 13. Marletta MA, Yoon PS, Iyengar R, Leaf CD, Wishnok JS Parasitol 160:107–115. 5. Rao JK, Bujacz G, Wlodawer A (1998) Crystal structure of (1988) Macrophage oxidation of L-arginine to nitrite 21. Zhao Y, Brandish PE, Ballou DP, Marletta MA (1999) A rabbit muscle creatine kinase. FEBS Lett 439:133–137. and nitrate: Nitric oxide is an intermediate. Bio- molecular basis for nitric oxide sensing by soluble gua- 6. Tannenbaum SR, Fett D, Young VR, Land PD, Bruce WR chemistry 27:8706–8711. nylate cyclase. Proc Natl Acad Sci USA 96:14753–14758. (1978) Nitrite and nitrate are formed by endogenous 14. White KA, Marletta MA (1992) Nitric oxide synthase is 22. Pellicena P, Karow DS, Boon EM, Marletta MA, synthesis in the human intestine. Science 200: a cytochrome P-450 type hemoprotein. Biochemistry Kuriyan J (2004) Crystal structure of an oxygen-bind- 1487–1489. 31:6627–6631. ing heme domain related to soluble guanylate cy- 7. Wagner DA, Young VR, Tannenbaum SR (1983) 15. Hevel JM, White KA, Marletta MA (1991) Purification clases. Proc Natl Acad Sci USA 101:12854–12859. Mammalian nitrate biosynthesis: Incorporation of of the inducible murine macrophage nitric oxide syn- 23. Karow DS, et al. (2004) Spectroscopic characterization 15NH3 into nitrate is enhanced by endotoxin treat- thase. Identification as a flavoprotein. J Biol Chem of the soluble -like heme domains ment. Proc Natl Acad Sci USA 80:4518–4521. 266:22789–22791. from Vibrio cholerae and Thermoanaerobacter teng- 8. Stuehr DJ, Marletta MA (1985) Mammalian nitrate 16. Pufahl RA, Nanjappan PG, Woodard RW, Marletta MA congensis. Biochemistry 43:10203–10211. biosynthesis: mouse macrophages produce nitrite and (1992) Mechanistic probes of N-hydroxylation of 24. Gray JM, et al. (2004) Oxygen sensation and social nitrate in response to Escherichia coli lipopolysaccha- L-arginine by the inducible nitric oxide synthase from feeding mediated by a C. elegans guanylate cyclase ride. Proc Natl Acad Sci USA 82:7738–7742. murine macrophages. Biochemistry 31:6822–6828. homologue. Nature 430:317–322.

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