February 2011

Ruma V. Banerjee and Stephen W. Ragsdale: deciphering sulfur and carbon metabolism

Ruma V. Banerjee and Stephen W. Ragsdale are professors in the department of biochemistry at the University of Michigan and partners in life as well. Below, we look at their research and how they balance life and the lab.


Scientists are used to expecting the unexpected in their research, but sometimes the surprising discoveries occur outside the petri dish.

Such is the case of Ruma V. Banerjee and Stephen W. Ragsdale, both professors in the department of biochemistry at the University of Michigan.

In the halls of Medical Science Research Building III, which houses the biochemistry department, Banerjee and Ragsdale are independent researchers who tackle intriguing biological problems. Banerjee, the Vincent Massey collegiate professor of biological chemistry, focuses on mammalian sulfur metabolism and its reliance on enzymes utilizing the cobalt-containing vitamin B12, while Ragsdale studies the microbial metabolism of one-carbon compounds, which also relies heavily on metalloenzymes.

However, their connection goes beyond cobalt, nickel and iron. Banerjee and Ragsdale are partners in life as well as in the department; they have been married for nearly 20 years and share the successes and challenges that come with a life in research that intertwines with life outside the lab.

Chemistry in action

Their initial encounter occurred in 1989, when Stephen Ragsdale was a young assistant professor who had just established his own lab at the University of Wisconsin-Milwaukee. In graduate school, he became fascinated by microbes and the vast and unusual types of chemistry they could carry out and he decided to take up research in acetogenesis, the metabolic process used by certain anaerobic bacteria to create energy by converting carbon sources into acetate.

In particular, Ragsdale was examining the enzymes involved in the Wood-Ljungdahl pathway, which fixes organic carbon from carbon dioxide with the aid of coenzyme A. (Considering he did his graduate and postdoctoral studies with Lars G. Ljungdahl and Harlan Wood, respectively, Ragsdale certainly had the credentials to tackle this pathway.)

One day, he received a call from Rowena Matthews, a colleague and fellow microbial biochemist at the University of Michigan. The two had conversed previously at a microbiology conference, where she described her frustration at not being able to obtain redox measurements for a particular enzyme because the cobalt-containing cofactor had such a low redox potential.

“I had told her I’m sure I could get the measurements because my lab was working on a corrinoid/iron-sulfur protein with a similarly very negative redox potential, but we had managed to develop some techniques to overcome that, so she should let me know if she wanted any assistance,” recalls Ragsdale.

In the phone call, Matthews took Ragsdale up on his offer and mentioned that she had just the perfect person, a bright and talented postdoctoral fellow, to come to Ragsdale’s lab and collaborate on this effort.

***
In Michigan, Ruma Banerjee was packing her bags for a trip. She had been working with Matthews for about a year and a half on methionine synthase, which adds a methyl group to homocysteine to complete the biosynthesis of the amino acid methionine.

Her initial experiments had gone quite well – Banerjee had managed to clone and characterize the gene from this enzyme in E. coli; however, her kinetic studies were stalling, as she was unable to analyze the transition of the vitamin B12 (cobalamin) cofactor from cobalt (II) to cobalt (I) during the reaction.

Matthews had arranged for Banerjee to do some measurements with a colleague in Milwaukee, Stephen Ragsdale. He had developed a new type of cell for spectroelectrochemical analysis (a method allowing spectroscopic detection of changes in oxidation state) that could analyze even minute changes.

After arriving, Banerjee and Ragsdale spent the next several days carrying out a host of experiments on methionine synthase. “It was intense,” she recalls. “We did all these electrochemical and spectroscopy studies, and were getting great data.

“At the same time, there was definitely more than spectroelectrical chemistry going on in that lab.”

Model describing redox and heme-dependent regulation of the Ca activated voltage gated potassium channel (BK channel) and heme oxygenase-2. When O2 levels are high, the redox switches in HO2 and the BK channel are in the disulfide state where HO2 binds heme tightly and has high heme degradation activity, producing CO which activates the BK channel. Under hypoxic conditions, the redox switches on both proteins are in the reduced state, which decreases HO2's ability to degrade heme lowering CO levels, while the increased affinity of the BK channel for heme leads to its inhibition. Reference: Yi, L., et al (2010) J. Biol. Chem. 285, 20117-20127.

Convergent evolutions

If one subscribes to the theory that opposites attract, then certainly Banerjee and Ragsdale were destined to find a spark from the moment they met, as their histories up to that point were a contrast in styles if ever one existed.

Banerjee, for her part, had quite a transient youth; as the daughter of a general in the Indian army, her family moved around quite a bit across the country, and she had attended 10 different schools by the time she was getting ready for college – a dizzying journey made even more astounding by the fact that Banerjee graduated high school at the age of 14.

Despite this constant fluidity, her goals solidified early on, and by the time Banerjee was 11, she had developed a strong desire to pursue scientific research, which she subsequently did, obtaining a bachelor’s and master’s degree in plant science from Delhi University.

“I can’t really put my finger on any event or influence that seemed to steer me to science,” she says. “It was more of a subconscious unfolding in that I always had a sense I was going down the right path.”

Ragsdale, by comparison, grew up in rural Rome, Ga., and spent his formative years in constant intellectual flux. He did enjoy science a great deal, but the manner in which science was typically taught – involving the rote memorization of terms and concepts – kept it as a secondary interest.

“I’ve always had a mind that was better at understanding things than memorizing things,” he says. “So in high school and college I was always drawn more to arts and humanities classes, though it was really hard to corral myself to any one discipline.”

Ragsdale’s other great passion was music; in fact, he took a few years off from full-time studies at the University of Georgia to pursue a music career, singing at various bars and coffeehouses while working odd jobs to support his dreams of folk stardom.

But some chance encounters steered these distant partners a little closer together, both in physical distance and in academic fields.

Banerjee came to the United States to conduct her doctoral work at Rensselaer Polytechnic Institute in upstate New York after she met someone affiliated with the university in India who encouraged her to attend, noting it was a fine scientific institute.

The structure of cystathionineb-synthase, one of two major generators of the gaseous signaling molecule, H2S, which was recently solved in a collaboration involving the Banerjee laboratory.

The only problem, Banerjee discovered, was that RPI did not have a botany program, so she switched her studies to chemistry, one of the school’s strengths. At first, she considered switching schools, but she quickly became enamored with medicinal chemistry and synthesis reactions and decided to stay.

Meanwhile, Ragsdale had reinvigorated his science interests through personal readings and eventually decided to return to university full time. Not long after, he bumped into renowned scientist Marion M. Bradford at a soda machine.

As it happens, Bradford (inventor of the Bradford protein assay) and Ragsdale shared the same hometown and knew each other from church, so Ragsdale mentioned that he needed work to help pay for school and wondered if Bradford had any jobs in his lab for an undergraduate.

“He said sure and told me to stop on by,” remembers Ragsdale.

Ragsdale took up a project studying the acrosome reaction in sperm, and immediately, the concept of research – using deductive skills and reasoning to solve a daunting biological problem – struck a chord. “It was like solving a puzzle,” Ragsdale says. “It only took one day for me to get hooked.”

Balancing life and lab

More than two decades later, Banerjee and Ragsdale still are hooked, both on each other and on their research in metabolism and enzymology, though they certainly have had to maneuver through the delicate balancing act of family and research.

Their first significant challenge was finding a suitable destination once Banerjee had finished her postdoc and was ready to start her independent career. The main goal was to find a place together, for, despite the relative proximity of Milwaukee and Ann Arbor, the constant travel between the cities was a hassle.

A gas channel between the active sites of CO dehydrogenase (C) and acetyl-CoA synthase (A). Ragsdale is studying this enzyme complex, which is responsible for reductive conversion of CO2 to acetyl-CoA. Revised from Doukov, T. I., et al. (2008). Biochem. 47, 3474-3483.

“We also made the conscious decision that we would not work in the same lab,” Banerjee says. “Steve was a little ahead of me in his career, so when I started my independent work, I did not want to be seen as riding his coattails or risk working in his shadow.”

They eventually found a suitable joint destination at the University of Nebraska-Lincoln, and both took positions there in 1991. “Initially, it was a compromise destination,” Banerjee says, “but we quickly felt right at home, and the time we spent there was extremely positive.”

Along the way, they also helped bolster Nebraska’s research reputation through their prolific research and numerous honors; Banerjee even helped establish the National Institutes of Health-funded Redox Biology Center at the university in 2002 to explore redox metabolism and its connection to disease.

Such outstanding work received notice, and the pair eventually was recruited back to Michigan in 2007 (though Ragsdale never officially attended Michigan, he says he felt like an adopted member of Matthews’ lab, so it felt like a return trip).

Today, they continue exploring the frontiers of redox enzymology and one-carbon metabolism, though in different ways – Banerjee through studying mammalian pathways and clinical applications and Ragsdale through his work on microbial chemistry and applications in biotechnology.

“We do have joint lab meetings, so our students benefit from the shared expertise in our groups,” Banerjee says. “But over the years we have managed to keep our research aims different and maintain scientific independence.”

There were a couple of moments when they considered running a lab in parallel, she notes, but in the end they thought the management involved would be a little too complex.

“It’s kind of funny,” Ragsdale adds. “We started our relationship with a scientific collaboration, but in the 20 years since, we’ve both had independent careers; we’ve only published one Annual Reviews article together.”

Vitamin B12 is an essential cofactor that is both reactive and rare. Research in the Banerjee laboratory is revealing how an intricate network of proteins tailor and escort the vitamin from its point of entry to its target enzymes in cells.

Molecular traffic patterns

Banerjee, who also serves as a member of the American Society for Biochemistry and Molecular Biology council, has focused her efforts on looking at how sulfur enzymes operate in the framework of a network. “What are the traffic lights that govern the flow of sulfur to help furnish cells with some very important reagents?”

In recent years, her group has been particularly interested in the trafficking of vitamin B12, an essential vitamin that requires 30 dedicated enzymes to synthesize in bacteria. Although humans only have two B12-requiring enzymes, both of which support sulfur metabolism (methylmalonyl-CoA mutase and methionine synthase), this rare vitamin is extremely important for every cell, as evidenced by a complex protein network involved in B12 trafficking.

Banerjee’s group has been busy identifying and assigning functions to the genes involved in B12 maintenance, which include chaperones that escort this highly reactive molecule to various destinations and some novel enzymes that tailor the cobalamin molecule to its enzyme-specific active form. For example, she recently solved a long-standing mystery by revealing that a B12 chaperone called MMACHC also was responsible for cleaving off the cyanide group in cyanocobalamin, the form that’s most prevalent in vitamin supplements.

Ragsdale has expanded his field of research to include methanogenesis in addition to acetogenesis, and his group recently elucidated the reaction for the final step in methane synthesis, demonstrating that the process is nickel-dependent.

“I’ve been getting excited about that area because not only does methane have many wonderful chemical properties, but it could be a great source of future energy,” Ragsdale says. “There’s lots of stored methane available, it’s got a great energy potential and it’s clean burning.”

Following an eye-opening Gordon Conference on metals in biology, the formerly inorganic-adverse Ragsdale also has become interested in other classes of metalloproteins. One intriguing area his lab has just started investigating involves a potentially novel type of metabolic regulation in which thiol/disulfide redox switches regulate a protein’s affinity for heme; these heme-regulatory motifs respond to conditions like oxidative stress and subsequently adjust protein function. The heme functionality also allows the protein to respond to gas-signaling molecules like carbon monoxide and nitric oxide.

And, in a discovery that definitely pleased his music-loving soul, Ragsdale even found one such thiol/disulfide redox switch on a key nuclear hormone receptor involved in the circadian cycle called Rev-erb. “It was not a planned occurrence, and I didn’t name the protein,” he says, “but it kind of highlights the wonders of science and how the right protein, or person, seems to find you.”

References

Banerjee, R. V., Harder, S. R., Ragsdale, S. W., and Matthews, R. G. (1990) Mechanism of reductive activation of cobalamin-dependent methionine synthase: an electron paramagnetic resonance spectroelectrochemical study. Biochemistry 29, 1129 – 1135.

Kim, J., Gherasim, C., and Banerjee, R. (2008) Decyanation of vitamin B12 by a trafficking chaperone. Proc. Nat. Acad. Sci. 105, 14551 – 14554.

Padovani, D., Labunska, T., Palfey, B. A., Ballou, D. P., and Banerjee, R. (2008) Adenosyltransferase tailors and delivers coenzyme B12. Nature Chem. Biol. 4, 194 – 196.

Li, X., Telser, J., Hoffman, B. M., Gerfen, G., and Ragsdale, S. W. (2010) Observation of organometallic and radical intermediates formed during the reaction of methyl-coenzyme M reductase with bromoethanesulfonate. Biochemistry 49, 6866 – 6876.

Gupta, N., and Ragsdale, S. W. (2010) Thiol-disulfide redox dependence of heme binding and heme ligand switching in a nuclear hormone receptor Rev-erb{beta}. J. Biol. Chem. [Epub Dec 1 ahead of print].

Nick Zagorski (nicozags@gmail.com) is a freelance science writer.


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