In 2000, when Dr. Rosianna Gray was a Stillman College student and a University of Alabama (UA) Howard Hughes Scholar, she made 82 mutants from previously cloned DNA of Delftia acidovorans bacteria and stored them in a UA freezer.  Dr. Gray eventually went on to graduate school at UA, where she completed her Ph.D. in molecular biology in only four and a half years and became the first Howard Hughes Scholar from Stillman to earn a doctoral degree.  She returned to Stillman as an assistant professor in 2008. Her neglected Delftia acidovorans DNA remained in a deep freeze.

Two years ago, when funding was resumed and the once defunct Howard Hughes Medical Institute Scholar’s program was resurrected by UA Professor Martha Powell, Dr. Gray was invited to serve as the coordinator and primary investigator on the Stillman campus.   Working closely with her mentee, Stillman junior Lyndsey Payne, who is both a Howard Hughes Scholar and a Lewis Stokes Alliance for Minority Participation Scholar (LSAMP), Dr. Gray thawed out a section of her Delftia acidovorans DNA that contained the histodine degradation pathway.  Armed with state-of-the-art laboratory tools, she and Lyndsey embarked upon a two-month journey that yielded exciting new findings about the enzyme histidine ammonia-lyase (HAL) that catalyzes the first reaction in the degradation pathway.

“Researchers have known for years that HAL is extremely important.  It is the first enzyme in the degradation pathway for histidine, which is an essential amino acid.    Enzymes help to move reactions in a biochemical pathway.  Recent research has found out how elaborate HAL is and how exact everything has to be when it comes to its protein folding mechanism, which is a highly electrophilic 5-methylene-3,5-dihydroimidazol-4-one (MIO) group,” says Dr. Gray.  She adds that an array of disorders such as degenerative diseases in humans and Mad Cow Disease may result when proteins do not fold properly. Although Lyndsey and Dr. Gray’s research is not disease-centered, the proper folding of proteins is critical in several disciplines of science, including microbiology.

“HAL has an elaborate center and it is believed that three specific amino acid residues have to be present for the proteins to fold properly.  If there are disruptions in the way protein folds, pathways can’t proceed so you can’t make the product—in this case, the histidine,” states Dr. Gray, who now serves as Chair of the Department of Natural Sciences at Stillman.   According to previous studies of HAL, the residues of the amino acids alanyl, seryl and glycyl would have to be present during protein folding in order for the pathways to proceed and for the degradation of histidine to occur.  

“We said, ‘Let’s see if that is true.  Let’s see what happens if there are changes in the amino acid residues?  Will HAL still work properly, or will a change in any of the three amino acid residues decrease or eliminate HAL activity? We also wanted to see if any other amino acids are also important,’” Dr. Gray states. “Using our piece of DNA from Delftia acidovorans bacteria that has a histodine degradation pathway, we conducted randomized mutagenesis, chopping up DNA into tiny pieces and using highly engineered genetic tools to test the pieces for enzyme activity and test each segment for HAL enzyme activity.  We wanted to test the theory and, if the theory were correct, we would see decreased HAL activity in some mutants and possibly no activity in others.” 

Their results were significant.  While the mutants did not exactly go wild, they definitely misbehaved.  And like a proud mother discussing her naughty but adorable children, Dr. Gray shows off diagrams of her mutants and questions their peculiar behavior.  “Notice a base is missing here,” she exclaims, pointing to her research findings. “A few of the pieces of mutants still had HAL activity even when a base from one of the three required amino acids was missing.    We also found that some pieces had a different amino acid residue present, yet still had some activity even if not full strength.”  

“This is a spinoff of research I began as an undergraduate student in the Howard Hughes program, so it’s so exciting to work on this project with Lyndsey, who is also a Howard Hughes Scholar.  She is an exceptional student, and because she is also a LSAMP scholar, she was required to present our findings at a competition.  She was able to discuss and explain our research so well that she won second place out of over fifty student who competed from schools throughout the region,” says Dr. Gray, whose initial project as an undergraduate was to study how Delftia acidovorans break down histidine. 

“This research is not going to cure cancer, but if you find out how things are regulated in one organism you may eventually find out how they are regulated in a another organism,” Dr. Gray states. “I’ve talked to other researchers who are conducting similar investigations.  Although we’re excited about what we have discovered, we cannot confirm any concrete statements until we have conducted more extensive studies.  We are off to a great start and, fortunately, there are 41 more mutants in the freezer.”