Alpha,beta-Amino acid aminomutases catalyze the isomerization of Alpha- to beta-amino acids. One class of aminomutases requires no co-factors to promote isomerization via heterolytic processes involving ionic reaction intermediates, while another requires multiple co-factors, homolytic processes, and radical intermediates. The phenylalanine aminomutase (PAM) isolated from Taxus plants is among the set of mutases that lacks co-factor dependency, and the reaction sequence is likely initiated by action of a 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) prosthetic group, derived by spontaneous cyclization of an ASG amino acid tandem in the active site. After transient removal of the NH3 and H+ from the substrate, these migratory elements are reattached to the phenylpropanoid with retention of configuration at the receiving carbons. The (3R)-configuration of the beta-amino product is opposite to that of the product made by the presumed mechanistically related tyrosine aminomutase (TAM) reaction at pre-equilibrium. The described retention of stereochemistry in the PAM reaction is uncommon, considering that all aminomutases for which the stereochemistry has been examined invert the configuration of carbon receiving the amine group. PAM also displays broad substrate specificity for ring-substituted beta-aryl- and beta-styryl-Alpha-alanines, and thus provides a potential source of unnatural beta-amino acids. Comparing the mechanisms and stereochemical fates of all the 2,3-aminomutases, PAM appears to sort it into a distinct sub-class based on its unique reaction processes. Thus, the long-range goal of this research project is to decipher the molecular and biochemical components of the determinants that govern the PAM mechanism. The specific objectives and timeframe follow: Yrs 1 and 2, assess requirement of the MIO prosthetic group for the function of phenylalanine aminomutase from Taxus sp. Site-specific mutagenesis of the ASG residues comprising the MIO, comparative kinetic assays between wild-type and mutant enzyme will be conducted. Yrs 2 and 3, determine catalytic base that removes the activated benzylic hydrogen. Interaction of the MIO with alpha-phenylalanine is hypothesized to lower the pKa and facilitate the removal of the benzylic hydrogens. Site-specific mutagenesis, comparative kinetics assays and isotopomers of (2S)-alpha-phenylalanine will be employed to address this aim. Yrs 3 and 4, establish the complete stereochemistry of the reverse reaction. The stereochemistry of the migratory hydrogen and amino group in the reverse reaction from (3R)-beta- to (2S)-alpha-phenylalanine is hypothesized to be consistent with that observed for the forward reaction, and will be examined with synthetically derived [2H]-labeled beta-phenylalanines. Yrs 4 and 5, assess kinetics of PAM mutants that have a less encumbered active site. Rational mutation of bulky aliphatic side chains to smaller groups in the PAM active site will be employed to increase active site volume, and the specificity of the mutants will be tested with variously 2', 3', and 4'-substituted aryl alpha-amino acids. Broader Impacts of activities: Educational: In addition to cross training a diverse, multicultural pool of graduate students, this project will integrate research and education through year-round opportunities for undergraduate students. Participants will assist in DNA informatics, biochemical analyses, in vitro assay development, chromatographic and mass spectrometry-based analytical chemistry, enzymology, and synthetic organic chemistry to prepare them for careers in academia and industry. Undergraduate participation and mentoring of ethnic majority and minority students will continue, and the PI continue to recruit students from his 2nd year Organic Chemistry course, which has been the source of previous undergraduate researchers (selected from a pool of the 1000 students). The ultimate goal is to provide fledgling scientists with necessary social context, personal integrity and responsibility to embolden their self-esteem, which provides a foundation of independent thinking and problem-solving skills in Science and Life. Scientific: The project will center on discovering the principles that govern substrate specificity of the Taxol pathway phenylalanine aminomutase (PAM) isolated from Taxus spp. Knowledge of the catalytic amino acids and residues involved in substrate binding will hone our ability to predict and test hypotheses of enzymatic function. Understanding these key elements that participate in substrate/enzyme recognition will lay the groundwork to rationally design biocatalysts that can stereoselectively isomerize readily accessible alpha-amino acids to chiral beta-amino acids. The development of such biocatalysts, in vivo or in vitro, would minimize the need to use harmful and environmentally unfriendly solvents that are currently used in synthetic procedures. Ultimately, data on the molecular and structural elements that control substrate specificity will provide invaluable insight into the genetic and biochemical origins of what moderates the retention of NH3 and H+ in the MIO-dependent aminomutase reactions and loss of these groups from the closely related ammonia lyase reactions.
We looked at the mechanisms of three aminomutases, one from the Taxus canadensis plant (TcPAM) and two from bacteria (CcTAM from Chondromyces crocatus and PaPAM from Pantoea agglomerans). These enzymes are on the biological routes to antibacterial agents, made by microorganisms, and on the pathway to the anticancer drug Taxol, made in the Yew tree. TcPAM catalytically converted several different aromatic α-amino acids to their β-amino acids, while α- to β-conversion by CcTAM was more limited. The mechanism for this aminomutase family begins with transfer of the amino group from the α-amino acid to a specialized acceptor group within the enzyme, called an MIO. The MIO moves the amino group from the α- to β-position to complete the reaction. Understanding these aminomutases not only told us how they work in Nature, but also provided a green way to make non-natural β-amino acids. The latter are integral building blocks of β-peptide polymers for antibacterial agents. It was also unknown if the intermediates on aminomutase pathways could affect the reaction stream for this recently growing family of enzymes. We used several labeled molecules to show that for the TcPAM reaction, 97% of the β-amino product received all of its atoms from the α-amino acid, and the intermediate did not interfere. Fewer times (3%), the amino group of the α-amino acid transferred to a new intermediate that we added, before the β-amino product was made. Motivated by these data, we assessed the 3% exchange mechanism of TcPAM and revealed an exponential increase in the transfer of the amino group from the MIO to amino group acceptors as the latter bound better to TcPAM. Next, we hypothesized that the short lifetime of the MIO-amine pair limited the amino transfer; weaker binding acceptors had less opportunity to harvest the amino group. The lifetime of the MIO-amino pair was unknown for MIO-enzymes, and this gap in knowledge likely stemmed from the lack of real-time detection of trace amounts of NH3 in enzyme systems. Thus, we designed a burst-phase kinetic analysis to calculate the lifetime of the MIO-amino pair. This highlighted a significant discovery in MIO-based enzyme chemistry. Our research on the aminomutases solved several points that remained unanswered over the last two decades for several members of the MIO-aminomutases. An advance came when we solved the crystal structures of TcPAM and PaPAM. This was the first report on the crystal structure of an aminomutase from plants. The two PAMs make the same product, a β-phenylalanine amino acid, but were mirror images of each other, despite the enzymes using almost identical reactions and chemical space. We showed that TcPAM rotates its reaction pathway intermediate 180°, while PaPAM keeps the same intermediate stationary, which explained the different stereochemical outcomes. This new evidence showed that seemingly similar enzymes from the same aminomutase family used distinct mechanisms. Nature hones the chemical space of enzymes to catalyze chemical reactions that benefit survival. Guided by the structure, we altered the chemical space (or active site) of TcPAM. To relieve unfavorable clashes between the active site and the incoming α-amino acid, we replaced a 4-carbon group with a 1-carbon group to increase the active site volume. After this change, TcPAM more efficiently converted larger α-amino acid substrates to their β-partners; we can now biosynthesize larger β-amino acids for making new anticancer Taxol analogs. The outcomes of this research also came from unexpected observations. We thought the mechanism catalyzed by aminomutases from higher plants, such as Taxus, would be different from related aminomutases isolated from bacteria. To answer this, we synthesized labeled tyrosine α-amino acids that involved making a chiral rhodium-metal catalyst. The structure of CcTAM is unknown; however, we used our synthetic products to dissect the chemistry of CcTAM; its mechanism was surprisingly similar to that of TcPAM. The β-tyrosine product made by CcTAM had the same spatial organization as the β-phenylalanine made by TcPAM. Broader Impacts: The research included the (2008, 2013) American Chemical Society Project SEED program to provide research experiences, new social context, and public speaking opportunities to minority high school students from East Lansing and Everett High Schools in central Michigan. Undergraduates from Southern University (Louisiana), Florida AMU (Tallahassee), Michigan State University (East Lansing, MI), Lake Superior State University (Sault Ste. Marie, MI), and Ohio Wesleyan University (Columbus, OH) joined in several MSU-sponsored or NSF REU-sponsored summer programs. These students are in graduate school or actively seeking admissions into a graduate program. Five undergraduate researchers from a pool of top-rate undergraduate Organic Chemistry students also did synthesis and biochemistry work. The PI started an active bridge program with HBCU institutions by organizing the Summer 2013 Bridge Program with A&M Universities in Alabama and Florida. We brought talented undergraduates and their academic advisors to engage in summer research with faculty in the Department of Chemistry.
