CAREER: Molecular and Biochemical Evaluation of a Phenylalanine Aminomutase

Michigan State University, East Lansing MI

Investigators

Abstract

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.