Honggao Yan Research Interests
The research in my laboratory is mainly in the area of protein biochemistry focusing on protein structure, function, and dynamics with applications to protein engineering and structure-based drug design. We study protein biochemistry from both structural and functional perspectives. Thus, a multidisciplinary approach, including molecular genetic, biochemical, bio-organic, biophysical, and computational methods, has been employed in our research. The main structural technique is multidimensional NMR spectroscopy, the most powerful spectroscopic technique that can be used not only to determine the three-dimensional structures of proteins but also to elucidate their chemical and dynamic properties in solution at atomic resolution. The thrust of our research is to understand the relationships between structure, function, and dynamics in enzymes, particularly enzymes in the folate biosynthetic pathway and nucleotide metabolism, because these enzymes are not only good model systems for addressing the important biochemical issues that we are interested in but also of great medical significance.
Folate Biosynthetic Enzymes.
Folates are essential for all organisms. Mammals are unable to synthesize folates but have an active transport system for deriving folates from the diet. In contrast, most microorganisms must synthesize folates de novo because they lack the active transport system. Thus, the folate biosynthetic pathway is an attractive target for developing antimicrobial agents. Our first target enzyme is 6-hydroxymethyl-7, 8-dihydropterin (HP) pyrophosphokinase (HPPK), which catalyzes the transfer of pyrophosphate from ATP to HP. Because of its small size and high thermal stability, E. coli HPPK is not only a new target for developing antimicrobial agents but also an excellent model system for studying the mechanisms of enzymatic pyrophosphoryl transfer, which takes place at β-phosphorus of ATP, instead of γ-phosphorus as in phosphoryl transfer reactions catalyzed by kinases. While kinases have been intensely studied for decades with respect to both structure and mechanism, structural and mechanistic studies of pyrophosphokinases just begun in earnest in recent years.
When we began to study E. coli HPPK, very little was known except some basic properties of the enzyme, such as amino acid sequence and steady-state kinetic parameters. Because of the strength and advantages of our combined molecular genetic, biochemical, and biophysical approaches, we have been able to make major contributions to the understanding of the structure and mechanism of HPPK. Our results have revealed that HPPK undergoes dramatic and unusualconformational changes during its catalytic cycle and the conformational changes play critical roles in its catalysis. The major goal of the ongoing research on HPPK is to elucidate the roles of conformational dynamics in HPPK catalysis. Multiple intermediates and conformations are general characteristics of enzymes, and conformational dynamics is coupled to enzymatic catalysis. However, very few enzymes have atomic structures determined for every stage in the catalytic cycle as in HPPK. While the coupling between conformational dynamics and enzymatic catalysis has been long recognized, correlating conformational dynamics with catalysis has been most challenging and direct evidence for the coupling is scarce. Our understanding of the role of protein dynamics in catalysis lags far behind our knowledge of the structures and chemical mechanisms of enzymes. Thus, the ongoing research on HPPK will have significant impact on the understanding of enzymatic catalysis on the atomic basis.
Our interest in the folate biosynthetic enzymes has driven us to expand our research to include dihydroneopterin aldolase (DHNA), whose product is the substrate for HPPK. DHNA is a unique aldolase, because it requires neither the formation of a Schiff base nor metal ions for catalysis. In addition, DHNA also catalyzes the epimerization of its substrate. Interestingly, the amino acid sequences of DHNAs from Gram-positive and Gram-negative bacteria are significantly different. Many differences are at or near their active centers. Although the structure of S. aureus DHNA, a representative of DHNAs from Gram-positive bacteria, has been reported, the most critical interaction between the enzyme and the substrate has not been revealed. The catalytic mechanism of DHNA is largely unknown. We are interested in elucidating the catalytic mechanism of DHNA and comparing the mechanisms and structures of S. aureus and E. coli DHNAs, the latter of which is DHNAs from Gram-negative bacteria.
Enzymes in Nucleotide Metabolism.
Our current research on enzymes in nucleotide metabolism focuses on yeast cytosine deaminase (yCD). The goals of the research are to correlate protein dynamics with catalysis in yCD by a combination of site-directed mutagenesis, biochemical analysis, NMR, and computational methods and to improve the catalytic efficiency and stability of yCD by directed evolution.The challenge in cancer therapy is to kill tumor cells without damaging normal cells. Gene-directed enzyme prodrug therapy (GDEPT) meets the challenge by activating a prodrug by an enzyme within the tumor and thereby minimizing damage to normal tissue. Cytosine deaminase (CD) catalyzes the conversion of the prodrug 5-fluorocytosine (5-FC) to the anticancer drug 5-fluorouracil. The CD and 5-FC combination is one of the most widely used enzyme/prodrug combinations for GDEPT. While yCD is preferred for CD-based GDEPT, the enzyme remains sub-optimal for GDEPT in two critical respects. First, the catalytic efficiency of yCD is much lower than those of the most proficient enzymes, and second, yCD loses its activity rapidly at the physiological temperature. An improved yCD derived from directed evolution experiments will significantly improve the efficacy of yCD as an activating enzyme for CD-based GDEPT for treatment of cancer.
The structure of yCD has been determined at high resolution both in the free form and in the complex with a transition state analogue. Surprisingly, the substrate-binding pocket of yCD is closed in both structures. Based on the crystal structures of yCD and our transient kinetic and NMR analyses, we hypothesize that yCD exists in a major form with the active center closed and a minor form with an open active center, and protein dynamics plays an important role throughout the catalytic cycle of yCD. To test the hypothesis, we will (1) compare the dynamic properties of the enzyme at the different stages of the catalytic cycle, (2) compare the dynamic properties of the mutants derived from site-directed mutagenesis and directed evolution with those of the wild-type enzyme, and (3) compare the kinetic properties of the mutants with those of the wild-type enzyme. Comparison of the dynamic properties of yCD at the different stages of the catalytic cycle will allow us to identify changes in protein dynamics along the reaction coordinate. Comparison of the dynamic and kinetic properties of the mutants with those of the wild-type enzyme will allow us to correlate protein dynamics with catalysis in yCD. To this end, we have made the total sequential resonance assignment of the homodimeric yCD (~35 kDa for the dimer) by multidimensional NMR spectroscopy. Furthermore, we have established both genetic selection and biochemical screening methods for directed evolution of yCD, the most critical steps in any directed evolution experiments.