Biosyntheses of sulfur-containing natural products, especially by selective C-H activation, are at the frontiers of mechanistic enzymology and biocatalysis. In this award (CHE-2004109), using enzymes from thermophilic organisms, the key C-S bond construction steps in ergothioneine and ovothiol biosynthetic pathways were characterized. Ergothioneine and ovothiol are thiol-histidine derivatives. In the last decades, four ergothioneine and one ovothiol biosynthetic pathways have been discovered. Among them, there are two aerobic and two anaerobic ergothioneine biosynthetic pathways. Recent biological function characterizations indicated that both ergothioneine and ovothiol are longevity compounds that significantly extend the median lifespan on mouse/worm models. In addition, both ergothioneine and ovothiol have protective effects against several aging associated diseases. Current chemical synthesis of ergothioneine and ovothiol is costly. Mechanistic studies of these pathway have led to innovative industrial scale production of these two compounds, allowing our students to have a smooth school-to-industry transition by solving real world problems. In addition, fundamental discoveries under this award (CHE-2004109) open up the opportunities for many follow up studies on related system in the coming years. Specifically, we have the following discoveries:

Biosynthetic C-S bond formation reactions can be roughly classified as ionic or radical type of reactions. For ionic-type of trans-sulfuration reactions, the protein-based persulfide or thiocarboxylate intermediates are the direct sulfur source in many biosynthetic C-S bond construction reactions. In general, a cysteine desulfurase converts cysteine to alanine and delivers the cysteine sulfur atom to produce one of these two intermediates. Under the support of this award (CHE-2004109), we demonstrated that a rhodanese-domain containing protein, EanB, catalyzes the production of a cysteine persulfide production using polysulfide as the substrate, which is the first case of involving polysulfide as the direct sulfur source in natural product biosynthesis. In addition, our subsequent biochemical and computational work suggest that EanB-catalysis may involve carbene intermediate, which adds EanB to the short list of carbene involving enzymes. Given the wide-distribution of rhodanese-domain containing enzymes, these discoveries open-up opportunities for follow-up studies of many more related biosynthetic pathways. Encouraged by this discovery, we characterized YcfA, an adenine nucleotide alpha hydrolase-like (AANH-like) superfamily member, and YcfA-catalysis indeed involves new trans-sulfuration mechanism.

For the aerobic radical-types C-S bond formation reactions, isopenicillin N synthase (IPNS) is a prototypical example. Similar to IPNS, the Egt1/EgtB/OvoA enzymes in the aerobic ergothioneine and ovothiol biosynthetic pathways are also mononuclear non-heme iron enzymes. These enzymes all catalyze oxidative C-S bond formation reactions, while they are distinct in substrate selectivity and product C-S bond regio-selectivity. Under the support of this award (CHE-2004109), we identified several of their homologs from thermophilic organisms, which enables their X-ray structure, pre-steady state and freeze-quench spectroscopic characterizations. Our work led to the successful OvoA crystal structure after 10-years of efforts. In addition, we have trapped and characterized a S = 1 Fe^IV=O species, which is the first time of trapping such a species in mononuclear NHFe enzymes after decades of efforts of searching for its existence.

The research program has been highly integrated with many education and outreach activities and involves graduate, undergraduate and high school students. First, because ergothioneine and ovothiol are longevity compounds, our studies led to the improvement of their production yield by orders of magnitude. The translational aspects of these project offer a platform to train our students with skills that are directly relevant to the industry needs. In addition, due to the complexities of the C-S bond formation reactions in ergothioneine and ovothiol biosynthesis, an interdisciplinary approach was used in our studies. For example, my lab is one of the labs at Boston University that offers wet lab training for bioinformatics students. In addition, we are establishing collaboration with data scientists so that our students exposed to data science at the early stage of their career.  Under the support of this award (CHE-2004109), a large number of undergraduate students (17 of them and 2 minority and one REU students) were part of our work. Seven of them are co-authors in our publications and nine of them received UROP awards.

Last Modified: 12/02/2024
Modified by: Pinghua Liu