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      Our laboratory uses mainly X-ray crystallographic technique to study the structure and function of proteins. We emphasizes on the application of  computational/bioinformatics methods on the genome sequence data in the discovery of the ‘missing links’ in important metabolic pathways. Subsequent structural characterizations of these newly annotated gene products are essential for understanding their function and for the structure-base drug design. Currently our interests are focused on several proteins in 1). the NAD biosynthesis and regulation pathways;  and 2). GHMP kinase superfamily..

1. Structural studies of NAD biosynthesis and regulation 

    One area of our research is focused on proteins involved in NAD biosynthesis and regulation in both bacteria and human. The central step of all NAD biosynthesis pathways converges to the step that is catalyzed by nicotinamide (or nicotinate) mononucleotide adenylyltransferase (NMNAT), an essential enzyme for cell survival in prokaryotes. Human NMNAT has been shown to be localized exclusively in the nucleus, interact specifically with the DNA repair protein poly(ADP-ribose) polymerase, and may be regulated by phosphorylation. Human NMNAT also catalyzes the rate-limiting step in the metabolic conversion of anti-cancer agent tiazofurin to its active form tiazofurin adenine dinucleotide (TAD). Low levels of NMNAT activity in tumor cells have been associated with the development of tiazofurin resistance. Both bacterial and human NMNAT genes were identified only very recently. We have now solved the crystal structures of both E.coli and human NMN adenylyltransferase and their complexes with ligands. These structures provided a structural basis for the different substrate specificity of the two enzymes, and reveal the structural mechanism of tiazofurin adenylation. 

    The cellular NAD level needs to be tightly controlled. In bacteria, a multifunctional protein NadR plays a key role in the transcriptional regulation of NAD biosynthesis genes in response to the cellular NAD levels, and in the uptake of NMN from the medium. We are currently working on the elucidation of the different conformational states of NadR and the three dimensional structure of its complex with cognate DNA.

2. GHMP kinases

    The GHMP class of small metabolite kinases participates in several essential metabolic processes, such as glycolysis, amino acid biosynthesis, and sterol biosynthesis. Currently, the GHMP superfamily contains more than 170 proteins, representing about 12-13 different functions, including galactokinases, homoserine kinases, mevalonate kinases, phosphomevalonate kinases (hence GHMP), mevalonate diphosphate decarboxylase, isopentenyl monophosphate kinase, and archaeal shikimate kinase. Deficiencies in GHMP enzyme activities cause auxotrophic phenotypes in bacteria and hereditary metabolic diseases in humans. Structural characterization of GHMP enzymes and their complexes with substrates is crucial for understanding the active site, catalytic mechanism, inhibition and regulation of these enzymes. The first three dimensional structure of a GHMP protein, the homoserine kinase (HK), revealed a novel nucleotide-binding fold and a unique ATP binding mode. The crystals of the ternary complex between HK and its substrates were also obtained. Structural analysis of the HK-substrate complexes will reveal the key catalytic residues in the phosphoryl transfer reaction and the residues responsible for the substrate specificity. To learn how GHMP-fold accommodates substrates of very different structures, other members in the GHMP superfamily are selected for the structural studies in a systematic approach. Diffracting crystals of the archaeal shikimate kinase have been obtained. Comparative analysis of the enzyme-substrate complexes from different members of the GHMP superfamily will reveal the structural determinants of the substrate specificity in each family, provide a foundation for the structure-base drug design, and further our understanding of the structure-function evolution of this important class of enzymes.