The overall goal of our research is to achieve a detailed understanding of the mechanism of biologically important enzymatic reactions. Our research strategy is multifaceted and encompasses numerous experimental approaches such as x-ray crystallography, recombinant protein expression, site-directed mutagenesis, use of mechanistic probes (e.g., suicide substrates) and rapid reaction kinetic techniques, including single turnover studies with ultra-fast spectroscopic detection. Several areas of current interest are described below.
Antibiotic Biosynthesis A powerful mandate for the development of better antifungal agents has been created by the emergence of new opportunistic fungal pathogens that sicken healthy individuals, a rapidly growing population of highly susceptible, immunocompromised patients, and the development of resistance against currently available drugs. We are investigating a remarkable aromatization reaction catalyzed by nikD, an unusual flavoprotein oxidase that plays a critical role in the biosynthesis of nikkomycin antibiotics. Nikkomycins are antifungal agents that act by blocking the biosynthesis of chitin. The nonribosomal peptide in nikkomycins contains an essential N-terminal pyridyl residue that is synthesized by nikD in a multistep reaction comprising two redox cycles, a dihydropicolinate intermediate (DHP), and at least one isomerization step. The nikD substrate, piperideine-2-carboxylate (P2C), is derived from L-lysine. The nikD crystal structure reveals two distinct substrate binding modes and a mobile cation-binding loop of unknown function. There are six possible DHP isomers. We have shown that nikD oxidizes P2C to a DHP isomer (DHPx) that cannot be directly oxidized to picolinate. Instead, a reduced form of nikD catalyzes the isomerization of DHPx to produce an isomer (DHPy) that can be converted to picolinate. Mutagenesis studies rule out amino acid residues that might play a role in DHP isomerization. The results strongly suggest that the reduced flavin cofactor in nikD acts as the acid-base catalyst required for DHP isomerization. The mechanism underlying the nikD reaction is likely to provide the first example of a flavin coenzyme acting as both a redox and an acid-base catalyst, a paradigm for related enzymatic aromatization reactions that produce important pharmacophores found in other nonribosomal peptide antibiotics and a springboard for future antifungal drug discovery studies.
Oxygen Activation The ability to activate molecular oxygen underpins all aerobic biology. However, the mechanism of oxygen activation is poorly understood and the pathways used by oxygen to access enzyme active sites are largely unknown. Flavoprotein oxidases activate the reduction of molecular oxygen to hydrogen peroxide, a highly reactive compound that, analogous to nitric oxide, is both a cytotoxin and a cell signaling molecule. Our current studies with several members of a novel superfamily of amino acid oxidases suggest that oxygen activation is mediated by basic residues at polar active sites, a model that challenges the prevailing dogma that hydrophobic cavities are likely to function as oxygen binding sites. A more diverse mechanistic landscape is emerging regarding oxygen transport where oxygen access to buried or solvent accessible active sites may require dynamic protein motions or be mediated by stable gas tunnels, respectively.
Substrate Channeling Metabolism is built of complex networks of enzymes that form links by sharing substrate and product molecules. Some of these shared molecules are reactive and not freely diffusing. Instead, their motion is directed or channeled from one active site to another in bi- or trifunctional enzymes by mechanisms which are poorly understood. We seek to understand how an imine intermediate in sarcosine (N-methylglycine) catabolism is channeled between two active sites that are 35 Å apart in a complex bifunctional enzyme, heterotetrameric sarcosine oxidase (TSOX). A high-resolution crystal structure of TSOX reveals a remarkable system of large internal cavities which are hypothesized to function in substrate channeling. The mechanism deduced for substrate channeling in TSOX will serve as a model for other bifunctional enzymes that channel charged hydrophilic organic metabolites that cannot be readily transported via classical narrow hydrophobic tunnels.
Selected Publications
"Structural characterization of the oxygen activation site in monomeric sarcosine oxidase"
M.S. Jorns, Z. Chen, F.S. Mathews,
Biochemistry, 49, 3631-3639 (2010).
"Factors that affect oxygen activation and coupling of the two redox cycles in the aromatization reaction catalyzed by nikD, an unusual amino acid oxidase"
P.R. Kommoju, R.C. Bruckner, P. Ferreira, C.J. Carrell, F.S. Mathews and M.S. Jorns
Biochemistry, 48, 9542-9555 (2009).
"Probing the role of active site residues in nikD, an unusual amino acid oxidase that catalyzes an aromatization reaction important in nikkomycin biosynthesis"
P.R. Kommoju, R.C. Bruckner, P.Ferreira and M.S. Jorns
Biochemistry, 48, 6951-6962 (2009).
"Spectral and Kinetic Characterization of Intermediates in the Aromatization Reaction Catalyzed by NikD, an Unusual Amino Acid Oxidase"
R.C. Bruckner, and M.S. Jorns
Biochemistry, 48, 4455-4465 (2009).
"Identification of the Oxygen Activation Site in Monomeric Sarcosine Oxidase: Role of Lys265 in Catalysis"
G. Zhao, R. C. Bruckner, and M.S. Jorns
Biochemistry, 47, 9124-9135 (2008).
"Covalent Flavinylation of Monomeric Sarcosine Oxidase: Identification of a Residue Essential for Holoenzyme Biosynthesis"
A. Hassan-Abdallah, G. Zhao and M.S. Jorns
Biochemistry, 47, 1136-1143 (2008).
"Arginine 49 is a Bifunctional Residue Important in Catalysis and Biosynthesis of Monomeric Sarcosine Oxidase: A Context-sensitive Model for the Electrostatic Impact of Arginine to Lysine Mutations"
A. Hassan-Abdallah, G. Zhao, Z. Chen, F.S. Mathews and M.S. Jorns
Biochemistry, 47, 2913-2922 (2008).
"NikD, an Unusual Amino Acid Oxidase Essential for Nikkomycin Biosynthesis"
C.J. Carrell, R.C. Bruckner, D. Venci, G. Zhao, M.S. Jorns and F.S. Mathews
Structure, 15, 928-941 (2007).
"A Mobile Tryptophan is the Intrinsic Charge Transfer donor in a Flavoenzyme Essential for Nikkomycin Antibiotic Biosynthesis"
R.C. Bruckner, G. Zhao, P. Ferreira and M.S. Jorns
Biochemistry, 46, 819-827 (2007).
"Heterotetrameric Sarcosine Oxidase: Structure of a Diflavin Metalloenzyme at 1.85 Å Resolution"
Z. Chen, A. Hassan-Abdallah, G. Zhao, M.S. Jorns and F.S. Mathews.
J. Mol.Biol., 360, 1000-1018 (2006).
"Biosynthesis of Covalently Bound Flavin: Isolation and in vitro Flavinylation of the Monomeric Sarcosine Oxidase Apoprotein"
A. Hassan-Abdallah, R.C. Bruckner, G.H. Zhao and M.S. Jorns
Biochemistry, 44, 6452-6462 (2005).
"Molecular Characterization of NikD, a New Flavoenzyme Important in the Biosynthesis of Nikkomycin Antibiotics"
D. Venci, G. Zhao and M.S. Jorns
Biochemistry, 41, 15795-15802 (2002).
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