Research Projects

Metallo-oxygenases are among nature’s most powerful catalysts-capable of driving highly selective oxidative transformations under mild conditions. The copper-dependent lytic polysaccharide monooxygenases (LPMOs) and heme-containing cytochrome P450s (CYP450s) showcase remarkable capabilities: activating inert C–H bonds, exhibiting exquisite regio- and stereoselectivity, and accepting a broad range of substrates. Despite their immense potential, these enzymes remain largely untapped in biotechnological applications. This proposal envisions a transformative approach: leveraging the oxidative chemistry of metallo-oxygenases to (1) dismantle biofilms that drive drug-resistant infections and (2) unlock a new biosynthetic route to anti-cardiac steroid drugs—two challenges with urgent clinical and global health relevance. (1) Harnessing LPMOs to disarm biofilms: Microbial antibiotic resistance is a global threat. Biofilms, the protective shields bacteria form to evade antibiotics and immune clearance, are rich in polysaccharides. Disrupting this barrier is critical for restoring antimicrobial efficacy. LPMOs evolved to cleave tough polysaccharides in biomass. They are largely overlooked in medical contexts, could be the key to degrading the polysaccharide-rich bacterial biofilms. We have identified, expressed and purified a novel LPMO (Vc-LPMO). In this study, we will explore the enzyme’s catalytic landscape by investigating its mechanism using a combination of spectroscopic, kinetic and computational investigations, optimize its stability and activity through protein engineering, and deploy it against biofilms formed by multidrug-resistant pathogens such as A. baumannii, K. pneumoniae, and Methicillin-resistant S. aureus. The goal is to degrade the biofilm architecture, enhance antibiotic penetration, and enable the host immune system to clear persistent infections. (2) Redefining steroid biosynthesis through a bacterial CYP450: Steroids hydroxylated at the C14 position represent a valuable class of pharmaceutical agents. However, conventional chemical synthetic routes to these molecules are inefficient, owing to the sterically hindered and chemically inert nature of the C14-carbon. While certain fungal CYP450s can perform this transformation, their membrane-bound nature and low yields make them impractical for biotechnological use. We recently discovered and purified a novel bacterial CYP450 enzyme (Bt-CYP450) that uniquely hydroxylates progesterone at the C14 position—a transformation previously unseen in bacterial systems. This breakthrough opens the door to a scalable, enzyme-driven method for producing C14-hydroxysteroids. In this project, we will dissect the mechanism of Bt-CYP450’s regioselectivity and engineer variants with improved efficiency and stereoselectivity. Through structure-guided mutagenesis and screening, we aim to develop a robust biocatalyst capable of generating pharmaceutically relevant steroids with unprecedented precision and yield. This requires a thorough understanding of the mechanism of Bt-CYP450 which distinguishes it from other CYP450s allowing it to catalyse this unique transformation. This will require understanding the enzyme substrate complex, trapping and characterizing of the reactive intermediate. These will require a combination of spectroscopic tools like resonance Raman, EPR, and rapid kinetics with a stopped flow in conjunction with theoretical simulations. By integrating modern bioinorganic chemistry and enzyme engineering, this dual approach addresses two major biomedical challenges: antimicrobial resistance and steroid drug accessibility. Enzyme-based strategies offer distinct advantages for resource-limited healthcare environments, where biofilm-driven infections are prevalent and access to complex drugs remains limited. This work not only reimagines the therapeutic potential of metallo-oxygenases—it lays the foundation for a new generation of enzyme-enabled biotechnological tools.