Academic Appointments

2023-24 Courses

All Publications

  • Discovery and Characterization of Antibody Probes of Module 2 of the 6-Deoxyerythronolide B Synthase. Biochemistry Guzman, K. M., Cogan, D. P., Brodsky, K. L., Soohoo, A. M., Li, X., Sevillano, N., Mathews, I. I., Nguyen, K. P., Craik, C. S., Khosla, C. 2023


    Fragment antigen-binding domains of antibodies (Fabs) are powerful probes of structure-function relationships of assembly line polyketide synthases (PKSs). We report the discovery and characterization of Fabs interrogating the structure and function of the ketosynthase-acyltransferase (KS-AT) core of Module 2 of the 6-deoxyerythronolide B synthase (DEBS). Two Fabs (AC2 and BB1) were identified to potently inhibit the catalytic activity of Module 2. Both AC2 and BB1 were found to modulate ACP-mediated reactions catalyzed by this module, albeit by distinct mechanisms. AC2 primarily affects the rate (kcat), whereas BB1 increases the KM of an ACP-mediated reaction. A third Fab, AA5, binds to the KS-AT fragment of DEBS Module 2 without altering either parameter; it is phenotypically reminiscent of a previously characterized Fab, 1B2, shown to principally recognize the N-terminal helical docking domain of DEBS Module 3. Crystal structures of AA5 and 1B2 bound to the KS-AT fragment of Module 2 were solved to 2.70 and 2.65 A resolution, respectively, and revealed entirely distinct recognition features of the two antibodies. The new tools and insights reported here pave the way toward advancing our understanding of the structure-function relationships of DEBS Module 2, arguably the most well-studied module of an assembly line PKS.

    View details for DOI 10.1021/acs.biochem.3c00156

    View details for PubMedID 37184546

  • Fragment antigen binding domains (Fabs) as tools to study assembly-line polyketide synthases. Synthetic and systems biotechnology Guzman, K. M., Khosla, C. 1800; 7 (1): 506-512


    The crystallization of proteins remains a bottleneck in our fundamental understanding of their functions. Therefore, discovering tools that aid crystallization is crucial. In this review, the versatility of fragment-antigen binding domains (Fabs) as protein crystallization chaperones is discussed. Fabs have aided the crystallization of membrane-bound and soluble proteins as well as RNA. The ability to bind three Fabs onto a single protein target has demonstrated their potential for crystallization of challenging proteins. We describe a high-throughput workflow for identifying Fabs to aid the crystallization of a protein of interest (POI) by leveraging phage display technologies and differential scanning fluorimetry (DSF). This workflow has proven to be especially effective in our structural studies of assembly-line polyketide synthases (PKSs), which harbor flexible domains and assume transient conformations. PKSs are of interest to us due to their ability to synthesize an unusually broad range of medicinally relevant compounds. Despite years of research studying these megasynthases, their overall topology has remained elusive. One Fab in particular, 1B2, has successfully enabled X-ray crystallographic and single particle cryo-electron microscopic (cryoEM) analyses of multiple modules from distinct assembly-line PKSs. Its use has not only facilitated multidomain protein crystallization but has also enhanced particle quality via cryoEM, thereby enabling the visualization of intact PKS modules at near-atomic (3-5A) resolution. The identification of PKS-binding Fabs can be expected to continue playing a key role in furthering our knowledge of polyketide biosynthesis on assembly-line PKSs.

    View details for DOI 10.1016/j.synbio.2021.12.003

    View details for PubMedID 34977395

  • Properties of a "Split-and-Stuttering" Module of an Assembly Line Polyketide Synthase JOURNAL OF ORGANIC CHEMISTRY Guzman, K. M., Yuet, K. P., Lynch, S. R., Liu, C. W., Khosla, C. 2021; 86 (16): 11100-11106


    Notwithstanding the "one-module-one-elongation-cycle" paradigm of assembly line polyketide synthases (PKSs), some PKSs harbor modules that iteratively elongate their substrates through a defined number of cycles. While some insights into module iteration, also referred to as "stuttering", have been derived through in vivo and in vitro analysis of a few PKS modules, a general understanding of the mechanistic principles underlying module iteration remains elusive. This report serves as the first interrogation of a stuttering module from a trans-AT subfamily PKS that is also naturally split across two polypeptides. Previous work has shown that Module 5 of the NOCAP (nocardiosis associated polyketide) synthase iterates precisely three times in the biosynthesis of its polyketide product, resulting in an all-trans-configured triene moiety in the polyketide product. Here, we describe the intrinsic catalytic properties of this NOCAP synthase module. Through complementary experiments in vitro and in E. coli, the "split-and-stuttering" module was shown to catalyze up to five elongation cycles, although its dehydratase domain ceased to function after three cycles. Unexpectedly, the central olefinic group of this truncated product had a cis configuration. Our findings set the stage for further in-depth analysis of a structurally and functionally unusual PKS module with contextual biosynthetic plasticity.

    View details for DOI 10.1021/acs.joc.1c00120

    View details for Web of Science ID 000687851300016

    View details for PubMedID 33755455

    View details for PubMedCentralID PMC8380650