Molecular Determinants of Efficient Cobalt-Substituted Hemoprotein Production in E. coli

Abstract

Exchanging the native iron of heme for other metals yields artificial metalloproteins with new properties for spectroscopic studies and biocatalysis. Recently, we reported a method for the biosynthesis and incorporation of a non-natural metallocofactor, cobalt protoporphyrin IX (CoPPIX), into hemoproteins using the common laboratory strain Escherichia coli BL21(DE3). This discovery inspired us to explore the determinants of metal specificity for metallocofactor biosynthesis in E. coli. Herein, we report detailed kinetic analysis of the ferrochelatase responsible for metal insertion, EcHemH (E. coli ferrochelatase). This enzyme exhibits a small, less than 2-fold preference for Fe 2+ over the non-native Co 2+ substrate in vitro. To test how mutations impact EcHemH, we used a surrogate metal specificity screen to identify variants with altered metal insertion preferences. This engineering process led to a variant with an ∼30-fold shift in specificity toward Co 2+ . When assayed in vivo, however, the impact of this mutation is small compared to the effects of alteration of the external metal concentrations. These data suggest that incorporation of cobalt into PPIX is enabled by the native promiscuity of EcHemH coupled with BL21's impaired ability to maintain transition-metal homeostasis. With this knowledge, we generated a method for CoPPIX production in rich media, which yields cobalt-substituted hemoproteins with >95% cofactor purity and yields comparable to standard expression protocols for the analogous native hemoproteins.

Directed evolution of Escherichia coli surface-displayed Vitreoscilla hemoglobin as an artificial metalloenzyme for the synthesis of 5-imino-1,2,4-thiadiazoles

“…37 These factors limit the throughput of genetic optimization schemes applied to ArMs and the applicability in vivo to expand natural metabolism. In recent decades, numerous studies have focused on making the assembly and screening of these biohybrid catalysts more convenient: (i) in vivo assembly of ArM with biosynthesized cobalt protoporphyrin IX (Co(ppIX)) under iron-limited, cobalt-rich growth conditions, 38,39 (ii) in vivo assembly of ArM and cofactor with a system to transport the cofactor into the cytoplasm ( e.g. , recombinant production of heme proteins in E. coli strain Nissle 1917, 40,41 ChuA 42 and the Hug 43 system for porphyrin transportation), and (iii) in vivo screening via cell surface display.…”