Evolution in a family of chelatases facilitated by the introduction of active site asymmetry and protein oligomerization

Abstract

The class II chelatases associated with heme, siroheme, and cobalamin biosynthesis are structurally related enzymes that insert a specific metal ion (Fe 2þ or Co 2þ ) into the center of a modified tetrapyrrole (protoporphyrin or sirohydrochlorin). The structures of two related class II enzymes, CbiX S from Archaeoglobus fulgidus and CbiK from Salmonella enterica, that are responsible for the insertion of cobalt along the cobalamin biosynthesis pathway are presented in complex with their metallated product. A further structure of a CbiK from Desulfovibrio vulgaris Hildenborough reveals how cobalt is bound at the active site. The crystal structures show that the binding of sirohydrochlorin is distinctly different to porphyrin binding in the protoporphyrin ferrochelatases and provide a molecular overview of the mechanism of chelation. The structures also give insights into the evolution of chelatase form and function. Finally, the structure of a periplasmic form of Desulfovibrio vulgaris Hildenborough CbiK reveals a novel tetrameric arrangement of its subunits that are stabilized by the presence of a heme b cofactor. Whereas retaining colbaltochelatase activity, this protein has acquired a central cavity with the potential to chaperone or transport metals across the periplasmic space, thereby evolving a new use for an ancient protein subunit.enzyme mechanism | tetrapyrrole biosynthesis

Characterization of the Enzyme CbiH60 Involved in Anaerobic Ring Contraction of the Cobalamin (Vitamin B12) Biosynthetic Pathway

“…B, the structure of cyano-cobalt(III) factor IV with the NOE contacts observed in the ROESY spectrum shown with blue arrows. C and D, two regions of the 13 C 1 H HSQC spectrum of the HSQC spectrum of cyano-cobalt(III) factor IV with major resonances labeled. C, methyl groups; D, methylene bridges.…”

“…All two-dimensional data sets were recorded with 2048 by 256 points in the direct and indirect dimensions, respectively. 13 (20) to allow the recombinant protein to be produced with a C-terminal His 6 tag and with 9 extra amino acid residues (MVQTSSKIW) incorporated into the N-terminal region of the protein. After transformation of B. megaterium DSM319, recombinant CbiH 60 was homologously overproduced and purified by metal ion affinity chromatography, allowing the isolation of ϳ20 mg liter Ϫ1 of CbiH 60 .…”

“…All NMR experiments were carried out using a 14.1 T Bruker Avance III spectrometer (600 MHz 1 H resonance frequency) equipped with a QCI (ϩ 19 F) cryoprobe). Resonance assignment was completed using a range of two-dimensional NMR experiments, including 13 C-1 H heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond correlation, as well as 1 H-1 H homonuclear correlation spectroscopy, rotating frame Overhauser effect (ROESY), and total correlation spectroscopy NMR experiments. All two-dimensional data sets were recorded with 2048 by 256 points in the direct and indirect dimensions, respectively.…”

“…Data sets of two-dimensional HSQC (supplemental Fig. S3), heteronuclear multiple bond correlation, total correlation spectroscopy, homonuclear correlation spectroscopy, and ROESY were collected measuring resonance of naturally abundant 13 C and 1 H. The uniqueness of cobalt(II) factor IV is the presence of the ␦-lactone ring. This was shown to be present as a major and a minor tautomeric form, both derived from the C-20 methyl group with NOE connections to the ring D acetate (C18a), observed in the ROESY spectrum.…”

“…This isobacteriochlorin acts as the substrate for cobalt insertion by the cobaltochelatase CbiX (or in some organisms CbiK) to give cobalt factor II (8,13). Methylation of cobalt factor II at C20 by CbiL produces cobalt factor III and this appears to be the substrate for the ring contraction step ( Fig.…”