The ChlD subunit links the motor and porphyrin binding subunits of magnesium chelatase

Abstract

Magnesium chelatase initiates chlorophyll biosynthesis, catalysing the MgATP2−-dependent insertion of a Mg2+ ion into protoporphyrin IX. The catalytic core of this large enzyme complex consists of three subunits: Bch/ChlI, Bch/ChlD and Bch/ChlH (in bacteriochlorophyll and chlorophyll producing species, respectively). The D and I subunits are members of the AAA+ (ATPases associated with various cellular activities) superfamily of enzymes, and they form a complex that binds to H, the site of metal ion insertion. In order to investigate the physical coupling between ChlID and ChlH in vivo and in vitro, ChlD was FLAG-tagged in the cyanobacterium Synechocystis sp. PCC 6803 and co-immunoprecipitation experiments showed interactions with both ChlI and ChlH. Co-production of recombinant ChlD and ChlH in Escherichia coli yielded a ChlDH complex. Quantitative analysis using microscale thermophoresis showed magnesium-dependent binding (Kd 331 ± 58 nM) between ChlD and H. The physical basis for a ChlD–H interaction was investigated using chemical cross-linking coupled with mass spectrometry (XL–MS), together with modifications that either truncate ChlD or modify single residues. We found that the C-terminal integrin I domain of ChlD governs association with ChlH, the Mg2+ dependence of which also mediates the cooperative response of the Synechocystis chelatase to magnesium. The interaction site between the AAA+ motor and the chelatase domain of magnesium chelatase will be essential for understanding how free energy from the hydrolysis of ATP on the AAA+ ChlI subunit is transmitted via the bridging subunit ChlD to the active site on ChlH.

Biosynthesis of the modified tetrapyrroles—the pigments of life

“…The ~140 kDa ChlH subunit binds the porphyrin substrate (182), and the ~40 kDa ChlI and ~80 kDa ChlD subunits belong to the ATPases Associated with various cellular Activities (AAA + ) superfamily. The current view is that ChlD is the physical and mechanistic link between the ChlI AAA+ motor and ChlH, which houses the site of metal ion chelation (183).…”

Hexameric structure of the ATPase motor subunit of magnesium chelatase in chlorophyll biosynthesis

“…The BchD/ChlD subunit is composed of a C‐terminal integrin I domain preceded by a proline‐rich region and an N‐terminal domain similar to BchI/ChlI, but possesses no ATPase activity . Recently, it has been demonstrated that ChlD links the ATPase activity with the ChlH active site primarily through the integrin I domain . Assembly of the BchD/ChlD hexamer is generally assumed to be mediated by the N‐terminal BchI/ChlI‐homologous domain, while contribution from the integrin I domain is unclear.…”

“…The ChlI subunit from the cyanobacterium Synechocystis sp. PCC 6803 has been extensively characterized . The ChlI protomers can self‐assemble into a ring structure without ATP.…”

“…[18][19][20][21][22][23][24][25][26][27][28] Recently, it has been demonstrated that ChlD links the ATPase activity with the ChlH active site primarily through the integrin I domain. 29 Assembly of the BchD/ ChlD hexamer is generally assumed to be mediated by the N-terminal BchI/ChlI-homologous domain, while contribution from the integrin I domain is unclear. Thus, structural study of the BchI/ChlI hexamer may also help understand the assembly of the BchD/ChlD ring.…”

“…PCC 6803 has been extensively characterized. 11,14,18,20,22,[26][27][28][29] The ChlI protomers can selfassemble into a ring structure without ATP. To uncover the interactions stabilizing the assembly, we crystallized and determined its hexameric structure.…”