Interplay between an AAA module and an integrin I domain may regulate the function of magnesium chelatase

In chlorophyll biosynthesis, insertion of Mg2+ into protoporphyrin IX is ca talysed in an ATP-dependent reaction by a three-subunit (BchI, BchD and Bch H) enzyme magnesium chelatase. In this work we present the three-dimensiona l structure of the ATP-binding subunit BchI. The structure has been solved by the multiple wavelength anomalous dispersion method and refined at 2.1 A ngstrom resolution to the crystallographic R-factor of 22.2 % (R-free = 24. 5 %). It belongs to the chaperone-like "ATPase associated with a variety of cellular activities" (AAA) family of ATPases, with a novel arrangement of domains: the C-terminal helical domain is located behind the nucleotide-bin ding site, while in other known AAA module structures it is located on the top. Examination by electron microscopy of BchI solutions in the presence o f ATP demonstrated that BchI, like other AAA proteins, forms oligomeric rin g structures. Analysis of the amino acid sequence of subunit BchD revealed an AAA module at the N-terminal portion of the sequence and an integrin I A n acidic, proline-rich region linking these two domains is suggested to con tribute to the association of BchI and BchD by binding to a positively char ged cleft at the surface of the nucleotide-binding domain of Bch. Analysis of the amino acid sequences of BchI and BchH revealed integrin I domain-bin ding sequence motifs. These are proposed to bind the integrin I domain of B chD during the functional cycle of magnesium chelatase, linking porphyrin m etallation by BchH to ATP hydrolysis by BchI. An integrin I domain and an a cidic and proline-rich region have been identified in subunit CobT of cobal t chelatase, clearly demonstrating its homology to BchD. These findings, fo r the first time, provide an insight into the subunit organisation of magne sium chelatase and the homologous colbalt chelatase. (C) 2001 Academic Pres s.