Tài liệu Báo cáo khoa học: ATPase activity of magnesium chelatase subunit I is required to maintain

ATPase activity of magnesium chelatase subunit I is required

to maintain subunit D in vivo

Vanessa Lake1,2, Ulf Olsson2

, Robert D. Willows1 and Mats Hansson2

1

Department of Biological Science, Macquarie University, North Ryde, Australia; 2

Department of Biochemistry, Lund University,

Sweden

During biosynthesis of chlorophyll, Mg2+ is inserted into

protoporphyrin IX by magnesium chelatase. This enzyme

consists of three different subunits of
40, 70 and

140 kDa. Seven barley mutants deficient in the 40 kDa

magnesium chelatase subunit were analysed and it was

found that this subunit is essential for the maintenance of

the 70 kDa subunit, but not the 140 kDa subunit. The

40 kDa subunit has been shown to belong to the family

of proteins called
ATPases associated with various cellu￾lar activities, known to form ring-shaped oligomeric

complexes working as molecular chaperones. Three of the

seven barley mutants are semidominant mis-sense muta￾tions leading to changes of conserved amino acid residues

in the 40 kDa protein. Using the Rhodobacter capsulatus

40 and 70 kDa magnesium chelatase subunits we have

analysed the effect of these mutations. Although having

no ATPase activity, the deficient 40 kDa subunit could

still associate with the 70 kDa protein. The binding was

dependent on Mg2+ and ATP or ADP. Our study dem￾onstrates that the 40 kDa subunit functions as a chaperon

that is essential for the survival of the 70 kDa subunit

in vivo. We conclude that the ATPase activity of the

40 kDa subunit is essential for this function and that

binding between the two subunits is not sufficient to

maintain the 70 kDa subunit in the cell. The ATPase

deficient 40 kDa proteins fail to participate in chelation in

a step after the association of the 40 and 70 kDa subunits.

This step presumably involves a conformational change of

the complex in response to ATP hydrolysis.

Keywords: AAA; barley; chlorophyll; magnesium chela￾tase; Rhodobacter capsulatus.

The first unique enzymatic reaction of the (bacterio)chloro￾phyll biosynthetic pathway is the insertion of Mg2+ into

protoporphyrin IX. Three different polypeptides participate

in the catalytic reaction and these constitute the subunits of

magnesium chelatase (Fig. 1). The subunits are designated

BchI, BchD and BchH in bacteriochlorophyll-synthesizing

organisms such as Rhodobacter and Chlorobium, while in

plants, algae and cyanobacteria, the homologous proteins

are generally named ChlI, ChlD and ChlH [1]. The average

molecular masses of BchI/ChlI, BchD/ChlD and BchH/

ChlH are 40, 70 and 140 kDa, respectively. The largest

subunit is red upon purification due to bound protopor￾phyrin IX [2–4] and binding studies of deuteroporphyrin IX

to the H-subunit show a Kd value of 0.53–1.2 lM [5]. The

large subunit has therefore been suggested to be the catalytic

subunit. The exact role of the other two subunits is not

understood. It is known that they form a complex in the

presence of Mg2+ and ATP [2,3,6,7]. The complex forma￾tion does not require hydrolysis of ATP, as ADP and

nonhydrolysable ATP analogues (but not AMP) allowed

complex formation [8]. It is clear, however, that the overall

magnesium chelatase reaction requires ATP hydrolysis. The

observations are consistent with earlier observations with

pea magnesium chelatase where the magnesium chelatase

reaction was demonstrated to be a two-step reaction,

consisting of an activation step followed by the actual Mg2+

insertion step [9]. The activation step could proceed with

ATP-c-S, whereas ATP was required for the chelation.

The three-dimensional structure of the Rhodobacter

capsulatus BchI has recently been determined and it was

found to belong to the large family of
ATPases associated

with various cellular activities, or AAA+ proteins [10].

AAA+ proteins are important mechanoenzymes that

transform chemical energy into biological events and they

are usually found in various multimeric states [11,12]. They

play essential roles in a broad range of cellular activities,

including DNA replication, membrane fusion, cytoskeletal

regulation, protein folding and proteolysis [11–13], and now

also in porphyrin metallation [10]. The N-terminal half

of the BchD subunit is homologous to BchI, while the

C-terminal half includes a metal ion coordination motif

characteristic for integrin I domains [10]. Integrins are

known to participate in cell–matrix and cell–cell interactions

[14,15]. They are involved in signalling to and from cells in

various physiological processes, including morphogenesis,

cell migration, immunity and wound healing [16,17]. The

proposed models for the magnesium chelatase reaction all

involve an Mg2+- and ATP-dependent complex formation

of the 40 and 70 kDa subunits. In a subsequent step the

complex triggers the Mg2+ insertion into protoporphyrin

by the 140 kDa subunit [2,4,18–20]. Our present model for

Correspondence to M. Hansson, Department of Biochemistry, Lund

University, Box 124, S-22100 Lund, Sweden. Fax: + 46 46 2224534,

Tel.: + 46 46 2220105, E-mail: [email protected]

Abbreviations: AAA+ proteins, ATPases associated with various

cellular activities.

(Received 1 December 2003, revised 20 February 2004,

accepted 2 April 2004)

Eur. J. Biochem. 271, 2182–2188 (2004)
FEBS 2004 doi:10.1111/j.1432-1033.2004.04143.x