Tài liệu Báo cáo khoa học: Kinetic basis for linking the first two enzymes of chlorophyll

Kinetic basis for linking the first two enzymes of

chlorophyll biosynthesis

Mark Shepherd, Samantha McLean and C. Neil Hunter

Robert Hill Institute for Photosynthesis and Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology,

University of Sheffield, UK

Magnesium chelatase lies at a branch point in tetrapyr￾role biosynthesis where insertion of Mg2+ eventually

results in the production of chlorophyll, or the insertion

of Fe2+ produces heme. Magnesium chelatase is com￾prised of three protein subunits, ChlI (38–42 kDa),

ChlD (60–74 kDa) and ChlH (gun5, 140–150 kDa)

(BchIDH in photosynthetic bacteria) [1–4]. ChlI is an

AAA+ ATPase [5,6], contains a Mg2+ binding site [7],

and forms a stable complex with ChlD [8]. The third

subunit, ChlH, binds porphyrins [9,10] and presumably

contains the active site for chelation. The steady-state

kinetic characterization of magnesium chelatase quanti￾fied the ATP hydrolysis required to complete a catalytic

cycle and revealed a cooperativity with respect to Mg2+,

which has important implications for regulation of chlo￾rophyll biosynthesis [11]. The structure of Gun4, a pro￾tein that binds to the tetrapyrrole substrate and product

of the magnesium chelatase, has recently been solved

[12]. Kinetic analysis revealed that Gun4 dramatically

enhances the magnesium chelatase reaction, and reduces

the threshold Mg2+ concentration required for chelatase

activity at low substrate concentrations, implying a

possible role for this protein in substrate delivery.

The next step in chlorophyll biosynthesis, catalysed

by magnesium protoporphyrin IX methyltransferase

(ChlM in Synechocystis), involves the transfer of a

methyl group from S-adenosyl-l-methionine (SAM) to

the propionate group on ring C of magnesium proto￾porphyrin IX (MgP) to form magnesium protopor￾phyrin IX monomethylester (MgPME). Steady-state

kinetic assays showed that the reaction proceeds via a

random binding mechanism forming a ternary complex

[13]. Stopped-flow fluorescence studies indicated that

a relatively slow (
70 s)1

) domain reorganization of

ChlM alters the conformation of the MgD binding

site and precedes rapid (> 600 s)1

) substrate binding

(Kd 3.36 lm) [14]. Rapid quenched-flow analysis

showed that a catalytic intermediate is formed and

Keywords

chlorophyll; chelatase; methyltransferase;

gun signalling

Correspondence

M. Shepherd, Department of Biochemistry

and Molecular Biology, A222 Life Sciences

Building, Green Street, University of

Georgia, Athens, GA 30602, USA

Fax: +1 706 5427567

Tel: +1 706 5427252

E-mail: [email protected]

(Received 25 February 2005, revised 5 July

2005, accepted 19 July 2005)

doi:10.1111/j.1742-4658.2005.04873.x

Purified recombinant proteins from Synechocystis PCC6803 were used to

show that the magnesium chelatase ChlH subunit stimulates magnesium

protoporphyrin methyltransferase (ChlM) activity. Steady-state kinetics

demonstrate that ChlH does not significantly alter the Km for the tetrapyr￾role substrate. However, quenched-flow analysis reveals that ChlH dramat￾ically accelerates the formation and breakdown of an intermediate in the

catalytic cycle of ChlM. In light of the profound effect that ChlH has on

the methyltransferase catalytic intermediate, the pre steady-state analysis in

the current study suggests that ChlH is directly involved in the reaction

chemistry. The kinetic coupling between the chelatase and methyltrans￾ferase has important implications for regulation of chlorophyll biosynthesis

and for the availability of magnesium protoporphyrin for plastid-to-nucleus

signalling.

Abbreviations

Mg chelatase, magnesium chelatase; MgD, magnesium deuteroporphyrin IX; MgDME, Mg deuteroporphyrin IX monomethyl ester;

MgP, magnesium protoporphyrin IX; MgPME, Mg protoporphyrin IX monomethyl ester; Mops, 4-morpholinepropanesulfonic acid; PIX,

protoporphyrin IX; SAH, S-adenosyl-L-homocysteine; SAM, S-adenosyl-L-methionine; Synechocystis, Synechocystis PCC6803.

4532 FEBS Journal 272 (2005) 4532–4539 ª 2005 FEBS