Reconstitution of a sequential reaction of two nitrogenase-like enzymes in the bacteriochlorophyll biosynthetic pathway of Rhodobacter capsulatus
Introduction
Chlorophylls (Chls) are tetrapyrrole pigments essential for photosynthesis. There are more than 100 different structures in various photosynthetic organisms. These various Chls are categorized into three types according to their basic skeletal structure: porphyrin, chlorin, and bacteriochlorin [1]. Chl a is a chlorin-type Chl with an absorbance maximum at 661 nm (Qy band in ether) that enables oxygenic photosynthesis using red light in plants and cyanobacteria. Bacteriochlorin-type Chls include bacteriochlorophyll a (Bchl a), which is distributed among anoxygenic photosynthetic bacteria. The absorbance maximum of the Qy band of Bchl a is 773 nm (in ether). This light-absorbing property of Bchl a enables the use of a longer-wavelength light than that used by Chl-a-containing photosynthetic organisms. In addition to some substitutions of side chain groups, such as the C-7 formyl group in Chl b and C-3 acetyl group in Bchl a, the basic skeletal structures exert a decisive influence on the absorption properties of Chl pigments.
In the biosynthesis of Bchl a, the bacteriochlorin structure is formed by a sequential operation of two nitrogenase-like enzymes. The first enzyme is dark-operative protochlorophyllide (Pchlide) oxidoreductase (DPOR) that converts a porphyrin-type precursor, Pchlide, to a chlorin-type precursor, chlorophyllide a (Chlide) by C17C18 double bond reduction (Fig. 1) [2]. The second enzyme is Chlide oxidoreductase (COR) that reduces the C7C8 double bond to produce the bacteriochlorin-type precursor, 3-vinyl bacteriochlorophyllide a (MV-Bchlide) (Fig. 1) [3]. The Qy band shifts from 625 nm (Pchlide) to 745 nm (MV-Bchlide) via 661 nm (Chlide) by the sequential reaction.
DPOR consists of two separable components, L-protein (a BchL dimer) as a reductase component and NB-protein (a BchN–BchB heterotetramer) as a catalytic component, which are structurally related to nitrogenase Fe protein and MoFe protein, respectively [2]. DPOR was the first enzyme whose reaction mechanism was proposed on the basis of X-ray crystal structures in (B)Chl biosynthesis [4]. L-Protein transfers electrons to NB-protein coupled with ATP hydrolysis, similar to the electron transfer by Fe protein of nitrogenase [5]. The electron transfer is mediated by a unique Fe–S cluster, NB-cluster, to reach the Pchlide molecules in the catalytic site of NB-protein [6], [7]. Although there have been extensive studies of DPOR from the purple bacterium Rhodobacter capsulatus [2], [4], [5], [6], [7], [8] and from a few other species [9], [10], [11], [12], [13], little is known about the second nitrogenase-like enzyme, COR. In vitro reconstitution using purified components indicated that COR also consists of two components, X-protein (a BchX dimer) and YZ-protein (a BchY–BchZ heterotetramer), which are cognates of L-protein and NB-protein, respectively, and that COR requires dithionite (reduced ferredoxin in vivo) and ATP for the catalysis, as do DPOR and nitrogenase [3]. Tsukatani et al. demonstrated that variants of COR are able to catalyze the formation of C8-ethylidene groups by reduction of the C7 and C82 carbons of 3,8-divinyl Chlide (DV-Chlide) in the biosynthetic pathways of Bchls b and g [14], [15], and that COR from R. capsulatus has the additional ability to reduce the C81C82 double bond of C8 vinyl group of DV-Chlide forming to 3-vinyl Chlide (MV-Chlide) [14].
We report the determination of kinetic parameters of COR and reconstitution of conversion of Pchlide (a porphyrin) to MV-Bchlide (a bacteriochlorin) by the sequential reaction of DPOR and COR. COR from R. capsulatus shows a significantly lower Vmax value than that of DPOR, which may be partly compensated by the lower Km value than that of DPOR. In addition, detailed analysis of the sequential reaction confirmed the 8-vinyl reductase activity of COR in addition to the C7C8 double bond reduction. We also discuss the plasticity of COR and the redundant role of the 8-vinyl reduction activity in Bchl biosynthesis in R. capsulatus.
Section snippets
Strains, culture conditions and purification of the four components
Escherichia coli JM105 and the transformants harboring pHAL1 [16], pHANB1 [16], pJN1X [14] and pJN1YZ [14] were used to overexpress four components: L-protein, NB-protein, X-protein, and YZ-protein, respectively. Details are described in Supplementary materials and methods. The Bradford method (Protein Assay; Bio-Rad) was used to determine protein concentrations, with bovine serum albumin as the standard. Protein purity was monitored using SDS–PAGE.
Preparation of Pchlide and Chlide
Pchlide was prepared from the spent medium of
Determination of kinetic parameters of COR with MV-Chlide
The two components of COR, X-protein and YZ-protein, were purified from E. coli JM105 (Fig. 2A, inset, lanes 3 and 4). As previously shown [3], X-protein is an ATP-dependent electron donor to YZ-protein. YZ-protein serves as a catalytic component providing the active site for Chlide reduction. In another nitrogenase-like enzyme, DPOR, NB-protein shows its maximal activity in the presence of a 3-fold molar excess of l-protein. To estimate the molar ratio of X-protein/YZ-protein required for
Discussion
The basic skeletal structure of Bchl a is formed from two reduction reactions: one on the D-ring (C17C18), catalyzed by DPOR, and the other on the B-ring (C7C8), catalyzed by COR. Both nitrogenase-like enzymes require ATP and dithionite for catalysis, allowing reconstitution of the sequential reactions by addition of the purified enzyme subunits and the substrate Pchlide to reaction mixtures containing ATP, dithionite, and the ATP-regeneration system [2], [3].
In this study, the basic enzymatic
Acknowledgments
We thank Yusuke Tsukatani for technical help on E. coli cultivation and valuable discussion. We thank Tadashi Mizoguchi and Hitoshi Tamiaki for the preparation of DV-Pchlide. We thank Jiro Nomata and Aya Tatematsu for the construction of pJN plasmids and technical help of the DPOR assay. We thank Tsuyoshi Matsumoto and Kazuyuki Tatsumi for arrangement of an anaerobic chamber. We also thank Tatsuo Omata and Kazuki Terauchi for valuable discussion. This work was supported by Grants-in-Aid for
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Cited by (0)
- 1
Present address: Institute for Protein Research, Osaka University, Suita 565-0871, Japan.
- 2
These authors contributed equally to this work.