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J. Biol. Chem., Vol. 281, Issue 21, 15021-15028, May 26, 2006

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A Second Nitrogenase-like Enzyme for Bacteriochlorophyll Biosynthesis

RECONSTITUTION OF CHLOROPHYLLIDE a REDUCTASE WITH PURIFIED X-PROTEIN (BchX) AND YZ-PROTEIN (BchY-BchZ) FROM RHODOBACTER CAPSULATUS^*

Jiro Nomata, Tadashi Mizoguchi, Hitoshi Tamiaki, and Yuichi Fujita¹

From the Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan and the Department of Bioscience and Biotechnology, Ritsumeikan University, Kusatsu 525-8577, Japan

In most photosynthetic organisms, the chlorin ring structure of chlorophyll a is formed by the reduction of the porphyrin D-ring by the dark-operative nitrogenase-like enzyme, protochlorophyllide reductase (DPOR). Subsequently, the chlorin B-ring is reduced in bacteriochlorophyll biosynthesis to form a bacteriochlorin ring structure. Phenotypic analysis of mutants lacking one of three genes, bchX, bchY, or bchZ, which show significant sequence similarity to the structural genes of nitrogenase, suggests that a second nitrogenase-like enzyme is involved in the chlorin B-ring reduction. However, there is no biochemical evidence for this. Here, we report the reconstitution of chlorophyllide a reductase (COR) with purified proteins. Two Rhodobacter capsulatus strains that overexpressed Strep-tagged BchX and BchY were isolated. Strep-tagged BchX was purified as a single polypeptide, and BchZ was co-purified with Strep-tagged BchY. When BchX and BchY-BchZ components were incubated with chlorophyllide a, ATP, and dithionite under anaerobic conditions, chlorophyllide a was converted to a new pigment with a Qy band of longer wavelength at 734 nm (P734) in 80% acetone. The formation of P734 was dependent on ATP and dithionite. High performance liquid chromatography and mass spectroscopic analysis indicated that P734 is 3-vinyl bacteriochlorophyllide a, which is formed by the B-ring reduction of chlorophyllide a. These results demonstrate that the B-ring of chlorin is reduced by a second nitrogenase-like enzyme and that the sequential actions of two nitrogenase-like enzymes, DPOR and COR, convert porphyrin to bacteriochlorin. The evolutionary implications of nitrogenase-like enzymes to determine the ring structure of (bacterio)chlorophyll pigments are discussed.

Received for publication, February 23, 2006 , and in revised form, March 28, 2006.

^* This work was supported by Grants-in-aid for Scientific Research Nos. 15570033, 14390051, and 13CE2005 and the 21st Century COE Program (to Y. F.); Nos. 17029065 and 15350107 (to H.T.); and No. 17750167 (to T. M.) and by an "Academic Frontier" Project for Private Universities (to H. T.), a matching fund subsidy, from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This article was selected as a Paper of the Week.

¹ To whom correspondence should be addressed. Tel.: 81-52-789-4105; Fax: 81-52-789-4107; E-mail: fujita{at}agr.nagoya-u.ac.jp.

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