Crystal structure of phytochromobilin synthase in complex with biliverdin IXα

Key enzyme in the biosynthesis of phytochrome

Significance 

Phytochromobilin (PΦB), a sensory pigment of red/far-red light is found in the photoreceptors in plants. The function of PΦB synthase is the catalysis of bilin’s site-specific reduction as well as the assistance of the production of PΦB from a tetrapyrrole pigment known as biliverdin. At the resolution of 1.95 Å, the tomato PΦB synthase having a crystal structure was found. It had structural similarities with FDBRs, the only difference is an extended C-terminal loop along with some small helices. It was revealed that this C-terminal loop plays a role in the interaction of biliverdin and ferredoxin. Whereas biliverdin that is bound to the tomato PΦB synthase possesses different arrangements as well as orientation compared to the biliverdin that is held together with other FDPRs. It was disclosed by the enzymatic and structural analysis that the residues of two aspartic-acid, Asp-123, and Asp-263, are vital for the biliverdin’s A-ring reduction.

To this account, Dr. Masakazu Sugishima and Professor Ken Yamamoto from Kurume University in collaboration with Dr. Kei Wada at University of Miyazaki and Emeritus Professor Keiichi Fukuyama at Osaka University explored in detail the mechanism of PΦB formation. The mechanism mediates the site-specific reduction in order to produce PΦB and a systematic process that underlies the product’s dissociation as well. The researchers examined the characteristics of recombinant tomato PΦB synthase by observing the enzymatic activity of PΦB synthase. The spectrum shifted before PΦB production which was comparable to Arabidopsis PΦB synthase. Their research work is published now in the Journal of Biological Chemistry,

The whole structure of the tomato PΦB synthase in complex with biliverdin determined the significance of extension of C-terminal. By the use of enzymatic assays and cross-linking methods, the residues taking part in the association of Ferredoxin 2 and Arabidopsis PΦB synthase were found. Being on the molecular surface the residues Lys263, Arg264, Lys183, and Arg200 are involved in the interaction with ferredoxin in a direct manner while Lys255 and Glu187 may be indirectly involved. The binding mode of biliverdin in the PΦB synthase is also observed to be located in the interior portion of enzymes.

By comparing the structure with other ‘FDBRs’, it was supported that PΦB synthase is evolved from the PebB. The long C-terminal loop interacts with biliverdin in PΦB. The two amino acids Asp123 and Asp263 seem to be involved in the reduction of A-ring and appear to be conserved in PΦB synthase of plants.

The researchers determined the crystal structure of “tomato PΦB synthase” in complex with biliverdin at the resolution of 1.95 Å. The PΦB synthase-specific sequence’s structure was found. The orientation and arrangement of biliverdin that is associated with PΦB synthase are wholly different from other known structures biliverdin associated with FDBRs. Relative to the biliverdin binding position in other FDBRs, biliverdin is slightly near the entry of biliverdin binding-pocket as the loss of association with pyrrole nitrogens present in biliverdin and residue of central aspartic-acid and product of interaction with the residue Arg316, that is definite to the PΦB synthase. It is suggested that residues of two aspartic-acids work as acidic catalysts in order to reduce A-ring.

The study by Masakazu Sugishima and colleagues is specifically important as it is first to explore the detailed mechanism of the formation of PΦB. The thorough and extensive study on the reaction mechanisms, structural comparisons and structural analysis of tomato PΦB synthase will serve to enhance the knowledge, develop better functioning synthetic enzymes and act as guide for future studies in the field.

Crystal structure of phytochromobilin synthase in complex with biliverdin IXα a key enzyme in the biosynthesis of phytochrome - Medicine Innovates

About the author

Masakazu Sugishima received his Ph.D. in biological sciences from Osaka University. Following three years post-doc, he was appointed to an assistant professor in Department of Medical Biochemistry, Kurume University School of Medicine in 2007. He also held a position of visiting scientist at Prof. Keith Moffat’s lab in the University of Chicago from 2010 to 2012. Currently he is appointed to an associate professor of the Kurume University School of Medicine.

His research has been focused on the structural biology in heme-metabolizing enzymes more than 20 years. Heme is catabolized by heme oxygenase (HO) using the reducing equivalents from cytochrome P450 reductase (CPR). He determined several reaction intermediate structure of HO, and the most notable his result is the structure determination of the CPR-HO complex. On the basis of the structure, he proposed the mechanism of the electron transfer from CPR to HO with the dynamic conformation change of CPR.

Following HO reaction, heme is catabolized to a green pigment, biliverdin. Biliverdin is reduced to a yellow pigment, bilirubin, in mammals, and phytobilins in plants and other oxygenic phototrophs.

Different enzymes are responsible for the reduction of biliverdin.

Biliverdin reductase (BVR) is responsible in mammals, whereas ferredoxin-dependent bilin reductases (FDBR) are responsible in oxygenic phototrophs. He determined the first crystal structure of the substrate complex of BVR in which two substrates were unexpectedly stacked in the active site. He also determined the first crystal structure of FDBR from cyanobacteria. He determined the crystal structure of FDBR from plants in this research.

He is a recipient of several awards, such as the young scientist awards from the Japanese Biochemical Society (2015), from the Crystallographic Society of Japan (2007), and from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (2015).

Reference

Sugishima, M., Wada, k., Fukuyama, K., Yamamoto, K. Crystal structure of phytochromobilin synthase in complex with biliverdin IXα a key enzyme in the biosynthesis of phytochrome, Journal of Biological Chemistry, (2019) 295 771-782 DOI: 10.1074/jbc.RA119.011431

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