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Applied Biochemistry and Biotechnology

, Volume 186, Issue 3, pp 613–632 | Cite as

Cytosolic Cysteine Synthase Switch Cysteine and Mimosine Production in Leucaena leucocephala

  • Md. Harun-Ur-Rashid
  • Hironori Iwasaki
  • Shahanaz Parveen
  • Shigeki Oogai
  • Masakazu Fukuta
  • Md. Amzad Hossain
  • Toyoaki Anai
  • Hirosuke Oku
Article

Abstract

In higher plants, multiple copies of the cysteine synthase gene are present for cysteine biosynthesis. Some of these genes also have the potential to produce various kinds of β-substitute alanine. In the present study, we cloned a 1275-bp cDNA for cytosolic O-acetylserine(thiol)lyase (cysteine synthase) (Cy-OASTL) from Leucaena leucocephala. The purified protein product showed a dual function of cysteine and mimosine synthesis. Kinetics studies showed pH optima of 7.5 and 8.0, while temperature optima of 40 and 35 °C, respectively, for cysteine and mimosine synthesis. The kinetic parameters such as apparent Km, kcat were determined for both cysteine and mimosine synthesis with substrates O-acetylserine (OAS) and Na2S or 3-hydroxy-4-pyridone (3H4P). From the in vitro results with the common substrate OAS, the apparent kcat for Cys production is over sixfold higher than mimosine synthesis and the apparent Km is 3.7 times lower, suggesting Cys synthesis is the favored pathway.

Keywords

α-aminoacrylate β-cyanoalanine synthase β-substituted alanine synthase Cysteine 2,5-dimethyl-3-pyridinol O-acetylserine(thiol)lyase Mimosine 

Notes

Acknowledgements

We are grateful to Dr. Shinichi Gima (Instrumental Research Center, University of the Ryukyus) and Dr. Michael Chandro Roy (Okinawa Institute of Science and Technology) for providing technical assistance during LC-MS/MS analyses. We sincerely thank Dr. Rafiq Islam (Professor, Department of Natural Sciences, Northwest Missouri State University) for useful discussion and necessary corrections of our manuscript, and Dr. Steven D. Aird (OIST Graduate University, Okinawa, Japan) for editing the manuscript. The authors also thank Dr. Takeshi Ishikawa (Nagasaki University Graduate School of Biomedical Sciences) for providing us the PAICS program.

Author Contributions

M.H.R. and H.I. contributed equally to this work; S.O. performed the molecular dynamics simulation; S.P. performed the experiments, analyzed the data and writing manuscript; F.M. supervised the student, developed the concepts and designed experiments, and edited the manuscript; M.A.H. and T.A. supervised the student; and H.O. supervised the student, performed the molecular dynamics simulation and analysis, and edited the manuscript.

Compliance with Ethical Standards

Conflicts of Interest

The authors declare that they have no conflict of interest.

Supplementary material

12010_2018_2745_MOESM1_ESM.pptx (689 kb)
Fig. S1 Purified L. leucocephala cytosolic OASTL from E. coli separated on a 10% polyacrylamide gel. Lane M is protein marker and lane P is purified Cy-OASTL of L. leucocephala from E. coli. The area circled in red is the target one (PPTX 688 kb)
12010_2018_2745_MOESM2_ESM.pptx (2.6 mb)
Fig. S2 Three-dimensional structure of the putative active site prediction of Cy-OASTL from L. leucocephala. (A) Green and the blue carbon skeleton denotes the active site along the substrate environment of the crystal structure protein, 1z7y, and 3vc3 respectively. The crystal structure of the 1z7y active site contains cysteine as a substrate, whereas 3vc3 contains methionine. (B) Putative active site of Cy-OASTL from L. leucocephala contains OAS intermediate (α-aminoacrylate) as a substrate. All three structures’ active sites are mostly similar in nature, except the substrate binding site. All figures were generated by PyMol (PPTX 2693 kb)
12010_2018_2745_MOESM3_ESM.pptx (3.3 mb)
Fig. S3 (A) The position of the Cy-OASTL active site forming a Schiff base with Lys 49 and PLP. The position of TSGN loop interacting residues are shown in magenta, cyan, deep blue, and yellow, respectively. All the residues are shown in stick form. (B) Residues interacting with the active sites corresponding to the Fig. 9 data are shown in stick form. All figures were generated by PyMol (PPTX 3367 kb)
12010_2018_2745_MOESM4_ESM.docx (14 kb)
Table S1 (DOCX 14 kb)

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Md. Harun-Ur-Rashid
    • 1
    • 2
  • Hironori Iwasaki
    • 3
  • Shahanaz Parveen
    • 1
    • 2
    • 3
  • Shigeki Oogai
    • 1
  • Masakazu Fukuta
    • 4
    • 5
  • Md. Amzad Hossain
    • 4
  • Toyoaki Anai
    • 6
  • Hirosuke Oku
    • 3
  1. 1.The United Graduate School of Agricultural SciencesKagoshima UniversityKagoshimaJapan
  2. 2.Faculty of AgricultureSher-e-Bangla Agricultural UniversityDhakaBangladesh
  3. 3.Tropical Biosphere Research CenterUniversity of the RyukyusOkinawaJapan
  4. 4.Graduate School of AgricultureUniversity of the RyukyusOkinawaJapan
  5. 5.Department of Subtropical Biochemistry and Biotechnology, Faculty of AgricultureUniversity of the RyukyusOkinawaJapan
  6. 6.Faculty of AgricultureSaga UniversitySagaJapan

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