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Molecular cloning of putative chloroplastic cysteine synthase in Leucaena leucocephala

Abstract

Cysteine biosynthesis is directed by the successive commitments of serine acetyltransferase, and O-acetylserine (thiol) lyase (OASTL) compounds, which subsequently frame the decameric cysteine synthase complex. The isoforms of OASTL are found in three compartments of the cell: the cytosol, plastid, and mitochondria. In this investigation, we first isolated putative chloroplastic OASTL (Ch-OASTL) from Leucaena leucocephala, and the Ch-OASTL was then expressed in BL21-competent Escherichia coli. The putative Ch-OASTL cDNA clone had 1,543 base pairs with 391 amino acids in its open reading frame and a molecular weight of 41.54 kDa. The purified protein product exhibited cysteine synthesis ability, but not mimosine synthesis activity. However, they both make the common α-aminoacrylate intermediate in their first half reaction scheme with the conventional substrate O-acetyl serine (OAS). Hence, we considered putative Ch-OASTL a cysteine-specific enzyme. Kinetic studies demonstrated that the optimum pH for cysteine synthesis was 7.0, and the optimum temperature was 40 °C. In the cysteine synthesis assay, the Km and kcat values were 838 ± 26 µM and 72.83 s−1 for OAS, respectively, and 60 ± 2 µM and 2.43 s−1 for Na2S, respectively. We can infer that putative Ch-OASTL regulatory role is considered a sensor for sulfur constraint conditions, and it acts as a forerunner of various metabolic compound molecules.

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Abbreviations

Acetyl-CoA:

Acetyl coenzymeA

AI:

Aliphatic index

AMBER:

Assisted model building with energy refinement

BSAS:

β-substituted alanine synthase

CAS:

β-cyanoalanine synthase

Ch-OASTL:

Chloroplastic OASTL

CSC:

Cysteine synthase complex

DMP:

2,5-dimethyl-3-pyridinol

FMO:

Fragment molecular orbital

GRAVY:

Grand average of hydropathicity

3H4P:

3-hydroxy-4-pyridone

IFIE:

Inter-fragment interaction energy

II:

Instability index

M9:

Minimal nutrient agar

NPAA:

Nonprotein amino acid

OAS:

O-acetyl serine

OASTL:

O-acetylserine (thiol) lyase

PAICS:

Parallelized ab initio calculation system

PDB:

Protein data bank

pI:

Theoretical isoelectric point

SAT:

Serine acetyltransferase

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Acknowledgements

We are thankful to Dr. Shinichi Gima of the Instrumental Research Center, University of the Ryukyus and Dr. Michael Chandra Roy, Okinawa Institute of Science and Technology for giving specialized help amid the LC–MS/MS analyses. We sincerely thank to Dr. Rafiq Islam (Professor, Department of Natural Sciences, Northwest Missouri State University) for useful discussion and necessary corrections of our manuscript. The author also thank Dr. Takeshi Ishikawa, Nagasaki University Graduate School of Biomedical Sciences for providing us the PAICS program.

Author information

MHR and SO equally contributed in this experiment. MHR performed all the experiment, data analysis and draft of the manuscript; SO performed the molecular dynamics simulation; SP performed the experiments and analyzed the data; FM supervised the student, developed the concepts and designed experiments, and edited the manuscript; MAH and HI supervised the student; MI guided MHR in the experiment and edited the manuscript, and HO supervised the student, performed the molecular dynamics simulation and analysis, and edited the manuscript.

Correspondence to Masakazu Fukuta.

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Accession numbers

The information of protein sequences can be found in Gene bank under the following accessions: 5JJC (crystal structure of double mutant (Q96A-Y125A) O-acetyl serine sulfhydralase from Brucella abortus), 1z7w (the crystal structure of O-acetylserine sulfhydrylase of Arabidopsis thaliana), LC342269 (Ch-OASTL of Leucaena leucocephala), BAK38374 (cysteine synthase, Mimosa pudica), LC306827 (Cy-OASTL, Leucaena leucocephala), and AHG97874 (Cy-O-acetylserine thiol lyase, Leucaena leucocephala).

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Harun-Ur-Rashid, M., Oogai, S., Parveen, S. et al. Molecular cloning of putative chloroplastic cysteine synthase in Leucaena leucocephala. J Plant Res 133, 95–108 (2020) doi:10.1007/s10265-019-01158-y

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Keywords

  • α-Aminoacrylate
  • Active site
  • 2,5-Dimethyl-3-pyridinol
  • O-acetylserine (thiol) lyase
  • Chloroplastic OASTL
  • Cysteine synthase complex