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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 K m and k cat values were 838 ± 26 µM and 72.83 s -1 for OAS, respectively, and 60 ± 2 µM and 2.43 s -1 for Na 2 S, 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.

Mimosinase degrades the non-protein amino acid mimosine and is thought to have evolved from cystathionine β-lyase (CBL) via gene duplication. However, no study has, to date, compared the molecular characteristics of mimosinase and CBL. We therefore cloned mimosinase and CBL from the Mimosoideae subfamily member Mimosa pudica (Mp) and explored the molecular relationship between mimosinase and CBL for the first time. The recombinant Mp mimosinase degraded both mimosine and cystathionine with a much higher turnover number (k cat ) for mimosine compared with cystathionine, and Mp CBL utilized only cystathionine as a substrate. The critical residues implicated in the substrate binding of Arabidopsis thaliana CBL (Tyr-127, Arg-129, Tyr-181, and Arg-440) were highly conserved in both Mp mimosinase and CBL. However, homology modeling and molecular simulation of these enzymes predicted variations in the residues that interact with substrates. A mutation experiment on Mp mimosinase revealed that the disruption of a disulfide bond in the vicinity of the pyridoxal-5'-phosphate domain increased the enzyme's preference toward cystathionine. Treatment of Mp mimosinase with a disulfide-cleavage agent also decreased mimosinase activity. Furthermore, mutation near the conserved binding residue altered the substrate preference between mimosine and cystathionine. Molecular dynamics simulations of Mp mimosinase suggested a closer coordination of the residues that interact with mimosine at the active site compared with cystathionine, indicating a more compact pocket size for mimosine degradation. This study thus may provide new insights into the molecular diversification of CBL, a C-S lyase, into the C-N lyase mimosinase in the Mimosoideae subfamily.

In the cysteine and mimosine biosynthesis process, O-acetyl-L-serine (OAS) is the common substrate. In the presence of O-acetylserine (thiol) lyase (OASTL, cysteine synthase) the reaction of OAS with sulfide produces cysteine, while with 3-hydroxy-4-pyridone (3H4P) produces mimosine. The enzyme OASTL can either catalyze Cys synthesis or both Cys and mimosine. A cDNA for cytosolic OASTL was cloned from M. pudica for the first time containing 1,410 bp nucleotides. The purified protein product from overexpressed bacterial cells produced Cys only, but not mimosine, indicating it is Cys specific. Kinetic studies revealed that pH and temperature optima for Cys production were 6.5 and 50 °C, respectively. The measured Km, Kcat, and Kcat Km -1 values were 159 ± 21 µM, 33.56 s -1 , and 211.07 mM -1 s -1 for OAS and 252 ± 25 µM, 32.99 s -1 , and 130.91 mM -1 s -1 for Na 2 S according to the in vitro Cys assay. The Cy-OASTL of Mimosa pudica is specific to Cys production, although it contains sensory roles in sulfur assimilation and the reduction network in the intracellular environment of M. pudica.

Three isoprene synthase (IspS) cDNA clones have been isolated from tropical trees (Ficus septica, F. virgata, and Casuarina equisetifolia), and their enzyme properties have been compared with those of Populus alba IspS. Phylogenetic analysis of the deduced amino acid sequences with known monoterpene synthase resolved IspS from F. septica and F. virgata and other IspSs in a clade together with TPS-b clade I, whereas IspS from C. equisetifolia was within another clade, sister to TPS-b clade II. The optimum reaction temperature was 40 °C for the IspSs isolated from the tropical trees, and 45 °C for P. alba IspS. The optimum pH of the IspSs from the tropical trees peaked between pH 8 and pH10 contrasting with the rather broad optimum pH (7.5-10.5) of P. alba IspS. IspSs from F. septica and F. virgata were activated solely by Mg(2+), whereas IspS from C. equisetifolia was dependent more on Mn(2+) than on Mg(2+). Michaelis constant (Km) values of IspSs from tropical trees were lower than that of P. alba IspS. Analysis of inter fragment interaction energy of IspS-substrate complex model and crystal structure of bornyl diphosphate synthase (1N20) found that the coordination geometry of amino acids with higher attraction force is similar at the active site of C. equisetifolia IspS and bornyl diphosphate synthase. These observations suggest the occurrence of another group of IspSs in TPS-b subfamily and extend the knowledge on biochemical regulatory mechanism of isoprene emission from tropical trees.