Structural insights into the catalysis and substrate specificity of cyanobacterial aspartate racemase McyF

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Highlights

  • We determined the crystal structure of McyF, the amino acids racemase.

  • We also determined structures in complex with different substrates.

  • Structures and biochemical assays enabled us to identify key residues.

  • L- and D-MeAsp were proved to be the substrates for the first time.

Abstract

L-amino acids represent the most common amino acid form, most notably as protein residues, whereas D-amino acids, despite their rare occurrence, play significant roles in many biological processes. Amino acid racemases are enzymes that catalyze the interconversion of L- and/or D-amino acids. McyF is a pyridoxal 5′-phosphate (PLP) independent amino acid racemase that produces the substrate D-aspartate for the biosynthesis of microcystin in the cyanobacterium Microcystis aeruginosa PCC7806. Here we report the crystal structures of McyF in complex with citrate, L-Asp and D-Asp at 2.35, 2.63 and 2.80 Å, respectively. Structural analyses indicate that McyF and homologs possess highly conserved residues involved in substrate binding and catalysis. In addition, residues Cys87 and Cys195 were clearly assigned to the key catalytic residues of “two bases” that deprotonate D-Asp and L-Asp in a reaction independent of PLP. Further site-directed mutagenesis combined with enzymatic assays revealed that Glu197 also participates in the catalytic reaction. In addition, activity assays proved that McyF could also catalyze the interconversion of L-MeAsp between D-MeAsp, the precursor of another microcystin isoform. These findings provide structural insights into the catalytic mechanism of aspartate racemase and microcystin biosynthesis.

Introduction

All α-amino acids but glycine exist in either of the two enantiomers, termed D- and L-enantiomers. L-amino acids represent the most common form of protein residues, whereas D-amino acids play significant roles in many biological processes despite their rare occurrence. For instance, the bacterial D-glutamic acid and D-alanine are essential components of cell-wall peptidoglycan [1,2]. The mammalian D-serine is a neuromodulator and D-aspartate is indispensable for fetal brain development [3,4]. Amino acid racemases catalyze the production of D-amino acid from its L-form, either dependent on or independent of the cofactor pyridoxal 5′-phosphate (PLP). For PLP-independent amino acid racemases, such as glutamate, proline, and aspartate racemases [5], it was proposed that two conserved cysteines work as the catalytic acids/bases [6]. Notably, recent reports of Escherichia coli aspartate/glutamate racemase crystal structures suggested that Thr83/Cys197, instead of two cysteines, function as the catalytic residues [7,8]. However, the fine catalytic mechanism and substrate specificity of amino acid racemases remain unclear.

The biosynthesis of microcystin in Microcystis aeruginosa PCC7806 is catalyzed by a series of proteins encoded by the mcy gene cluster [9,10]. McyF, which is encoded by mcyF gene, was reported to be an aspartate racemase [11]. In fact, M. aeruginosa PCC7806 produces two isoforms of microcystin, which employ either D-Asp or d-β-methyl-aspartate (D-MeAsp) as the precursor [12]. D-Asp has been proved to be the product of McyF from L-Asp [11]; however, whether McyF contributes to the generation of another precursor D-MeAsp remains unclear. Moreover, there is no direct evidence for the substrate specificity and catalytic mechanism of McyF.

Here we determined the crystal structures of McyF in complex with citrate, L-Asp and D-Asp, respectively. These structures enabled us to assign Cys87 and Cys195 to function as the previously proposed “two bases” [6]. In addition, the highly conserved residue Glu197 was identified to assist Cys195 in the racemase reaction of the interconversion of L-Asp and D-Asp. Furthermore, with biochemical assays we proved that McyF is capable of catalyzing the interconversion of L-MeAsp and D-MeAsp.

Section snippets

Cloning, expression and purification

The coding sequence of McyF was amplified from the genomic DNA of M. aeruginosa PCC 7806, cloned into a pET-28a-derived expression vector (Novagen) with an N-terminal 6 × His tag and overexpressed in E. coli strain BL21 (DE3) (Novagen) using 2 × YT culture medium with 30 μg/mL kanamycin. Bacteria were grown at 37 °C to an absorbance of 0.8 at 600 nm and then induced with 0.2 mM isopropyl-β-D-1-thiogalactopyranoside for an additional 4 h. The bacteria were harvested and resuspended with lysis

Overall structure of McyF

We determined the crystal structures of seleno-methionine substituted McyF (SeMet-McyF) from M. aeruginosa PCC7806 in complex with a citrate at 2.35 Å (Table 1). The full-length McyF was applied for crystallization; however, only the segment of residues Lys4–Lys232 could be traced in the electron density map. The crystal belongs to the space group P3221 and each asymmetric unit of structure contains one molecule (Fig. 1A), although gel-filtration profile indicated that McyF exists as a dimer in

Discussion

We solved three crystal structures of McyF in complex with the substrates or analogues. Structural analyses combined with activity assays enabled us to clearly assign the substrate-binding and catalytic residues. The racemization activity assays combined with structural analyses illustrated the substrate specificity of McyF and provide insight into the catalytic mechanism of the aspartate racemase. The biochemical assays proved that McyF is capable of catalyzing the interconversion of L-MeAsp

Statement of conflict of interest

The authors declare no conflict of interest.

Author contributions

D.D.C., C.P.Z, Y.C., and C.Z.Z. designed the study; D.D.C. and C.P.Z performed experiments; D.D.C., K.Z., Y.L.J., X.F.T., J.X., Y.C., C.Z.Z. and W.T.H analyzed the data; Y.M.R. provided reagents; D.D.C., Y.C., C.Z.Z. and W.T.H wrote the manuscript.

Acknowledgments

We thank Rui-Han Dai from School of Chemistry and Materials Science, University of Science and Technology of China for the synthesis of L-/D-MetAsp. This work was supported by National Natural Science Foundation of China (Grants No. 31630001, 31370757, and 31500602), and Ministry of Education of the People’s Republic of China (Grant No. 20133402110023). We appreciate the assistance of the staff at the Shanghai Synchrotron Radiation Facility (SSRF) and the Core Facility Center for Life Sciences

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    • Microcystin biosynthesis and toxic effects

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      Citation Excerpt :

      Cys-87 and Cys-195 are two key catalytic residues, in addition to Glu-197 [19]. Furthermore, McyF participates in the reversible conversion of L-MeAsp into D-MeAsp [82]. In Anabaena strain 90, mcyF (756 bp) is located 42 bp downstream of the mcyE stop codon and encodes a polypeptide with similarity to an aspartic racemase that consists of 252 amino acids and has a predicted molecular weight of about 28.4 kDa [63].

    1

    These authors contribute equally to this work.

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