Structural basis for substrate specificity of meso-diaminopimelic acid decarboxylase from Corynebacterium glutamicum

https://doi.org/10.1016/j.bbrc.2017.11.097Get rights and content

Highlights

  • We determined a crystal structure of meso-Diaminopimelic acid decarboxylase from Corynebacterium glutamicum (CgDAPDC).

  • Binding mode of the PLP cofactor of CgDAPDC were elucidated.

  • Substrate binding mode of CgDAPDC were elucidated.

Abstract

l-lysine is an essential amino acid that is widely used as a food supplement for humans and animals. meso-Diaminopimelic acid decarboxylase (DAPDC) catalyzes the final step in the de novo l-lysine biosynthetic pathway by converting meso-diaminopimelic acid (meso-DAP) into l-lysine by decarboxylation reaction. To elucidate its molecular mechanisms, we determined the crystal structure of DAPDC from Corynebacterium glutamicum (CgDAPDC). The PLP cofactor is bound at the center of the barrel domain and forms a Schiff base with the catalytic Lys75 residue. We also determined the CgDAPDC structure in complex with both pyridoxal 5′-phosphate (PLP) and the l-lysine product and revealed that the protein has an optimal substrate binding pocket to accommodate meso-DAP as a substrate. Structural comparison of CgDAPDC with other amino acid decarboxylases with different substrate specificities revealed that the position of the α15 helix in CgDAPDC and the residues located on the helix are crucial for determining the substrate specificities of the amino acid decarboxylases.

Introduction

Corynebacterium glutamicum is a gram-positive, aerobic, and rod-shaped bacterium. It has been traditionally used in biotechnological research, because it is easy to handle, safe, and capable of large-scale fermentation [1], [2], [3], [4]. C. glutamicum has been widely used for producing amino acids such as l-glutamate and l-lysine; recently, the use of C. glutamicum has been extended to the production of other chemicals such as cadaverine, ethylene glycol, and isobutanol [5], [6], [7], [8].

l-Lysine is an essential amino acid that has been widely used as a feed additive with l-threonine, l-tryptophan, and l-methionine. Because of its potential to be converted into other high value bio-molecules such as diaminopentane and poly-ε-lysine [9], [10], [11], l-lysine is the fastest growing amino acid in global market [4], [12]. The de novo l-lysine biosynthetic pathway is classified into four pathways, namely, succinylase, acetylase, dehydrogenase, and aminotransferase pathways [13], [14], [15]. In bacteria, the succinylase pathway is commonly used, and C. glutamicum synthesizes l-lysine through the succinylase and dehydrogenase pathways [16]. All four de novo l-lysine biosynthetic pathways lead to production of meso-diaminopimelic acid (meso-DAP), which is converted into l-lysine by meso-DAP decarboxylase (DAPDC) through a decarboxylation reaction (Fig. 1A).

Here, we report the crystal structure of DAPDC from C. glutamicum (CgDAPDC) in complex with the PLP cofactor and the l-lysine product. Using the identified crystal structures, the binding mode of the PLP cofactor and substrate was elucidated. Biochemical studies using the DAPDC mutants were used to confirm the results. These results could be used in protein engineering to increase the activity of CgDAPDC to improve l-lysine productivity.

Section snippets

Cloning, expression and purification of CgDAPDC

The CgDAPDC coding gene (Gene accession code: X07563) was amplified from Corynebacterium glutamicum chromosomal DNA by a polymerase chain reaction (PCR). The PCR products were then sub-cloned into pET30a (Novagen) with 6x-His tag at the C-terminus. The resulting expression vector pET30a:CgDAPDC was transformed into E. coli BL21(DE3)-T1R strain, which was grown to an OD600 of 0.6 in LB medium containing 50 mg L−1 of kanamycin at 310 K and CgDAPDC protein expression was induced by adding 1 mM

Overall structure of CgDAPDC

To elucidate the molecular mechanism of CgDAPDC, we determined its crystal structure at a 2.40 Å resolution. The refined structure was in good agreement with the X-ray crystallographic statistics for bond angles, bond lengths, and other geometric parameters (Table 1). In an asymmetric unit, there are two CgDAPDC molecules forming a dimer (Fig. 2A). The size-exclusion chromatography experiment also showed that CgDAPDC exists as a dimer in solution (Fig. 2B), indicating that the protein functions

Acknowledgement

This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean Government (MSIP) (NRF-2014M1A2A2033626 and NRF-2017R1A2B4003809) and was also supported by the New & Renewable Energy Core Technology Program of the Korea Institute of Energy Technology Evaluation and Planning (KETEP) granted financial resource from the Ministry of Trade, Industry & Energy, Republic of Korea (20153030091360). H-F Son was supported by the NRF-2015-Global PhD Fellowship Program of

References (25)

  • B. Blombach et al.

    Corynebacterium glutamicum tailored for efficient isobutanol production

    Appl. Environ. Microbiol.

    (2011)
  • J. Huang et al.

    Cofactor recycling for co-production of 1,3-propanediol and glutamate by metabolically engineered Corynebacterium glutamicum

    Sci. Rep.

    (2017)
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