Biochemical and Biophysical Research Communications
Structural basis for substrate specificity of meso-diaminopimelic acid decarboxylase from Corynebacterium glutamicum
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)
- et al.
Metabolic engineering of Corynebacterium glutamicum for the de novo production of ethylene glycol from glucose
Metab. Eng.
(2016) - et al.
Processing of X-ray diffraction data collected in oscillation mode
Method Enzymol.
(1997) Solvent content of protein crystals
J. Mol. Biol.
(1968)- et al.
Crystal structure of Mycobacterium tuberculosis diaminopimelate decarboxylase, an essential enzyme in bacterial lysine biosynthesis
J. Biol. Chem.
(2003) - et al.
Inference of macromolecular assemblies from crystalline state
J. Mol. Biol.
(2007) - et al.
Assessment of robustness against dissolved oxygen/substrate oscillations for C-glutamicum DM1933 in two-compartment bioreactor
Bioprocess Biosyst. Eng.
(2014) - et al.
CO2/HCO3 (-) perturbations of simulated large scale gradients in a scale-down device cause fast transcriptional responses in Corynebacterium glutamicum (vol 98, pg 8563, 2014)
Appl. Microbiol. Biot.
(2014) - et al.
Handbook of Corynebacterium Glutamicum
(2005) - et al.
A giant market and a powerful metabolism: L-lysine provided by Corynebacterium glutamicum
Appl. Microbiol. Biotechnol.
(2015) - et al.
Metabolic engineering of Corynebacterium glutamicum for production of 1,5-diaminopentane from hemicellulose
Biotechnol. J.
(2011)
Corynebacterium glutamicum tailored for efficient isobutanol production
Appl. Environ. Microbiol.
Cofactor recycling for co-production of 1,3-propanediol and glutamate by metabolically engineered Corynebacterium glutamicum
Sci. Rep.
Cited by (5)
RNA-Seq comparative analysis reveals the response of Enterococcus faecalis TV4 under fluoride exposure
2020, GeneCitation Excerpt :However, sugar ABC transporter (orf0161) was down-regulated. Meanwhile, the genes of arginine deiminase (orf2081), serine dehydratase (orf2086 and orf2087), pyrroline-5-carboxylate reductase (orf1041), dipeptidase (orf1560), diaminopimelate decarboxylase (orf0356), oligoendopetidase (orf1550), M23 peptidase (orf0703) and amino acid dehydrogenase (orf0039) were all up-regulated, and all of them were directly involved in amino acid metabolism (Cheng et al., 2015; Thoden et al., 2014; Paes et al., 2013; Hellendoorn et al., 1997; Son and Kim, 2018; Jasilionis and Kuisiene, 2015; Yang et al., 2016; Häusler et al., 2014). These results indicated that the TV4 strain began to use the amino acids and peptides as a source for carbon and energy metabolism when its PEP-PTS system was inhibited by fluoride exposure.
Structure-function analyses of two plant mesodiaminopimelate decarboxylase isoforms reveal that active-site gating provides stereochemical control
2019, Journal of Biological ChemistryCitation Excerpt :Therefore, we sought to investigate the quaternary structure of the recombinant At-DAPDC enzymes in solution. Although monomeric (5, 17), dimeric (6, 7, 9), and tetrameric (8) assemblies have been observed in crystal structures of prokaryotic orthologs of DAPDC, the quaternary structure has not yet been investigated for any plant orthologs. To address this, the quaternary structure of the two At-DAPDC enzymes were characterized by analytical ultracentrifugation.
Thermostable D-amino acid decarboxylases derived from Thermotoga maritima diaminopimelate decarboxylase
2021, Protein Engineering, Design and Selection