Characterization and Molecular Mechanism of AroP as an Aromatic Amino Acid and Histidine Transporter in Corynebacterium glutamicum

Abstract

bCorynebacterium glutamicum is equipped with abundant membrane transporters to adapt to a changing environment. Many amino acid transporters have been identified in C. glutamicum, but histidine uptake has not been investigated in detail. Here, we identified the aromatic amino acid transporter encoded by aroP as a histidine transporter in C. glutamicum by a combination of the growth and histidine uptake features. Characterization of histidine uptake showed that AroP has a moderate affinity for histidine, with a K m value of 11.40 ؎ 2.03 M, and histidine uptake by AroP is competitively inhibited by the aromatic amino acids. Among the four substrates, AroP exhibits a stronger preference for tryptophan than for tyrosine, phenylalanine, and histidine. Homology structure modeling and molecular docking were performed to predict the substrate binding modes and conformational changes during substrate transport. These results suggested that tryptophan is best accommodated in the binding pocket due to shape compatibility, strong hydrophobic interactions, and the lowest binding energy, which is consistent with the observed substrate preference of AroP. Furthermore, the missense mutations of the putative substrate binding sites verified that Ser24, Ala28, and Gly29 play crucial roles in substrate binding and are highly conserved in the Gram-positive bacteria. Finally, the expression of aroP is not significantly affected by extracellular histidine or aromatic amino acids, indicating that the physiological role of AroP may be correlated with the increased fitness of C. glutamicum to assimilate extracellular amino acid for avoiding the high energy cost of amino acid biosynthesis.

A novel pyruvate kinase and its application in lactic acid production under oxygen deprivation in Corynebacterium glutamicum

“…The cDNA from approximately 300 ng of RNA was prepared using the specific primers listed in Table S4 (Additional file 1: Table S4) and the FastQuant RT Kit (Tiangen, China). The rpoB gene, which encodes RNA polymerase β subunit, was used as the reference gene [43]. The primer pair WZ1171 and WZ1156 was used to amplify the ldhA-pyk2 region in the RT-PCR.…”

YjeH Is a Novel Exporter of l -Methionine and Branched-Chain Amino Acids in Escherichia coli

“…It is a general phenomenon that multiple transport systems share responsibility for the same substrate. For instance, in C. glutamicum, both PheP and AroP are responsible for L-phenylalanine uptake (6,52). In E. coli, YdeD, YfiK, CydDC, and Bcr are involved in…”

“…Cells were cultured as described above. Membrane proteins were extracted as in the method reported previously (6). The total protein concentration in the membrane fraction was ana- lyzed by bicinchoninic acid (BCA) assay according to the manufacturer's instructions (Solarbio).…”

Reconstruction of tricarboxylic acid cycle in Corynebacterium glutamicum with a genome‐scale metabolic network model for trans‐4‐hydroxyproline production

“…The E. coli strain EC135 was used as the cloning host and pXMJ19 and pK18mobsacB were applied for gene overexpression with the induction of isopropyl-β-D-thiogalactopyranoside (IPTG) and gene deletion, respectively (Shang et al, 2013). The E. coli strain EC135 was used as the cloning host and pXMJ19 and pK18mobsacB were applied for gene overexpression with the induction of isopropyl-β-D-thiogalactopyranoside (IPTG) and gene deletion, respectively (Shang et al, 2013).…”

“…The wild-type (WT) C. glutamicum ATCC13032 (American Type and Culture Collection, Manassas, VA) was used as the parent strain for strain engineering. The E. coli strain EC135 was used as the cloning host and pXMJ19 and pK18mobsacB were applied for gene overexpression with the induction of isopropyl-β-D-thiogalactopyranoside (IPTG) and gene deletion, respectively (Shang et al, 2013). All of the strains and plasmids used in this study are listed in Table 1.…”