Corynebacterium glutamicum contains 3-deoxy-D-arabino-heptulosonate 7-phosphate synthases that display novel biochemical features

Abstract

3-Deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (EC 2.5.1.54) catalyzes the first step of the shikimate pathway that finally leads to the biosynthesis of aromatic amino acids phenylalanine (Phe), tryptophan (Trp), and tyrosine (Tyr). In Corynebacterium glutamicum ATCC 13032, two chromosomal genes, NCgl0950 (aroF) and NCgl2098 (aroG), were located that encode two putative DAHP synthases. The deletion of NCgl2098 resulted in the loss of the ability of C. glutamicum RES167 (a restriction-deficient strain derived from C. glutamicum ATCC 13032) to grow in mineral medium; however, the deletion of NCgl0950 did not result in any observable phenotypic alteration. Analysis of DAHP synthase activities in the wild type and mutants of C. glutamicum RES167 indicated that NCgl2098, rather than NCgl0950, was involved in the biosynthesis of aromatic amino acids. Cloning and expression in Escherichia coli showed that both NCgl0950 and NCgl2098 encoded active DAHP synthases. Both the NCgl0950 and NCgl2098 DAHP synthases were purified from recombinant E. coli cells and characterized. The NCgl0950 DAHP synthase was sensitive to feedback inhibition by Tyr and, to a much lesser extent, by Phe and Trp. The NCgl2098 DAHP synthase was slightly sensitive to feedback inhibition by Trp, but not sensitive to Tyr and Phe, findings that were in contrast to the properties of previously known DAHP synthases from C. glutamicum subsp. flavum. Both Co2+ and Mn2+ significantly stimulated the NCgl0950 DAHP synthase's activity, whereas Mn2+ was much more stimulatory than Co2+ to the NCgl2098 DAHP synthase's activity.

FIG. 1.

Confirmation of gene disruption of genes NCgl0950 and NCgl2098 by PCR amplification using chromosomal DNAs from the wild type or mutants as templates (lanes 1 to 4), expression of NCgl0950 and NCgl2098 in E. coli, and purification of NCgl0950 DAHP synthase and NCgl2098 DAHP synthase from recombinant E. coli cells (lanes 5 to 9). Lanes: 1, NCgl0950; 2, ΔNCgl0950; 3, ΔNCgl2098; 4, NCgl2098; 5, cellular lysate of BL21(DE3)/pET28a; 6, cellular lysate of BL21(DE3)/pET28a-NCgl0950; 7, cellular lysate of BL21(DE3)/pET28a-NCgl2098; 8, purified NCgl0950 product; 9, purified NCgl2098 product; M₁, DNA ladder (5,000, 3,000, 2,000, 1,000, 750, and 500 bp); M₂, protein molecular mass standards (90, 62, 40, 30, 20, and 12 kDa).

FIG. 2.

Growth curves of wild-type C. glutamicum RES167 and C. glutamicum RES167ΔNCgl2098 mutants. To observe the phenotypes after gene disruption or complementation, strains were cultivated in minimal medium with 10 mM glucose as the only source of carbon and energy. Symbols: ♦, RES167; ▴, RES167ΔNCgl2098; ⋄, RES167ΔNCgl2098 supplemented with NCgl2098. Data are averages ± standard deviations of the results from three parallel cultures. OD₆₀₀, optical density at 600 nm.

FIG. 3.

Feedback inhibition of NCgl0950 and NCgl2098 DAHP synthases from C. glutamicum RES167 by aromatic amino acids (Phe, Tyr, and Trp). Symbols: ♦, NCgl0950 DAHP synthase inhibited by Phe; ▪, NCgl0950 DAHP synthase inhibited by Tyr; ▴, NCgl0950 DAHP synthase inhibited by Trp; ⋄, NCgl2098 DAHP synthase inhibited by Phe; □, NCgl2098 DAHP synthase inhibited by Tyr; ▵, NCgl2098 DAHP synthase inhibited by Trp. DAHP synthase activities without the addition of aromatic amino acids were determined to be 0.0081 ± 0.0012 and 0.089 ± 0.009 μmol mg⁻¹ min⁻¹, respectively. These average activities were calculated as 100%. Data are averages of the results from three parallel determinations.

FIG. 4.

Detection of NCgl0950 transcription by RT-PCR. Lanes: M, DNA ladder (1,000, 750, 500, and 250 bp); 1, PCR with cDNAs as template; 2, PCR with genome of RES167 as template; 3, negative control.