scholarly journals Production of aromatic amino acids by microorganisms. VIII. Regulatory properties of chorismate mutase from Corynebacterium glutamicum.

1975 ◽  
Vol 39 (2) ◽  
pp. 331-342 ◽  
Author(s):  
Hiroshi HAGINO ◽  
Kiyoshi NAKAYAMA
Keyword(s):  
Amino Acids ◽  
Corynebacterium Glutamicum ◽  
Aromatic Amino Acids ◽  
Chorismate Mutase ◽  
Regulatory Properties

scholarly journals Biochemical and Structural Characterization of the Secreted Chorismate Mutase (Rv1885c) from Mycobacterium tuberculosis H37Rv: an *AroQ Enzyme Not Regulated by the Aromatic Amino Acids

2006 ◽  
Vol 188 (24) ◽  
pp. 8638-8648 ◽  
Author(s):  
Sook-Kyung Kim ◽  
Sathyavelu K. Reddy ◽  
Bryant C. Nelson ◽  
Gregory B. Vasquez ◽  
Andrew Davis ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mycobacterium Tuberculosis ◽  
Active Site ◽  
Signal Sequence ◽  
Aromatic Amino Acids ◽  
Helical Structure ◽  
Data Bank ◽  
Chorismate Mutase ◽  
Site Directed Mutagenesis ◽  
Single Chain

ABSTRACT The gene Rv1885c from the genome of Mycobacterium tuberculosis H37Rv encodes a monofunctional and secreted chorismate mutase (*MtCM) with a 33-amino-acid cleavable signal sequence; hence, it belongs to the *AroQ class of chorismate mutases. Consistent with the heterologously expressed *MtCM having periplasmic destination in Escherichia coli and the absence of a discrete periplasmic compartment in M. tuberculosis, we show here that *MtCM secretes into the culture filtrate of M. tuberculosis. *MtCM functions as a homodimer and exhibits a dimeric state of the protein at a concentration as low as 5 nM. *MtCM exhibits simple Michaelis-Menten kinetics with a Km of 0.5 ± 0.05 mM and a k cat of 60 s−1 per active site (at 37°C and pH 7.5). The crystal structure of *MtCM has been determined at 1.7 Å resolution (Protein Data Bank identifier 2F6L). The protein has an all alpha-helical structure, and the active site is formed within a single chain without any contribution from the second chain in the dimer. Analysis of the structure shows a novel fold topology for the protein with a topologically rearranged helix containing Arg134. We provide evidence by site-directed mutagenesis that the residues Arg49, Lys60, Arg72, Thr105, Glu109, and Arg134 constitute the catalytic site; the numbering of the residues includes the signal sequence. Our investigation on the effect of phenylalanine, tyrosine, and tryptophan on *MtCM shows that *MtCM is not regulated by the aromatic amino acids. Consistent with this observation, the X-ray structure of *MtCM does not have an allosteric regulatory site.

scholarly journals Altered Oligomerization Properties of N316 Mutants of Escherichia coli TyrR

2008 ◽  
Vol 190 (24) ◽  
pp. 8238-8243 ◽  
Author(s):  
Takashi Koyanagi ◽  
Takane Katayama ◽  
Hideyuki Suzuki ◽  
Hidehiko Kumagai
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Amino Acids ◽  
Gel Filtration ◽  
Aromatic Amino Acids ◽  
Transcriptional Regulator ◽  
Wild Type ◽  
Oligomer Formation ◽  
Regulatory Properties

ABSTRACT The transcriptional regulator TyrR is known to undergo a dimer-to-hexamer conformational change in response to aromatic amino acids, through which it controls gene expression. In this study, we identified N316D as the second-site suppressor of Escherichia coli TyrRE274Q, a mutant protein deficient in hexamer formation. N316 variants exhibited altered in vivo regulatory properties, and the most drastic changes were observed for TyrRN316D and TyrRN316R mutants. Gel filtration analyses revealed that the ligand-mediated oligomer formation was enhanced and diminished for TyrRN316D and TyrRN316R, respectively, compared with the wild-type TyrR. ADP was substituted for ATP in the oligomer formation of TyrRN316D.

scholarly journals Corynebacterium glutamicum Contains 3-Deoxy-d-Arabino-Heptulosonate 7-Phosphate Synthases That Display Novel Biochemical Features

2008 ◽  
Vol 74 (17) ◽  
pp. 5497-5503 ◽  
Author(s):  
Ya-Jun Liu ◽  
Pan-Pan Li ◽  
Ke-Xin Zhao ◽  
Bao-Jun Wang ◽  
Cheng-Ying Jiang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Corynebacterium Glutamicum ◽  
Feedback Inhibition ◽  
Shikimate Pathway ◽  
Aromatic Amino Acids ◽  
Cloning And Expression ◽  
Dahp Synthase ◽  
E Coli ◽  
Phenotypic Alteration ◽  
In The Wild

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.

scholarly journals The Two Chorismate Mutases from both Mycobacterium tuberculosis and Mycobacterium smegmatis: Biochemical Analysis and Limited Regulation of Promoter Activity by Aromatic Amino Acids

2007 ◽  
Vol 190 (1) ◽  
pp. 122-134 ◽  
Author(s):  
Cristopher Z. Schneider ◽  
Tanya Parish ◽  
Luiz A. Basso ◽  
Diógenes S. Santos
Keyword(s):  
Amino Acids ◽  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Sequence Similarity ◽  
Aromatic Amino Acids ◽  
Open Reading Frames ◽  
Chorismate Mutase ◽  
Structural Class ◽  
Promising Target ◽  
Translational Start

ABSTRACT Chorismate mutase (CM) catalyzes the rearrangement of chorismate to prephenate in the biosynthetic pathway that forms phenylalanine and tyrosine in bacteria, fungi, plants, and apicomplexan parasites. Since this enzyme is absent from mammals, it represents a promising target for the development of new antimycobacterial drugs, which are needed to combat Mycobacterium tuberculosis, the causative agent of tuberculosis. Until recently, two putative open reading frames (ORFs), Rv0948c and Rv1885c, showing low sequence similarity to CMs have been described as “conserved hypothetical proteins” in the M. tuberculosis genome. However, we and others demonstrated that these ORFs are in fact monofunctional CMs of the AroQ structural class and that they are differentially localized in the mycobacterial cell. Since homologues to the M. tuberculosis enzymes are also present in Mycobacterium smegmatis, we cloned the coding sequences corresponding to ORFs MSMEG5513 and MSMEG2114 from the latter. The CM activities of both ORFs was determined, as well as their translational start sites. In addition, we analyzed the promoter activities of three M. tuberculosis loci related to phenylalanine and tyrosine biosynthesis under a variety of conditions using M. smegmatis as a surrogate host. Our results indicate that the aroQ (Rv0948c), *aroQ (Rv1885c), and fbpB (Rv1886c) genes from M. tuberculosis are constitutively expressed or subjected to minor regulation by aromatic amino acids levels, especially tryptophan.

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