scholarly journals Genetic and biochemical identification of the chorismate mutase from Corynebacterium glutamicum

Microbiology ◽  
2009 ◽  
Vol 155 (10) ◽  
pp. 3382-3391 ◽  
Author(s):  
Pan-Pan Li ◽  
Ya-Jun Liu ◽  
Shuang-Jiang Liu
Keyword(s):  
Corynebacterium Glutamicum ◽  
Shikimate Pathway ◽  
Hypothetical Protein ◽  
Chorismate Mutase ◽  
Cloning And Expression ◽  
Physical Interaction ◽  
Structural Class ◽  
Important Amino Acid ◽  
Key Enzyme ◽  
Biochemical Identification

Chorismate mutase (CM) catalyses the rearrangement of chorismate to prephenate and is also the first and the key enzyme that diverges the shikimate pathway to either tryptophan (Trp) or phenylalanine (Phe) and tyrosine (Tyr). Corynebacterium glutamicum is one of the most important amino acid producers for the fermentation industry and has been widely investigated. However, the gene(s) encoding CM has not been experimentally identified in C. glutamicum. In this study, the ncgl0819 gene, which was annotated as ‘conserved hypothetical protein’ in the C. glutamicum genome, was genetically characterized to be essential for growth in minimal medium, and a mutant deleted of ncgl0819 was a Phe and Tyr auxotroph. Genetic cloning and expression of ncgl0819 in Escherichia coli resulted in the formation of a new protein (NCgl0819) having CM activity. It was concluded that ncgl0819 encoded the CM of C. glutamicum (CM0819). CM0819 was demonstrated to be a homodimer and is a new member of the monofunctional CMs of the AroQ structural class. The CM0819 activity was not affected by Phe, Tyr or Trp. Two 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthases (DS0950 and DS2098, formerly NCgl0950 and NCgl2098) had been previously identified from C. glutamicum. CM0819 significantly stimulated DAHP synthase (DS2098) activity. Physical interaction between CM0819 and DS2098 was observed. When CM0819 was present, DS2098 activity was subject to allosteric inhibition by chorismate and prephenate. Conserved hypothetical proteins homologous to CM0819 were identified in all known Corynebacterium genomes, suggesting a universal occurrence of CM0819-like CMs in the genus Corynebacterium.

scholarly journals Functional Identification of Novel Genes Involved in the Glutathione-Independent Gentisate Pathway in Corynebacterium glutamicum

2005 ◽  
Vol 71 (7) ◽  
pp. 3442-3452 ◽  
Author(s):  
Xi-Hui Shen ◽  
Cheng-Ying Jiang ◽  
Yan Huang ◽  
Zhi-Pei Liu ◽  
Shuang-Jiang Liu
Keyword(s):  
Corynebacterium Glutamicum ◽  
Hypothetical Protein ◽  
Cloning And Expression ◽  
Functional Identification ◽  
E Coli ◽  
Transporter Protein ◽  
Genome Wide ◽  
Genes Encoding ◽  
Gentisate Pathway ◽  
Genome Wide Data

ABSTRACT Corynebacterium glutamicum used gentisate and 3-hydroxybenzoate as its sole carbon and energy source for growth. By genome-wide data mining, a gene cluster designated ncg12918-ncg12923 was proposed to encode putative proteins involved in gentisate/3-hydroxybenzoate pathway. Genes encoding gentisate 1,2-dioxygenase (ncg12920) and fumarylpyruvate hydrolase (ncg12919) were identified by cloning and expression of each gene in Escherichia coli. The gene of ncg12918 encoding a hypothetical protein (Ncg12918) was proved to be essential for gentisate-3-hydroxybenzoate assimilation. Mutant strain RES167Δncg12918 lost the ability to grow on gentisate or 3-hydroxybenzoate, but this ability could be restored in C. glutamicum upon the complementation with pXMJ19-ncg12918. Cloning and expression of this ncg12918 gene in E. coli showed that Ncg12918 is a glutathione-independent maleylpyruvate isomerase. Upstream of ncg12920, the genes ncg12921-ncg12923 were located, which were essential for gentisate and/or 3-hydroxybenzoate catabolism. The Ncg12921 was able to up-regulate gentisate 1,2-dioxygenase, maleylpyruvate isomerase, and fumarylpyruvate hydrolase activities. The genes ncg12922 and ncg12923 were deduced to encode a gentisate transporter protein and a 3-hydroxybenzoate hydroxylase, respectively, and were essential for gentisate or 3-hydroxybenzoate assimilation. Based on the results obtained in this study, a GSH-independent gentisate pathway was proposed, and genes involved in this pathway were identified.

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 Xanthomonas oryzae pv. oryzae XKK.12 Contains an AroQγ Chorismate Mutase That Is Involved in Rice Virulence

2010 ◽  
Vol 100 (3) ◽  
pp. 262-270 ◽  
Author(s):  
Giuliano Degrassi ◽  
Giulia Devescovi ◽  
Joseph Bigirimana ◽  
Vittorio Venturi
Keyword(s):  
Pathogenic Bacteria ◽  
Shikimate Pathway ◽  
Aromatic Amino Acids ◽  
Xanthomonas Oryzae ◽  
Chorismate Mutase ◽  
In Planta ◽  
Knock Out ◽  
Key Enzyme ◽  
Precise Role

Chorismate mutase (CM) is a key enzyme in the shikimate pathway which is responsible for the synthesis of aromatic amino acids. There are two classes of CMs, AroQ and AroH, and several pathogenic bacteria have been reported to possess a subgroup of CMs designated AroQγ. These CMs are usually exported to the periplasm or outside the cell; in a few cases, they have been reported to be involved in virulence and their precise role is currently unknown. Here, we report that the important rice pathogen Xanthomonas oryzae pv. oryzae XKK.12 produces an AroQγ CM which we have purified and characterized from spent supernatants. This enzyme is synthesized in planta and X. oryzae pv. oryzae knock-out mutants are hypervirulent to rice. The role of this enzyme in X. oryzae pv. oryzae rice virulence is discussed.

(De)regulation of key enzyme steps in the shikimate pathway and phenylalanine-specific pathway of the actinomycete Amycolatopsis methanolica

Microbiology ◽  
2003 ◽  
Vol 149 (11) ◽  
pp. 3321-3330 ◽  
Author(s):  
H. Kloosterman ◽  
G. I. Hessels ◽  
J. W. Vrijbloed ◽  
G. J. Euverink ◽  
L. Dijkhuizen
Keyword(s):  
Amino Acid ◽  
Protein Complex ◽  
Feedback Inhibition ◽  
Shikimate Pathway ◽  
Chorismate Mutase ◽  
Primary Metabolism ◽  
Amino Acid Residues ◽  
Dahp Synthase ◽  
Key Enzyme ◽  
Resistant Mutants

Prephenate dehydratase (PDT), chorismate mutase (CM) and 3-deoxy-d-arabino-7-heptulosonate 7-phosphate (DAHP) synthase are key regulatory enzymes in aromatic amino acid biosynthesis in the actinomycete Amycolatopsis methanolica. Deregulated, feedback-control-resistant mutants were isolated by incubation of A. methanolica on glucose mineral agar containing the toxic analogue p-fluoro-dl-phenylalanine (pFPhe). Several of these mutants had completely lost PDT sensitivity to Phe inhibition and Tyr activation. Mutant characterization yielded new information about PDT amino acid residues involved in Phe and Tyr effector binding sites. A. methanolica wild-type cells grown on glucose mineral medium normally possess a bifunctional CM/DAHP synthase protein complex (with DS1, a plant-type DAHP synthase). The CM activity of this protein complex is feedback-inhibited by Tyr and Phe, while DS1 activity is mainly inhibited by Trp. Isolation of pFPhe-resistant mutants yielded two feedback-inhibition-resistant CM mutants. These were characterized as regulatory mutants, derepressed in (a) synthesis of CM, now occurring as an abundant, feedback-inhibition-resistant, separate protein, and (b) synthesis of an alternative DAHP synthase (DS2, an E. coli-type DAHP synthase), only inhibited by Tyr and Trp. DS1 and DS2 thus are well integrated in A. methanolica primary metabolism: DS1 and CM form a protein complex, which stimulates CM activity and renders it sensitive to feedback inhibition by Phe and Tyr. Synthesis of CM and DS2 proteins appears to be controlled co-ordinately, sensitive to Phe-mediated feedback repression.

Disruption of genes for the enhanced biosynthesis of α-ketoglutarate in Corynebacterium glutamicum

2012 ◽  
Vol 58 (3) ◽  
pp. 278-286 ◽  
Author(s):  
Jae-Hyung Jo ◽  
Hye-Young Seol ◽  
Yun-Bom Lee ◽  
Min-Hong Kim ◽  
Hyung-Hwan Hyun ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Biosynthetic Pathway ◽  
Isocitrate Lyase ◽  
Glutamate Synthase ◽  
Flask Culture ◽  
Control Strain ◽  
Enhanced Production ◽  
Key Enzyme ◽  
Microbial Strains ◽  
Glyoxylate Pathway

The development of microbial strains for the enhanced production of α-ketoglutarate (α-KG) was investigated using a strain of Corynebacterium glutamicum that overproduces of l-glutamate, by disrupting three genes involved in the α-KG biosynthetic pathway. The pathways competing with the biosynthesis of α-KG were blocked by knocking out aceA (encoding isocitrate lyase, ICL), gdh (encoding glutamate dehydrogenase, l-gluDH), and gltB (encoding glutamate synthase or glutamate-2-oxoglutarate aminotransferase, GOGAT). The strain with aceA, gltB, and gdh disrupted showed reduced ICL activity and no GOGAT and l-gluDH activities, resulting in up to 16-fold more α-KG production than the control strain in flask culture. These results suggest that l-gluDH is the key enzyme in the conversion of α-KG to l-glutamate; therefore, prevention of this step could promote α-KG accumulation. The inactivation of ICL leads the carbon flow to α-KG by blocking the glyoxylate pathway. However, the disruption of gltB did not affect the biosynthesis of α-KG. Our results can be applied in the industrial production of α-KG by using C. glutamicum as producer.

scholarly journals Evidence that a Linear Megaplasmid Encodes Enzymes of Aliphatic Alkene and Epoxide Metabolism and Coenzyme M (2-Mercaptoethanesulfonate) Biosynthesis in XanthobacterStrain Py2

2001 ◽  
Vol 183 (7) ◽  
pp. 2172-2177 ◽  
Author(s):  
Jonathan G. Krum ◽  
Scott A. Ensign
Keyword(s):  
Ring Opening ◽  
Methanogenic Archaea ◽  
Hypothetical Protein ◽  
Epoxide Ring ◽  
Coenzyme M ◽  
Epoxide Ring Opening ◽  
Propylene Oxidation ◽  
Key Enzymes ◽  
Genes Encoding ◽  
Key Enzyme

ABSTRACT The bacterial metabolism of propylene proceeds by epoxidation to epoxypropane followed by a sequence of three reactions resulting in epoxide ring opening and carboxylation to form acetoacetate. Coenzyme M (2-mercaptoethanesulfonic acid) (CoM) plays a central role in epoxide carboxylation by serving as the nucleophile for epoxide ring opening and the carrier of the C3 unit that is ultimately carboxylated to acetoacetate, releasing CoM. In the present work, a 320-kb linear megaplasmid has been identified in the gram-negative bacterium Xanthobacter strain Py2, which contains the genes encoding the key enzymes of propylene oxidation and epoxide carboxylation. Repeated subculturing of Xanthobacter strain Py2 under nonselective conditions, i.e., with glucose or acetate as the carbon source in the absence of propylene, resulted in the loss of the propylene-positive phenotype. The propylene-negative phenotype correlated with the loss of the 320-kb linear megaplasmid, loss of induction and expression of alkene monooxgenase and epoxide carboxylation enzyme activities, and the loss of CoM biosynthetic capability. Sequence analysis of a hypothetical protein (XecG), encoded by a gene located downstream of the genes for the four enzymes of epoxide carboxylation, revealed a high degree of sequence identity with proteins of as-yet unassigned functions in the methanogenic archaeaMethanobacterium thermoautotrophicum andMethanococcus jannaschii and in Bacillus subtilis. The M. jannaschii homolog of XecG, MJ0255, is located next to a gene, MJ0256, that has been shown to encode a key enzyme of CoM biosynthesis (M. Graupner, H. Xu, and R. H. White, J. Bacteriol. 182: 4862–4867, 2000). We propose that the propylene-positive phenotype of Xanthobacter strain Py2 is dependent on the selective maintenance of a linear megaplasmid containing the genes for the key enzymes of alkene oxidation, epoxide carboxylation, and CoM biosynthesis.

scholarly journals Regulation of Expression of Genes Involved in Quinate and Shikimate Utilization in Corynebacterium glutamicum

2009 ◽  
Vol 75 (11) ◽  
pp. 3461-3468 ◽  
Author(s):  
Haruhiko Teramoto ◽  
Masayuki Inui ◽  
Hideaki Yukawa
Keyword(s):  
Corynebacterium Glutamicum ◽  
Aromatic Compounds ◽  
Sole Carbon Source ◽  
Shikimate Pathway ◽  
Transcriptional Regulator ◽  
Shikimate Dehydrogenase ◽  
Regulation Of Expression ◽  
Expression Of Genes ◽  
High Level ◽  
Utilization Pathway

ABSTRACT The utilization of the hydroaromatic compounds quinate and shikimate by Corynebacterium glutamicum was investigated. C. glutamicum grew well with either quinate or shikimate as the sole carbon source. The disruption of qsuD, encoding quinate/shikimate dehydrogenase, completely suppressed growth with either substrate but did not affect growth with glucose, indicating that the enzyme encoded by qsuD catalyzes the first step of the catabolism of quinate/shikimate but is not involved in the shikimate pathway required for the biosynthesis of various aromatic compounds. On the chromosome of C. glutamicum, the qsuD gene is located in a gene cluster also containing qsuA, qsuB, and qsuC genes, which are probably involved in the quinate/shikimate utilization pathway to form protocatechuate. Reverse transcriptase PCR analyses revealed that the expression of the qsuABCD genes was markedly induced during growth with either quinate or shikimate relative to expression during growth with glucose. The induction level by shikimate was significantly decreased by the disruption of qsuR, which is located immediately upstream of qsuA in the opposite direction and encodes a LysR-type transcriptional regulator, suggesting that QsuR acts as an activator of the qsuABCD genes. The high level of induction of qsuABCD genes by shikimate was still observed in the presence of glucose, and simultaneous consumption of glucose and shikimate during growth was observed.

The shikimate pathway. Part 7. Chorismate mutase: towards an enzyme model

1987 ◽  
pp. 2765 ◽  
Author(s):  
Trevor I. Richards ◽  
Keith Layden ◽  
Edward E. Warminski ◽  
Peter J. Milburn ◽  
Edwin Haslam
Keyword(s):  
Shikimate Pathway ◽  
Chorismate Mutase ◽  
Enzyme Model
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