A novel noncovalent complex of chorismate mutase and DAHP synthase from<i>Mycobacterium tuberculosis</i>: protein purification, crystallization and X-ray diffraction analysis

Ökvist, Mats; Sasso, Severin; Roderer, Kathrin; Kast, Peter; Krengel, Ute

doi:10.1107/s1744309109035878

Cited by 8 publications

(12 citation statements)

References 22 publications

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“…A similar functional reliance between the DAH7PS and CM is observed in the noncovalent DAH7PS-CM complex found in Corynebacterium glutamicum ( Cgl DAH7PS-CM) (27) and Mycobacterium tuberculosis ( Mtu DAH7PS-CM) (28). In the Mtu DAH7PS-CM complex, CM is activated upon complexation with DAH7PS and inhibited by the binding of allosteric inhibitors, Tyr and Phe, at allosteric binding sites within each DAH7PS subunit (29-31).…”

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confidence: 54%

“…In the MtuDAH7PS-CM complex, CM is activated upon complexation with DAH7PS and inhibited by the binding of allosteric inhibitors, Tyr and Phe, at allosteric binding sites within each DAH7PS subunit (29)(30)(31). The structure of the MtuDAH7PS-CM complex showed that the DAH7PS and CM subunits interact with each other extensively and form a stable interface (Figure 1B) (28,30). Molecular dynamics simulations suggest that the interface between DAH7PS and CM subunits is noticeably more flexible when the inhibitors are bound, which may result in a less stable DAH7PS-CM interaction (32), suggesting a crucial role of this stable interface for delivering CM function.…”

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confidence: 99%

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Reciprocal allostery arising from a bi-enzyme assembly controls aromatic amino acid biosynthesis inPrevotella nigrescens

Bai

Parker

2021

Preprint

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Modular protein assembly has been widely reported as a mechanism for constructing allosteric machinery. Recently, a distinctive allosteric system has been identified in a bi-enzyme assembly comprising a 3-deoxy-D-arabino heptulosonate-7-phosphate synthase (DAH7PS) and chorismate mutase (CM). These enzymes catalyze the first and branch point reactions of aromatic amino acid biosynthesis in the bacterium Prevotella nigrescens (PniDAH7PS), respectively. The interactions between these two distinct catalytic domains support functional inter-reliance within this bifunctional enzyme. The binding of prephenate, the product of CM-catalyzed reaction, to the CM domain is associated with a striking rearrangement of overall protein conformation that alters the interdomain interactions and allosterically inhibits the DAH7PS activity. In this study, we observed allosteric activation of CM activity in the presence of all DAH7PS substrates. Using small angle X-ray scattering (SAXS) experiments we show that changes in overall protein conformations and dynamics are associated with the presence of different DAH7PS substrates and the allosteric inhibitor prephenate. Furthermore, we have identified an extended interhelix loop located in CM domain, loopC320-F333, as a crucial segment for the interdomain structural and catalytic communications. Our results suggest that the dual function enzyme PniDAH7PS contains a reciprocal allosteric system between the two enzymatic moieties, as a result of this bidirectional interdomain communication. This arrangement allows for a complex feedback and feedforward system for control of pathway flux by connecting the initiation and branch point of aromatic amino acid biosynthesis.

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confidence: 54%

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confidence: 99%

Reciprocal allostery arising from a bi-enzyme assembly controls aromatic amino acid biosynthesis inPrevotella nigrescens

Bai

Parker

2021

Preprint

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“…Native MtCM (encoded on plasmid pKTCMM-H), native MtDS (pKTDS-H), His 6 -MtDS (pKTDS-HN), and His 6 -MtDS-R256A (pKTDS-HN-R256A) were produced and purified by essentially following published protocols [28,67], with the variations described in the Supplementary Information to optimize yields.…”

Section: Protein Production and Purificationmentioning

confidence: 99%

Remote Control by Inter-Enzyme Allostery: A Novel Paradigm for Regulation of the Shikimate Pathway

Munack

Roderer

Ökvist

et al. 2016

Journal of Molecular Biology

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DAHP synthase and chorismate mutase catalyze key steps in the shikimate biosynthetic pathway en route to aromatic amino acids. In Mycobacterium tuberculosis, chorismate mutase (MtCM; Rv0948c), located at the branch point toward phenylalanine and tyrosine, has poor activity on its own. However, it is efficiently activated by the first enzyme of the pathway, DAHP synthase (MtDS; Rv2178c), through formation of a non-covalent MtCM-MtDS complex. Here, we show how MtDS serves as an allosteric platform for feedback regulation of both enzymes, using X-ray crystallography, small-angle X-ray scattering, size-exclusion chromatography, and multi-angle light scattering. Crystal structures of the fully inhibited MtDS and the allosterically down-regulated MtCM-MtDS complex, solved at 2.8 and 2.7Å, respectively, reveal how effector binding at the internal MtDS subunit interfaces regulates the activity of MtDS and MtCM. While binding of all three metabolic end products to MtDS shuts down the entire pathway, the binding of phenylalanine jointly with tyrosine releases MtCM from the MtCM-MtDS complex, hence suppressing MtCM activation by 'inter-enzyme allostery'. This elegant regulatory principle, invoking a transient allosteric enzyme interaction, seems to be driven by dynamics and is likely a general strategy used by nature.

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“…Binary combinations that involve Trp (Trp+Phe and Trp+Tyr) result in a significant loss of enzyme activity [ 10 ], and ternary combination of all three aromatic amino acids completely abolish the enzyme activity [ 11 ]. Adding an additional layer of sophisticated pathway flux regulation, Mtu DAH7PS forms a complex with chorismate mutase from the same organism [ 12 , 13 ]. Chorismate mutase is an important branchpoint enzyme downstream of the shikimate pathway, catalyzing the transformation of chorismate to prephenate, the first committed precursor for phenylalanine and tyrosine biosynthesis.…”

Section: Introductionmentioning

confidence: 99%

Probing the Sophisticated Synergistic Allosteric Regulation of Aromatic Amino Acid Biosynthesis in Mycobacterium tuberculosis Using ᴅ-Amino Acids

et al. 2016

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Chirality plays a major role in recognition and interaction of biologically important molecules. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) is the first enzyme of the shikimate pathway, which is responsible for the synthesis of aromatic amino acids in bacteria and plants, and a potential target for the development of antibiotics and herbicides. DAH7PS from Mycobacterium tuberculosis (MtuDAH7PS) displays an unprecedented complexity of allosteric regulation, with three interdependent allosteric binding sites and a ternary allosteric response to combinations of the aromatic amino acids l-Trp, l-Phe and l-Tyr. In order to further investigate the intricacies of this system and identify key residues in the allosteric network of MtuDAH7PS, we studied the interaction of MtuDAH7PS with aromatic amino acids that bear the non-natural d-configuration, and showed that the d-amino acids do not elicit an allosteric response. We investigated the binding mode of d-amino acids using X-ray crystallography, site directed mutagenesis and isothermal titration calorimetry. Key differences in the binding mode were identified: in the Phe site, a hydrogen bond between the amino group of the allosteric ligands to the side chain of Asn175 is not established due to the inverted configuration of the ligands. In the Trp site, d-Trp forms no interaction with the main chain carbonyl group of Thr240 and less favourable interactions with Asn237 when compared to the l-Trp binding mode. Investigation of the MtuDAH7PSN175A variant further supports the hypothesis that the lack of key interactions in the binding mode of the aromatic d-amino acids are responsible for the absence of an allosteric response, which gives further insight into which residues of MtuDAH7PS play a key role in the transduction of the allosteric signal.

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A novel noncovalent complex of chorismate mutase and DAHP synthase fromMycobacterium tuberculosis: protein purification, crystallization and X-ray diffraction analysis

Cited by 8 publications

References 22 publications

Reciprocal allostery arising from a bi-enzyme assembly controls aromatic amino acid biosynthesis inPrevotella nigrescens

Reciprocal allostery arising from a bi-enzyme assembly controls aromatic amino acid biosynthesis inPrevotella nigrescens

Remote Control by Inter-Enzyme Allostery: A Novel Paradigm for Regulation of the Shikimate Pathway

Probing the Sophisticated Synergistic Allosteric Regulation of Aromatic Amino Acid Biosynthesis in Mycobacterium tuberculosis Using ᴅ-Amino Acids

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