X-ray structure of MalY from Escherichia coli: a pyridoxal 5′-phosphate-dependent enzyme acting as a modulator in mal gene expression

Clausen, Tim; Schlegel, Anja; Peist, Ralf; Schneider, Erich; Steegborn, Clemens; Chang, Yuh-Shin; Haase, Andrea; Bourenkov, Gleb; Bartunik, H.D.; Boos, Winfried

doi:10.1093/emboj/19.5.831

Cited by 47 publications

(59 citation statements)

References 43 publications

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“…This activity is not required for repression; a mutant lacking the enzymatic activity still shows repressor activity (44). On the other hand, mutants can be isolated that exhibit normal cystathionase activity but are defective in their repressor activity (10). MalY is a negative effector of MalT that competes with maltotriose binding, thereby inhibiting its transcriptional activity.…”

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The N Terminus of the Escherichia coli Transcription Activator MalT Is the Domain of Interaction with MalY

et al. 2002

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The maltose system of Escherichia coli consists of a number of genes encoding proteins involved in the uptake and metabolism of maltose and maltodextrins. The system is positively regulated by MalT, its transcriptional activator. MalT activity is controlled by two regulatory circuits: a positive one with maltotriose as effector and a negative one involving several proteins. MalK, the ATP-hydrolyzing subunit of the cognate ABC transporter, MalY, an enzyme with the activity of a cystathionase, and Aes, an acetyl esterase, phenotypically act as repressors of MalT activity. By in vivo titration assays, we have shown that the N-terminal 250 amino acids of MalT contain the interaction site for MalY but not for MalK. This was confirmed by gel filtration analysis, where MalY was shown to coelute with the N-terminal MalT structural domain. Mutants in MalT causing elevated mal gene expression in the absence of exogenous maltodextrins were tested in their response to the three repressors. The different MalT mutations exhibited a various degree of sensitivity towards these repressors, but none was resistant to all of them. Some of them became nearly completely resistant to Aes while still being sensitive to MalY. These mutations are located at positions 38, 220, 243, and 359, most likely defining the interaction patch with Aes on the three-dimensional structure of MalT.The Escherichia coli maltose system consists of 10 genes encoding proteins dedicated to the uptake and the metabolism of maltose and maltodextrins (4). These genes are under the control of MalT, a specific transcriptional activator of 901 amino acids (aa). MalT belongs to a class of bacterial transactivators, the MalT or LAL family (11, 42). They are large proteins (Ͼ90 kDa), possess an ATP binding site near their N terminus, and share homology with LuxR near their C terminus. MalT binds and activates its target promoters (29) only in the presence of ATP (34) and the inducer maltotriose (28). MalT consists of four structural domains (11): domain 1 (DT1, aa 1 to 241) binds ATP, domains 2 (DT2, aa 250 to 436) and 3 (DT3, aa 437 to 806) bind the inducer, and domain 4 (DT4, aa 807 to 901) harbors the DNA binding site (11,43). MalT exists in an equilibrium between a monomeric (inactive) and a monomeric (active) form and is prone to multimerize. This equilibrium is shifted to the active form by the inducer maltotriose, which triggers a conformational change involving DT1, -2, and -3 and the linkers in between. The conformational change, which also requires ATP, is a step towards the formation of a high-order oligomer, the transcriptionally competent form of the protein (11, 36). Point mutations in malT (malT c ) have been isolated that confer a constitutive expression of the maltose regulon when maltodextrin is not present in the growth medium (12, 13) The in vitro analysis of two corresponding MalT c proteins revealed that, in contrast to the wild-type MalT, they could activate transcription from a MalT-dependent promoter in the absence of maltotriose but could st...

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“…MalY most likely stabilizes MalT in its inactive (monomeric) form (37). The three-dimensional structure of the MalY dimer has been determined, revealing its interaction site with MalT (10).…”

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The N Terminus of the Escherichia coli Transcription Activator MalT Is the Domain of Interaction with MalY

et al. 2002

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“…In addition, MalT is negatively controlled by at least three proteins: MalY, MalK, and Aes. MalY is an enzyme with cystathionine ␤-lyase activity (9,10), which interacts directly with the amino terminus of MalT (11,12), seemingly through a patch made up of a hydrophobic core surrounded by highly polar residues (13). MalK, the ATP-hydrolyzing subunit of the maltose transport system (14), has been reported to interact directly with MalT (15), probably through some residues localized in its C-terminal domain (16).…”

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“…The one or more sites of interaction on MalT of Aes and MalY are probably superimposed, whereas MalK would appear to interact at a different location (12). It is possible that MalY and Aes share similar structural determinants, but these cannot be easily predicted on the basis of sequences analysis only (13).…”

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The Aes Protein and the Monomeric α-Galactosidase fromEscherichia coli Form a Non-covalent Complex

Mandrich¹,

Caputo²,

Martin³

et al. 2002

Journal of Biological Chemistry

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Aes, a 36-kDa acetylesterase from Escherichia coli, belongs to the hormone-sensitive lipase family, and it is involved in the regulation of MalT, the transcriptional activator of the maltose regulon. The activity of MalT is depressed through a direct protein-protein interaction with Aes. Although the effect is clear-cut, the meaning of this interaction and the conditions that trigger it still remain elusive. To perform a comparative thermodynamic study between the mesophilic Aes protein and two homologous thermostable enzymes, Aes was overexpressed in E. coli and purified. At the last step of the purification procedure the enzyme was eluted from a Mono Q HR 5/5 column as a major form migrating, anomalously, at 56 kDa on a calibrated Superdex 75 column. A minor peak that contains the Aes protein and a polypeptide of 50 kDa was also detected. By a combined analysis of size-exclusion chromatography and surface-enhanced laser desorption ionization-time of flight mass spectrometry, it was possible to demonstrate the presence in this peak of a stable 87-kDa complex, containing the Aes protein itself and the 50-kDa polypeptide in a 1:1 ratio. The homodimeric molecular species of Aes and of the 50-kDa polypeptide were also detected. The esterase activity associated with the 87-kDa complex, when assayed with p-nitrophenyl butanoate as substrate, proved 6-fold higher than the activity of the major Aes form of 56 kDa. Amino-terminal sequencing highlighted that the 50-kDa partner of Aes in the complex was the ␣-galactosidase from E. coli. The E. coli cells harboring plasmid pT7-SCII-aes and, therefore, expressing Aes were hampered in their growth on a minimal medium containing raffinose as a sole carbon source. Because ␣-galactosidase is involved in the metabolism of raffinose, the above findings suggest a potential role of Aes in the regulation of carbohydrate metabolism in E. coli.The Escherichia coli aes (or ybac) gene encodes the 36-kDa cytoplasmic protein Aes (1), a carboxylesterase belonging to the hormone-sensitive lipase family (HSL) 1 (2). Although the physiological role of this enzyme is still obscure, it has recently received greater attention due to the discovery that Aes is involved in the regulation of the maltose regulon in E. coli (reviewed in Ref. 3). The E. coli maltose system consists of 10 genes encoding proteins for the uptake and metabolism of maltodextrin and maltose (3). MalT is the transcriptional activator of the maltose regulon and the prototype of a new family of transcription factors (4, 5) and integrates several signals through its amino-terminal three domains (6). To activate mal genes transcription MalT acts together with the cyclic AMPcatabolite protein complex, the inducer maltotriose, and ATP (7). Several regulatory circuits modulate the activity of MalT, e.g. MalT expression is regulated by Mlc, a glucose-inducible repressor (8). In addition, MalT is negatively controlled by at least three proteins: MalY, MalK, and Aes. MalY is an enzyme with cystathionine ␤-lyase activity (9, 10), which ...

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“…The interconversion between the inactive and active states involves DT1, DT2, and DT3 based on the observation that the transition toward the active state causes a change in the sensitivity of both the DT1-DT2 and the DT2-DT3 linkers to protease attack (21). Given that the three inhibitory proteins are unrelated at the sequence level and that MalK and MalY do not share any obvious structural similarity (29,32), the molecular details of the interaction between the inactive form of MalT and each of the negative effectors is likely to be different. Consistent with this idea, the T38R substitution interferes with the DT1/Aes interaction (20) in the DT1 context, whereas it has no effect on the DT1/MalK interaction.…”

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confidence: 99%

MalK, the ATP-binding Cassette Component of the Escherichia coli Maltodextrin Transporter, Inhibits the Transcriptional Activator MalT by Antagonizing Inducer Binding

Joly

Böhm

Boos

et al. 2004

Journal of Biological Chemistry

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X-ray structure of MalY from Escherichia coli: a pyridoxal 5′-phosphate-dependent enzyme acting as a modulator in mal gene expression

Cited by 47 publications

References 43 publications

The N Terminus of the Escherichia coli Transcription Activator MalT Is the Domain of Interaction with MalY

The N Terminus of the Escherichia coli Transcription Activator MalT Is the Domain of Interaction with MalY

The Aes Protein and the Monomeric α-Galactosidase fromEscherichia coli Form a Non-covalent Complex

MalK, the ATP-binding Cassette Component of the Escherichia coli Maltodextrin Transporter, Inhibits the Transcriptional Activator MalT by Antagonizing Inducer Binding

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