Identification of a common protease‐sensitive region in <scp>d</scp>‐alanyl‐<scp>d</scp>‐alanine and <scp>d</scp>‐alanyl‐<scp>d</scp>‐lactate ligases and photoaffinity labeling with 8‐azido ATP

Wright, Gerard D.; Walsh, Christopher T.

doi:10.1002/pro.5560021020

Cited by 15 publications

(8 citation statements)

References 30 publications

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“…Taken together, the data comply with the assumption of substrate-induced conformational changes similar to those seen in other amide bond-forming ligases (29,(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41). Whereas the free enzyme is readily cleaved by trypsin or factor Xa at Arg-556 in the presumed flexible ⍀-loop, substrate binding induces a compact form, in which the ⍀-loop is no longer attacked by proteases.…”

Section: Role Of Arginines Located In the Putative ⍀-Loop And The Glysupporting

confidence: 83%

“…N-terminal Edman degradation confirmed that the larger fragment corresponded to the N terminus of Cf-TryS sequence ( 1 MGHHHHHHHHH-HSSGHIDDDDKH 23 MASAERVPVS, normal and italic letters mark the tag and the first 10 N-terminal amino acids, respectively; construct pMC10 -15) whereas the smaller one started at Asn-557, as predicted. synthetase, and E. coli GspS, were reported to have a flexible "⍀-loop" that is prone to proteolysis when unliganded but protected from cleavage by binding of substrates or inhibitors (29,31,32). Analogous experiments with Cf-TryS revealed that all individual substrates alone or in combination inhibited cleavage of the enzyme, although to a different extent (Fig.…”

Section: Table II Comparison Of Specific Activities Of Recombinant Cfmentioning

confidence: 98%

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Trypanothione Synthesis in Crithidia Revisited

Comini

Menge

Wissing

et al. 2005

Journal of Biological Chemistry

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In Crithidia fasciculata the biosynthesis of trypanothione (N 1 ,N 8 -bis(glutathionyl)spermidine; reduced trypanothione), a redox mediator unique to and essential for pathogenic trypanosomatids, was assumed to be achieved by two distinct enzymes, glutathionylspermidine synthetase and trypanothione synthetase (TryS), and only the first one was adequately characterized. We here report that the TryS of C. fasciculata, like that of Trypanosoma species, catalyzes the entire synthesis of trypanothione, whereas its glutathionylspermidine synthetase appears to be specialized for Gsp synthesis. A gene (GenBank TM accession number AY603101) implicated in reduced trypanothione synthesis of C. fasciculata was isolated from genomic DNA and expressed in Escherichia coli as His-tagged or Nus fusion proteins. The expression product proved to be a trypanothione synthetase (Cf-TryS) that also displayed a glutathionylspermidine synthetase, an amidase, and marginal ATPase activity. The dual specificity of the Cf-TryS preparations was not altered by removal of the tags. Steady-state kinetic analysis of Cf-TryS yielded a pattern that was compatible with a concerted substitution mechanism, wherein the enzyme forms a ternary complex with Mg 2؉ -ATP and GSH to phosphorylate GSH and then ligates the glutathionyl residue to glutathionylspermidine. Limiting K m values for GSH, Mg 2؉ -ATP, and glutathionylspermidine were 407, 222, and 480 M, respectively, and the k cat was 8.7 s ؊1 for the TryS reaction. Mutating Arg-553 or Arg-613 to Lys, Leu, Gln, or Glu resulted in marked reduction or abrogation (R553E) of activity. Limited proteolysis with factor Xa or trypsin resulted in cleavage at Arg-556 that was accompanied by loss of activity. The presence of substrates, in particular of ATP and GSH alone or in combination, delayed proteolysis of wild-type Cf-TryS and Cf-TryS R553Q but not in Cf-TryS R613Q, which suggests dynamic interactions of remote domains in substrate binding and catalysis.

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Section: Role Of Arginines Located In the Putative ⍀-Loop And The Glysupporting

confidence: 83%

Section: Table II Comparison Of Specific Activities Of Recombinant Cfmentioning

confidence: 98%

Trypanothione Synthesis in Crithidia Revisited

Comini

Menge

Wissing

et al. 2005

Journal of Biological Chemistry

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“…The apo structure from S. aureus does not contain the loop, as the residues were too disordered in the electron density to be modeled, implying they were highly mobile. The flexibility of this region has also been confirmed previously for DdlA and DdlB from E. coli and the VanA vancomycin-resistant determinant of Enterococcus faecium by limited proteolysis (44). Therefore, apo M. tuberculosis Ddl has a conformation distinct from that of other apo structures in that Ddl directly blocks the ATP binding site.…”

Section: Fig 1 M Tuberculosissupporting

confidence: 69%

“…S1 and S2 in the supplemental material). Two binding sites for DCS and one binding site for ATP per monomer were assumed for the binding assays, as reported by previous studies (44,47):…”

Section: Resultsmentioning

confidence: 99%

Structure of the Mycobacterium tuberculosis d -Alanine: d -Alanine Ligase, a Target of the Antituberculosis Drug d -Cycloserine

Bruning

Murillo

Chacón

et al. 2011

Antimicrob Agents Chemother

128

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“…5a). However, biochemical evidence for the presence of such a loop in VanA has been established (28). These amino acid sequence differences in the -loop region highlight the unique nature of VanA and VanB and place these enzymes in a separate class that is distinct from other known Ddls.…”

Section: Resultsmentioning

confidence: 99%

d -Ala- d -Ala ligases from glycopeptide antibiotic-producing organisms are highly homologous to the enterococcal vancomycin-resistance ligases VanA and VanB

Marshall

Broadhead

Leskiw

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

139

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The crisis in antibiotic resistance has resulted in an increasing fear of the emergence of untreatable organisms. Resistance to the glycopeptide antibiotic vancomycin in the enterococci, and the spread of these pathogens throughout the environment, has shown that this scenario is a matter of fact rather than fiction. Enterococci are frequent causes of nosocomial infections and are intrinsically resistant to many antibiotics, being susceptible only to synergistic penicillin͞aminoglycoside therapy or to the glycopeptide antibiotic vancomycin (1). The recent emergence of resistance to vancomycin in these organisms (2, 3) has been greeted with grave alarm, not only because of the immediate problem of how to treat an infection when no other good drugs are available, but also because vancomycin-resistant enterococci (VRE) may serve as a reservoir for Van R genes, which will no doubt eventually make their way into more virulent organisms such as those of the genus Staphylococcus. Highlevel resistance to vancomycin in VRE (minimal inhibitory concentration Ͼ 1,000 g͞ml) is conferred by five genes located on a transposable element (4). These genes include vanR and vanS, which encode a two-component regulatory system that directs the transcription of three structural genes: The origin of the Van R genes has been the subject of some speculation. Related to this question is the increasing debate concerning the prudence of the continued agricultural use of glycopeptide antibiotics such as avoparcin (8-12) in view of the capacity for selection of VRE in the environment (13-15). That environmental VRE isolates have not been observed yet in North America where glycopeptides are not used in agriculture (16), unlike the situation in Europe, adds another dimension to the debate.The amino acid sequence of the vancomycin-resistance enzyme VanA (17) and the related VanB (18) shows between 20% and 35% sequence homology to other Ddls, including the D-Ala-D-Lac ligases from organisms that are intrinsically resistant to vancomycin such as Leuconostoc spp. and Lactobacillus spp. (19). As part of an ongoing study of the biosynthesis of glycopeptide antibiotics, we have cloned the complete ddl gene from the glycopeptide-producing organism Streptomyces toyocaensis NRRL 15009, which produces the aglycopeptide A47934, and a partial ddl gene from Amycolatopsis orientalis, which produces vancomycin (Fig. 1). These genes encode proteins that show a high degree of homology to the VanA and VanB ligases and suggest that clinical vancomycin resistance may have originated in glycopeptide-producing organisms.

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Identification of a common protease‐sensitive region in d‐alanyl‐d‐alanine and d‐alanyl‐d‐lactate ligases and photoaffinity labeling with 8‐azido ATP

Cited by 15 publications

References 30 publications

Trypanothione Synthesis in Crithidia Revisited

Trypanothione Synthesis in Crithidia Revisited

Structure of the Mycobacterium tuberculosis d -Alanine: d -Alanine Ligase, a Target of the Antituberculosis Drug d -Cycloserine

d -Ala- d -Ala ligases from glycopeptide antibiotic-producing organisms are highly homologous to the enterococcal vancomycin-resistance ligases VanA and VanB

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