Purification and characterization of VanXY_C, a d,d‐dipeptidase/d,d‐carboxypeptidase in vancomycin‐resistant Enterococcus gallinarum BM4174

Abstract

VanXY C , a bifunctional enzyme from VanC-phenotype Enterococcus gallinarum BM4174 that catalyses D,D-peptidase and D,D-carboxypeptidase activities, was purified as the native protein, as a maltose-binding protein fusion and with an N-terminal tag containing six histidine residues. The kinetic parameters of His 6 -VanXY C were measured for a variety of precursors of peptidoglycan synthesis involved in resistance: for D-Ala-D-Ala, the K m was 3.6 mM and k cat , 2.5 s, K m was 18.8 mM and k cat 6.2 s )1 ; for D-Ala-D-Ser, K m was 15.5 mM and k cat 0.35 s )1 . His 6 -VanXY C was inactive against the peptido- [7,8]. The VanA and VanB gene clusters are contained on transposons that are integrated either into self-transferable plasmids or the host chromosome [8,9].VanC-type resistance is defined as intrinsic low-level resistance to vancomycin (2-32 lgAEmL )1 ), but not to teicoplanin [10]. It has been identified in Enterococcus gallinarum, E. casseliflavus and E. flavescens [6,17] [20,21]. In this study the substrate specificity of purified VanXY C , a cytoplasmic bifunctional D,D-peptidase and D,D-carboxypeptidase, was characterized kinetically and an investigation initiated of the role of specific residues in determining the substrate selectivity. E X P E R I M E N T A L P R O C E D U R E S Strains, plasmids and growth conditionsEscherichia coli strains were grown in Luria-Bertani medium, and maintained on Luria-Bertani agar (1.5%), with the exception of E. coli JM83 [22] containing deriva-

Recombinant protein expression and purification: A comprehensive review of affinity tags and microbial applications

“…In addition, there are also commercial vectors for the expression of MBP fusion proteins to the non-reducing environment of the E. coli periplasm (e.g., New England Biolabs pMAL), addition of which may improve the folding of disulfide bond-containing proteins. Although not generally required, MBP has been used in conjunction with a small affinity tag to improve purification purity [36,37].…”

Structural basis for the evolution of vancomycin resistance D , D -peptidases

“…and its spread to methicillin-resistant Staphylococcus aureus is a serious threat to public health (1) (7,8). Thus, Van D,D-peptidases demonstrate variation in peptidoglycan substrate selectivity that correlates with the specific resistance mechanism.…”

Vancomycin Resistance in Enterococci Due to Synthesis of Precursors Terminating in d -Alanyl- d -Serine

“…The k cat /K m ratio is ca. 25 to 30 times lower for D-Ala-D-Ser than for D-Ala-D-Ala (32). It has even stricter specificity as a DD-carboxypeptidase, having no activity against pentapeptide[D-Ser].…”

“…It does not have the specific binding sites for the N or C termini of D-Ala-D-Ala that are present in VanX-but not VanY-type enzymes, and it has a specific glutamine residue at position 67 that is essential for activity of a VanY-but not a VanX-type enzyme (35). The aspartic acid residue at position 59 that is vital in VanX (mutation to Ala or Ser results in an enormous decrease in the catalytic efficiency [27]) is unimportant in VanXY C , where mutation to Ala or Ser hardly affects the k cat /K m ratio (32). Other aspects of the amino acid sequence in close proximity to the active-site regions also suggest that VanXY C has evolved from a VanY-type enzyme rather than from a strict DD-dipeptidase.…”