Enzymes of vancomycin resistance: the structure of D-alanine-D-lactate ligase of naturally resistant Leuconostoc mesenteroides.
BACKGROUND
The bacterial cell wall and the enzymes that synthesize it are targets of glycopeptide antibiotics (vancomycins and teicoplanins) and beta-lactams (penicillins and cephalosporins). Biosynthesis of cell wall peptidoglycan requires a crosslinking of peptidyl moieties on adjacent glycan strands. The D-alanine-D-alanine transpeptidase, which catalyzes this crosslinking, is the target of beta-lactam antibiotics. Glycopeptides, in contrast, do not inhibit an enzyme, but bind directly to D-alanine-D-alanine and prevent subsequent crosslinking by the transpeptidase. Clinical resistance to vancomycin in enterococcal pathogens has been traced to altered ligases producing D-alanine-D-lactate rather than D-alanine-D-alanine.


RESULTS
The structure of a D-alanine-D-lactate ligase has been determined by multiple anomalous dispersion (MAD) phasing to 2.4 A resolution. Co-crystallization of the Leuconostoc mesenteroides LmDdl2 ligase with ATP and a di-D-methylphosphinate produced ADP and a phosphinophosphate analog of the reaction intermediate of cell wall peptidoglycan biosynthesis. Comparison of this D-alanine-D-lactate ligase with the known structure of DdlB D-alanine-D-alanine ligase, a wild-type enzyme that does not provide vancomycin resistance, reveals alterations in the size and hydrophobicity of the site for D-lactate binding (subsite 2). A decrease was noted in the ability of the ligase to hydrogen bond a substrate molecule entering subsite 2.


CONCLUSIONS
Structural differences at subsite 2 of the D-alanine-D-lactate ligase help explain a substrate specificity shift (D-alanine to D-lactate) leading to remodeled cell wall peptidoglycan and vancomycin resistance in Gram-positive pathogens.
Kuzin
A P
AP
Department of Molecular and Cell Biology, The University of Connecticut, Storrs, CT 06269-3125, USA.
Sun
T
T
Jorczak-Baillass
J
J
Healy
V L
VL
Walsh
C T
CT
Knox
J R
JR
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000409:Alanine / Q000031:analogs & derivatives / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000502:physiology; D019729:Carbon-Oxygen Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain / Q000502:physiology; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D007934:Leuconostoc / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity / Q000502:physiology; D020713:Vancomycin Resistance
D001426:Bacterial Proteins; C071437:VanA ligase, Bacteria; C064315:alanylphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D019729:Carbon-Oxygen Ligases; D000409:Alanine