Crystallization and structure determination of...

Details Crystallization and structure determination of... Crystallization and structure determination of...

2000-08-04

2002-03-12

Marschel, Ardin H. (Department: 1631)

Data processing: measuring, calibrating, or testing

Measurement system in a specific environment

Biological or biochemical

C435S183000, C702S027000

Reexamination Certificate

active

06356845

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to the crystallization and structure determination of
Staphylococcus aureus
UDP-N-acetylenolpyruvylglucosamine reductase (
S. aureus
MurB).
BACKGROUND OF THE INVENTION
Reports of an increase in antibiotic resistant bacteria have stimulated efforts to find new classes of therapeutic agents that will prevent society from entering a “post-antibiotic age.” Historically, three important cellular functions have been the major targets of antibiotics—cell wall biosynthesis, DNA replication, and protein translation. The biosynthesis of the bacterial cell wall, in particular the peptidoglycan polymer, is a particularly attractive target since this flexible structure provides protection for the cell against osmotic lysis. To date, most of the therapeutic agents discovered that target cell wall biosynthesis inhibit the later stages of peptidoglycan biosynthesis at the point where interstrand cross linking occurs between the peptide chains. Recent efforts have been directed toward purifying and characterizing all the enzymes in the peptidoglycan biosynthetic pathway with an eye toward designing novel enzyme inhibitors of these essential targets.
Bacterial peptidoglycan is a polymer which includes a repeating disaccharide subunit of N-acetylglucosamine and N-acetylmuramic acid and an extended four to five residue amino acid chain. The first step toward creating this peptidoglycan polymer involves the formation of UDP-N-acetylmuramic acid from UDP-N-acetylglucosamine by the enzymes MurA and MurB. MurA catalyzes the first stage of this transformation by transferring the enolpyruvate moiety of phosphoenolpyruvate to the 3′ hydroxyl of UDP-N-acetylglucosamine with the release of inorganic phosphate. The resulting product, enolpyruvyl-UDP-N-acetylglucosamine (EP-UDPGlcNAc), undergoes a reduction catalyzed by the MurB enzyme by utilizing one equivalent of NADPH and a solvent derived proton. This two electron reduction creates the lactyl ether of UDP-N-acetylmuramic acid upon which a five residue peptide chain is built. Construction of this pentapeptide is catalyzed in a nonribosomal fashion by the enzymes MurC, MurD, MurE, and MurF (
FIG. 1
) in both Gram negative bacteria such as
Escherichia coli
and Gram positive bacteria such as
Staphylococcus aureus.
The resulting UDP-N-acetylmuramyl pentapeptide is subsequently attached to an undecaprenyl lipid moiety by MraY and joined to another sugar, UDP-N-acetylglucosamine by MurG. In Staphylococci the next steps of peptidoglycan biosynthesis involve another family of enzymes, FemX, FemA, and FemB which create a pentaglycine strand in a stepwise fashion on the amino terminus of the lysine side chain. This extended Lys-Gly
5
chain serves as the interstrand bridge between nearby peptide strands. Crosslinking between strands can then occur between the lysine-pentapeptide bridge and the carbonyl of the fourth residue (D-Ala) with release of the terminal D-Ala in a transpeptidation step catalyzed by penicillin binding proteins.
While several laboratories have characterized some of the peptidoglycan biosynthetic enzymes for
E. coli
little biochemistry or structural biology has been carried out on these enzymes in a clinically relevant Gram positive organism. Interest in the molecular mechanisms of peptidoglycan biosynthesis in Gram positive organisms has increased in recent years as methicillin resistant
S. aureus
strains have surfaced that have acquired resistance to the antibiotic vancomycin.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides a method for crystallizing an
S. aureus
MurB molecule or molecular complex that includes preparing purified
S. aureus
MurB at a concentration of about 1 mg/ml to about 50 mg/ml and crystallizing
S. aureus
MurB from a solution comprising about 1 wt. % to about 50 wt. % PEG, 0 wt. % to about 40 wt. % DMSO, about 100 mM to about 1 M ammonium or lithium sulfate, about 0 mM to about 20 mM 2-mercaptoethanol, about 0.005 mM to about 40 mM EP-UDPGlcNAc substrate, and buffered to a pH of about 5 to about 8.
In another aspect, the present invention provides crystalline forms of an
S. aureus
MurB molecule. In one embodiment, a crystal of an
S. aureus
MurB is provided having the trigonal space group symmetry I2
1
3.
In another aspect, the present invention provides a scalable three dimensional configuration of points derived from structure coordinates of at least a portion of an
S. aureus
MurB molecule or molecular complex. In one embodiment, the scalable three dimensional set of points is derived from structure coordinates of at least the backbone atoms of the amino acids representing a FAD and/or substrate binding pocket of an
S. aureus
MurB molecule or molecular complex. In another embodiment, the scalable three dimensional set of points is derived from structure coordinates of at least a portion of a molecule or a molecular complex that is structurally homologous to an
S. aureus
MurB molecule or molecular complex. On a molecular scale, the configuration of points derived from a homologous molecule or molecular complex have a root mean square deviation of less than about 1.0 Å from the structure coordinates of the molecule or complex.
In another aspect, the present invention provides a molecule or molecular complex that includes at least a portion of an
S. aureus
MurB FAD and/or substrate binding pocket. In one embodiment, the
S. aureus
MurB FAD binding pocket includes the amino acids listed in Table 1, preferably the amino acids listed in Table 2, and more preferably the amino acids listed in Table 3, the FAD binding pocket being defined by a set of points having a root mean square deviation of less than about 1.7 Å, preferably less than about 1.0 Å, from points representing the backbone atoms of the amino acids. In another embodiment, the
S. aureus
MurB substrate binding pocket includes the amino acids listed in Table 4, preferably the amino acids listed in Table 5, and more preferably the amino acids listed in Table 6, the substrate binding pocket being defined by a set of points having a root mean square deviation of less than about 1.0 Å from points representing the backbone atoms of the amino acids.
TABLE 1
Residues near the FAD binding site in
S. aureus
MurB
Identified residues 4Å away from the FAD
TYR
42
LEU
 98
TYR
149
VAL
199
TYR
77
SER
115
MET
150
ARG
225
LEU
78
ILE
140
ALA
152
GLN
229
GLY
79
PRO
141
GLY
153
LEU
231
ASN
80
GLY
142
ALA
154
SER
235
GLY
81
SER
143
ARG
188
GLY
237
SER
82
GLY
145
ILE
192
PHE
274
ASN
83
GLY
146
LEU
197
ARG
310
ILE
84
ALA
147
VAL
198
TABLE 2
Residues near the FAD binding site in
S. aureus
MurB
Identified residues 7Å away from FAD
THR
41
LEU
 99
VAL
148
LEU
200
TYR
42
SER
115
TYR
149
GLU
201
THR
43
GLY
116
MET
150
ARG
225
THR
76
ALA
117
ASN
151
GLN
229
TYR
77
ILE
119
ALA
152
PRO
230
LEU
78
PHE
136
GLY
153
LEU
231
GLY
79
GLY
139
ALA
154
TYR
233
ASN
80
ILE
140
TYR
155
PRO
234
GLY
81
PRO
141
ARG
188
SER
235
SER
82
GLY
142
ILE
192
CYS
236
ASN
83
SER
143
GLN
193
GLY
237
ILE
84
ILE
144
HIS
196
SER
238
ILE
85
GLY
145
LEU
197
PHE
274
ILE
96
GLY
146
VAL
198
ARG
310
LEU
98
ALA
147
VAL
199
ILE
312
TABLE 3
Residues near the FAD binding site in
S. aureus
MurB
Identified residues 10Å away
LEU
37
SER
100
TYR
155
ARG
225
TYR
40
LEU
101
GLY
156
GLU
226
THR
41
ALA
113
GLY
157
LYS
228
TYR
42
GLY
114
GLU
158
GLN
229
THR
43
SER
115
VAL
159
PRO
230
LYS
44
GLY
116
LYS
160
LEU
231
THR
45
ALA
117
ALA
166
GLU
232
TYR
52
ALA
118
LEU
167
TYR
233
PRO
55
ILE
119
CYS
168
PRO
234
VAL
61
ILE
120
VAL
169
SER
235
VAL
65
GLU
135
ASN
170
CYS
236
VAL
75
PHE
136
LEU
183
GLY
237
THR
76
ALA
137
ASP
186
SER
238
TYR
77
CYS
138
TYR
187
VAL
239
LEU
78
GLY
139
ARG
188
SER
268
GLY
79
ILE
140
ASN
189
LYS
270
ASN
80
PRO
141
SER
190
HIS
271
GLY
81
GLY
142
ILE
191
GLY
273
SER
82
SER
143
ILE
192
PHE
274
ASN
83
ILE
144
GLN
193
MET
275
ILE
84
GLY
145
LYS
194
VAL
276
ILE
85
GLY
146
GLU
195
ASN
277
ILE
86
ALA
147
HIS
196
TYR
286
ILE
91
VAL
148
LEU
197
GLU
308
ILE
94
TYR
149
VAL
198
VA

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