Crystallization and structure determination of...

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

2000-08-04

2004-02-10

Brusca, John S. (Department: 1631)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

C435S015000, C117S011000, C702S019000, C702S027000, C530S350000, C530S355000, C530S820000, C530S825000

Reexamination Certificate

active

06689595

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to the crystallization and structure determination of thymidylate kinase (TMK) from Staphylococcus aureus.
BACKGROUND
Thymidylate kinase (TMK) catalyzes the synthesis of (deoxy)thymidine diphosphate (dTDP) from (deoxy)thymidine monophosphate (dTMP) and ATP along the pathway leading to the synthesis of (deoxy)thymidine triphosphate (dTTP) necessary for DNA synthesis (FIG.
1
). Since the phosphorylation of dTDP to dTTP is conducted by a nonspecific diphosphate kinase, TMK is a key player in the regulation of DNA synthesis and is a potential antibacterial target. Interest in thymidylate kinase biochemistry increased when it was recently discovered that this enzyme serves as one of the activators for the AIDS drug, 3′-azido-3′-deoxythymidine (AZT) (L. W. Frick et al.,
Biochem. Biophys. Res. Comm.
154:124-9 (1988); A. Fridland et al.,
Mol. Pharmacol.
37:665-70 (1990)). Activation of AZT to azidothymidine triphosphate (AZT-TP) proceeds along cellular phosphorylation pathways to produce the species which is incorporated into growing DNA chains by HIV reverse transcriptase. Similar to its role in serving as a control point for the production of dTTP, thymidylate kinase catalyzes the rate limiting phosphorylation of AZT-monophosphate to AZT-diphosphate (AZT-DP). AZT-DP phosphorylation to AZT-TP is then catalyzed by a nonspecific diphosphate kinase.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides a method for crystallizing an
S. aureus
thymidylate kinase molecule or molecular complex that includes preparing purified
S. aureus
thymidylate kinase at a concentration of about 1 mg/ml to about 50 mg/ml and crystallizing
S. aureus
thymidylate kinase from a solution including about 5 wt. % to about 50 wt. % PEG (preferably having a number average molecular weight between about 200 and about 20,000), about 0.05 M to about 0.5 M MgCl
2
, and about 0 wt. % to about 20 wt. % DMSO, wherein the solution is buffered to a pH of about 6 to about 7. In another aspect, the present invention provides a method for crystallizing an
S. aureus
thymidylate kinase molecule or molecular complex that includes preparing purified
S. aureus
thymidylate kinase at a concentration of about 1 mg/ml to about 50 mg/ml and crystallizing
S. aureus
thymidylate kinase from a solution including about 2 mM to about 20 mM &bgr;,&ggr;-difluoromethylene-bisphosphonate adenosine monophosphate and about 0 wt. % to about 20 wt. % DMSO, wherein the solution is buffered to a pH of about 6 to about 7
In another aspect, the present invention provides crystalline forms of an
S. aureus
thymidylate kinase molecule. In one embodiment, a crystal of
S. aureus
thymidylate kinase is provided having the trigonal space group symmetry P2
1
.
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
thymidylate kinase 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 TMP and/or TMP/ATP substrate binding pocket of an
S. aureus
thymidylate kinase molecule or molecular complex. In another embodiment, the scalable three dimensional configuration 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
thymidylate kinase 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 2.1 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
thymidylate kinase TMP and/or TMP/ATP substrate binding pocket. In one embodiment, the
S. aureus
thymidylate kinase TMP substrate 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 substrate binding pocket being defined by a set of points having a root mean square deviation of less than about 2.1 Å, preferably less than about 1.5 Å, more preferably less than about 1.0 Å, and most preferably less than about 0.5 Å from points representing the backbone atoms of the amino acids. In another embodiment, the
S. aureus
thymidylate kinase TMP/ATP 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 2.1 Å, preferably less than about 1.5 Å, more preferably less than about 1.0 Å, and most preferably less than about 0.5 Å from points representing the backbone atoms of the amino acids.
TABLE 1
Residues within about 4 Å of the TMP
binding pocket of
S. aureus
TMK
GLU
12
LEU
53
ARG
 93
ARG
37
LEU
66
SER
 97
ILE
48
PHE
67
SER
 98
ARG
49
SER
70
TYR
101
VAL
52
ARG
71
TABLE 2
Residues within about 7 Å of the TMP
binding pocket of
S. aureus
TMK
GLY
10
VAL
52
TYR
 94
GLU
12
LEU
53
ILE
 95
ARG
37
GLU
63
ASP
 96
GLU
38
MET
65
SER
 97
PRO
39
LEU
66
SER
 98
GLY
45
PHE
67
LEU
 99
GLU
38
MET
65
SER
 97
GLY
45
PHE
67
LEU
 99
GLU
46
ALA
68
ALA
100
GLU
47
ALA
69
TYR
101
ILE
48
SER
70
GLN
102
ARG
49
ARG
71
ASN
117
LYS
50
ASP
92
PHE
160
ILE
51
ARG
93
TYR
168
TABLE 3
Residues within about 10 Å of the TMP
binding pocket of
S. aureus
TMK
PHE
 8
ILE
51
TYR
 94
GLU
 9
VAL
52
ILE
 95
GLY
10
LEU
53
ASP
 96
PRO
11
GLU
54
SER
 97
GLU
12
GLY
55
SER
 98
GLY
13
MET
58
LEU
 99
SER
14
ILE
60
ALA
100
LYS
16
THR
62
TYR
101
THR
17
GLU
63
GLN
102
ARG
37
ALA
64
GLY
103
GLU
38
MET
65
TYR
104
PRO
39
LEU
66
ALA
105
GLY
40
PHE
67
ARG
106
GLY
41
ALA
68
VAL
113
VAL
42
ALA
69
LEU
116
PRO
43
SER
70
ASN
117
THR
44
ARG
71
ILE
143
GLY
45
ARG
72
PHE
160
GLU
46
GLU
73
HIS
161
GLU
47
HIS
74
VAL
164
ILE
48
CYS
91
TYR
168
ARG
49
ASP
92
LYS
50
ARG
93
TABLE 4
Residues within about 4 Å of the TMP/ATP
binding pocket of
S. aureus
TMK
GLU
12
GLU
38
SER
 98
GLY
15
PHE
67
TYR
101
LYS
16
ARG
71
GLN
102
THR
17
ASP
92
ARG
142
THR
18
ARG
93
LEU
188
ARG
37
SER
97
TABLE 5
Residues within about 7 Å of the TMP/ATP
binding pocket of
S. aureus
TMK
GLY
10
ARG
 49
TYR
101
PRO
11
GLU
 63
GLN
102
GLU
12
ALA
 64
ARG
106
GLY
13
PHE
 67
ASN
117
SER
14
ALA
 68
LEU
132
GLY
15
ARG
 71
GLU
141
LYS
16
ASP
 92
ARG
142
THR
17
ARG
 93
ILE
143
THR
18
TYR
 94
PHE
160
VAL
19
ILE
 95
ALA
184
ILE
20
ASP
 96
GLN
186
ASN
21
SER
 97
PRO
187
ARG
37
SER
 98
LEU
188
GLU
38
LEU
 99
GLU
189
ALA
100
VAL
191
TABLE 6
Residues within about 10 Å of the TMP/ATP
binding pocket of
S. aureus
TMK
PHE
 8
ALA
 64
VAL
113
GLU
 9
MET
 65
LEU
116
GLY
10
LEU
 66
ASN
117
PRO
11
PHE
 67
ALA
120
GLU
12
ALA
 68
LEU
132
GLY
13
ALA
 69
VAL
134
SER
14
SER
 70
VAL
138
GLY
15
ARG
 71
GLY
139
LYS
16
ARG
 72
ARG
140
THR
17
HIS
 74
GLU
141
THR
18
CYS
 91
ARG
142
VAL
19
ASP
 92
ILE
143
ILE
20
ARG
 93
ASP
157
ASN
21
TYR
 94
PHE
160
GLU
22
ILE
 95
HIS
161
MET
35
ASP
 96
VAL
164
THR
36
SER
 97
TYR
168
ARG
37
SER
 98
ASN
183
GLU
38
LEU
 99
ALA
184
PRO
39
ALA
100
ASP
185
GLY
40
TYR
101
GLN
186
GLU
46
GLN
102
PRO
187
ARG
49
GLY
103
LEU
188
VAL
52
TYR
104
GLU
189
LEU
53
ALA
105
ASN
190
ILE
60
ARG
106
VAL
191
GLU
63
ILE
108
VAL
192
In another aspect, the present invention provides molecules or molecular complexes that are structurally homologous to an
S. aureus
thymidylate kinase molecule or molecular complex.
In another aspec

Affiliated with

Also associated with

Rating

Crystallization and structure determination of... does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Crystallization and structure determination of..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Crystallization and structure determination of... will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3331715