L-alanine-p-nitroanilide is not a substrate for VanX.
L-alanine-p-nitroanilide is not a substrate for VanX.
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L-丙氨酸-对硝基苯胺不是 VanX 的底物。
DOI：
10.1016/j.ab.2004.05.026
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发表时间：
2004
期刊：
影响因子：
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通讯作者：
Crowder,MichaelW
中科院分区：
文献类型：
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作者：
Golich,FrankC;Sigdel,Tara;Breece,RobertM;Detar,Lisa;Herron,LissaR;Crowder,MichaelW
VanX is a D-D-specific dipeptidase that is essential for high-level vancomycin resistance and has been identified as an excellent target for inhibitors, which may be given in combination with vancomycin as a treatment for vancomycin-resistant bacterial infections. The crystal structure of VanX has been reported and revealed that VanX is a mononuclear Zn (II)-containing aminopeptidase that has a substrate binding pocket of very limited size [1]. Initially, a ninhydrin-based assay was used to conduct steady state kinetic and inhibition studies on VanX [2–4]. Due to the ninhydrin assay being diYcult to perform and to its being noncontinuous and not very useful in probing mechanism, a number of research groups have reported alternative activity assays. These assays include the use of capillary electrophoresis to monitor the formation of D-ala [5], a coupled enzyme assay in which the VanX-generated D-ala is oxidized to pyruvate that is then reduced (with oxidation of NADH) to lactate [6], and an assay in which a VanX-generated product is quantitated by using Ellman’s reagent [7]. We reported that L-ala-p-nitroanilide is a substrate for VanX, and one of the resulting products, p-nitroaniline, could be continuously monitored by Vis spectrophotometry [8]. Recently, efforts to prepare Co (II)-substituted VanX in our lab for spectroscopic studies using a published pET-5b-based overexpression system [8] required an alternative elution gradient for the Q-Sepharose chromatography step to obtain highly purified samples of Co (II)-substituted VanX (RM Breece and MW Crowder, unpublished results). When using this modified protocol, two distinct fractions were recovered from theQ-Sepharose column:(1) a highly purified, concentrated, pink-colored sample that is Co (II)-substituted VanX and (2) an orange-colored sample that contains Co (II)-substituted VanX and several other proteins. When these two fractions were tested for activity, the latter sample is the only one that hydrolyzed L-ala-p-nitroanilide. These results suggested that the continuous UV-Vis assay on which we previously reported [8] may have been based on the activity of a contaminating protein. To further probe this possibility, Escherichia coli BL21 (DE3) cells were transformed with pET-5bvanX, and the normal overexpression [8] and modified purification protocols were used to prepare Zn (II)–VanX. We found that the fractions that eluted earliest from the column were purest; however, these samples did not have any L-ala-p-nitroanilide hydrolase activity. On the other hand, fractions that eluted at higher salt concentrations did exhibit L-ala-p-nitroanilide hydrolase activity, and an SDS–PAGE gel of the fraction with highest activity is shown in Fig. 1c. Although this fraction clearly has significant amounts of a protein with a mass of ca. 23 kDa, there are other contaminating proteins in the lane. E. coli BL21 (DE3) cells were then transformed with pET-5b (empty vector), and the resulting cells were used in our modified purification protocol. A parallel experiment was run with untransformed E. coli BL21 (DE3) cells and yielded the same results. An SDS–PAGE gel was run on the fractions that eluted at the same salt concentrations as those with the fraction with highest L-ala-p-nitroanilide activity as described above (Fig. 1d). Surprisingly, there was a band that corresponded to ca. 23kDa and several other bands were present in the gel. This fraction exhibited significant L-ala-p-nitroanilide activity. This result indicates that a constitutively expressed E. coli enzyme exhibits L-ala-p-nitroanilide hydrolase activity.
基于机制的 D-丙氨酰-D-丙氨酸二肽酶 VanX 抑制剂的设计和评估
DOI：
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发表时间：
2002
期刊：
影响因子：
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作者：
L. Yaouancq;M. Anissimova;M. Badet‐Denisot;B. Badet
通讯作者：
B. Badet
连续测定 VanX，即高水平万古霉素耐药性所需的 D-丙氨酰-D-丙氨酸二肽酶。
DOI：
10.1006/abio.1999.4166
发表时间：
1999
期刊：
Analytical biochemistry.
影响因子：
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作者：
Brandt,JJ;Chatwood,LL;Yang,KW;Crowder,MW
通讯作者：
Crowder,MW