Structures of Mycobacterium tuberculosis Anthranilate Phosphoribosyltransferase Variants Reveal the Conformational Changes That Facilitate Delivery of the Substrate to the Active Site
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- 作者：Tammie V. M. Cookson ; Genevieve L. Evans ; Alina Castell ; Edward N. Baker ; J. Shaun Lott ; Emily J. Parker
- 刊名：Biochemistry
- 出版年：2015
- 出版时间：October 6, 2015
- 年：2015
- 卷：54
- 期：39
- 页码：6082-6092
- 全文大小：650K
- ISSN：1520-4995
文摘
Anthranilate phosphoribosyltransferase (AnPRT) is essential for the biosynthesis of tryptophan in Mycobacterium tuberculosis (Mtb). This enzyme catalyzes the second committed step in tryptophan biosynthesis, the Mg2+-dependent reaction between 5鈥?phosphoribosyl-1鈥?pyrophosphate (PRPP) and anthranilate. The roles of residues predicted to be involved in anthranilate binding have been tested by the analysis of six Mtb-AnPRT variant proteins. Kinetic analysis showed that five of six variants were active and identified the conserved residue R193 as being crucial for both anthranilate binding and catalytic function. Crystal structures of these Mtb-AnPRT variants reveal the ability of anthranilate to bind in three sites along an extended anthranilate tunnel and expose the role of the mobile 尾2鈭捨? loop in facilitating the enzyme鈥檚 sequential reaction mechanism. The 尾2鈭捨? loop moves sequentially between a 鈥渇olded鈥?conformation, partially occluding the anthranilate tunnel, via an 鈥渙pen鈥?position to a 鈥渃losed鈥?conformation, which supports PRPP binding and allows anthranilate access via the tunnel to the active site. The return of the 尾2鈭捨? loop to the 鈥渇olded鈥?conformation completes the catalytic cycle, concordantly allowing the active site to eject the product PRA and rebind anthranilate at the opening of the anthranilate tunnel for subsequent reactions. Multiple anthranilate molecules blocking the anthranilate tunnel prevent the 尾2鈭捨? loop from undergoing the conformational changes required for catalysis, thus accounting for the unusual substrate inhibition of this enzyme.