Vos S, Parry R J, Burns M R, de Jersey J, Martin J L
Department of Biochemistry, The University of Queensland, Brisbane, Qld 4072, Australia.
J Mol Biol. 1998 Oct 2;282(4):875-89. doi: 10.1006/jmbi.1998.2051.
Structures of free, substrate-bound and product-bound forms of Escherichia coli xanthine-guanine phosphoribosyltransferase (XGPRT) have been determined by X-ray crystallography. These are compared with the previously determined structure of magnesium and sulphate-bound XPRT. The structure of free XGPRT at 2.25 A resolution confirms the flexibility of residues in and around a mobile loop identified in other PRTases and shows that the cis-peptide conformation of Arg37 at the active site is maintained in the absence of bound ligands. The structures of XGPRT complexed with the purine base substrates guanine or xanthine in combination with cPRib-PP, an analog of the second substrate PRib-PP, have been solved to 2.0 A resolution. In these two structures the disordered phosphate-binding loop of uncomplexed XGPRT becomes ordered through interactions with the 5'-phosphate group of cPRib-PP. The cyclopentane ring of cPRib-PP has the C3 exo pucker conformation, stabilised by the cPRib-PP-bound Mg2+. The purine base specificity of XGPRT appears to be due to water-mediated interactions between the 2-exocyclic groups of guanine or xanthine and side-chains of Glu136 and Asp140, as well as the main-chain oxygen atom of Ile135. Asp92, together with Lys115, could help stabilise the N7-protonated tautomer of the incoming base and could act as a general base to remove the proton from N7 when the nucleotide product is formed. The 2.6 A resolution structure of XGPRT complexed with product GMP is similar to the substrate-bound complexes. However, the ribose ring of GMP is rotated by approximately 24 degrees compared with the equivalent ring in cPRib-PP. This rotation results in the loss of all interactions between the ribosyl group and the enzyme in the product complex.
通过X射线晶体学确定了大肠杆菌黄嘌呤-鸟嘌呤磷酸核糖转移酶（XGPRT）的游离形式、底物结合形式和产物结合形式的结构。将这些结构与先前确定的镁和硫酸盐结合的XPRT结构进行了比较。分辨率为2.25 Å的游离XGPRT结构证实了在其他PRTases中鉴定出的可移动环及其周围残基的灵活性，并表明在没有结合配体的情况下，活性位点处Arg37的顺式肽构象得以维持。已将XGPRT与嘌呤碱底物鸟嘌呤或黄嘌呤结合cPRib-PP（第二种底物PRib-PP的类似物）的复合物结构解析到2.0 Å分辨率。在这两种结构中，未复合的XGPRT中无序的磷酸结合环通过与cPRib-PP的5'-磷酸基团相互作用而变得有序。cPRib-PP的环戊烷环具有C3外向褶皱构象，由结合cPRib-PP的Mg2+稳定。XGPRT的嘌呤碱特异性似乎归因于鸟嘌呤或黄嘌呤的2-外环基团与Glu136和Asp140的侧链以及Ile135的主链氧原子之间的水介导相互作用。Asp92与Lys115一起可以帮助稳定进入碱基的N7-质子化互变异构体，并在形成核苷酸产物时作为通用碱从N7上去除质子。与产物GMP复合的XGPRT的2.6 Å分辨率结构与底物结合复合物相似。然而，与cPRib-PP中的等效环相比，GMP的核糖环旋转了约24度。这种旋转导致产物复合物中核糖基与酶之间的所有相互作用丧失。
