Mechanism of product chain length determination and the role of a flexible loop in Escherichia coli undecaprenyl-pyrophosphate synthase catalysis

被引:79
作者
Ko, TP
Chen, YK
Robinson, H
Tsai, PC
Gao, YG
Chen, APC
Wang, AHJ [1 ]
Liang, PH
机构
[1] Acad Sinica, Inst Biol Chem, Taipei 115, Taiwan
[2] Natl Taiwan Univ, Inst Biochem Sci, Taipei 106, Taiwan
关键词
D O I
10.1074/jbc.M106747200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The Escherichia coli undecaprayl-pyrophosphate synthase (UPPs) structure has been solved using the single wavelength anomalous diffraction method. The putative substrate-binding site is located near the end of the betaA-strand with Asp-26 playing a critical catalytic role. In both subunits, an elongated hydrophobic tunnel is found, surrounded by four beta -strands (betaA-betaB-betaD-betaC) and two helices (alpha2 and alpha3) and lined at the bottom with large residues Ile-62, Leu-137, Val-105, and His-103. The product distributions formed by the use of the I62A, V105A, and H103A mutants are similar to those observed for wild-type UPPs. Catalysis by the L137A UPPs, on the other hand, results in predominantly the formation of the C-70 polymer rather than the C-55 polymer. Ala-69 and Ala-143 are located near the top of the tunnel. In contrast to the A143V reaction, the C-30 intermediate is formed to a greater extent and is longer lived in the process catalyzed by the A69L mutant. These findings suggest that the small side chain of Ala-69 is required for rapid elongation to the C-55 product, whereas the large hydrophobic side chain of Leu-137 is required to limit the elongation to the C-55 product. The roles of residues located on a flexible loop were investigated. The S71A, N74A, or R77A mutants displayed 25-200-fold decrease in k(cat) values. W75A showed an 8-fold increase of the FPP K-m value, and 22-33-fold increases in the IPP K-m values were observed for E81A and S71A. The loop may function to bridge the interaction of IPP with FPP, needed to initiate the condensation reaction and serve as a hinge to control the substrate binding and product release.
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页码:47474 / 47482
页数:9
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