分类: 生物学 >> 生物物理学 提交时间: 2016-05-12
摘要: The FAOD/FPOD family of proteins has the potential to be useful for the longterm detection of blood glucose levels in diabetes patients. A bottleneck for this application is to find or engineer a FAOD/FPOD family enzyme that is specifically active towards alpha-fructosyl peptides but is inactive towards other types of glycated peptides. Here, the crystal structure of fructosyl peptide oxidase from Eupenicillium terrenum (EtFPOX) is reported at 1.9 angstrom resolution. In contrast to the previously reported structure of amadoriase II, EtFPOX has an open substrate entrance to accommodate the large peptide substrate. The functions of residues critical for substrate selection are discussed based on structure comparison and sequence alignment. This study reveals the first structural details of group I FPODs that prefer alpha-fructosyl substrates and could provide significant useful information for uncovering the mechanism of substrate specificity of FAOD/FPODs and guidance towards future enzyme engineering for diagnostic purposes.
分类: 生物学 >> 生物物理学 提交时间: 2016-05-11
摘要: Electron transfer (ET) is widely used for driving the processes that underlie the chemistry of life. However, our abilities to probe electron transfer mechanisms in proteins and design redox enzymes are limited, due to :the lack of methods to site-specifically insert electron acceptors into proteins in vivo. Here we describe the synthesis and genetic incorporation of 4-fluoro-3-nitrophenylalanine (FNO(2)Phe), which has similar reduction potentials to NAD(P)H and ferredoxin, the most important biological reductants. Through the genetic incorporation of FNO2Phe into green fluorescent:protein (GFP) and femtosecond transient absorption measurement, we show that photoinduced electron transfer (PET) from the GFP chromophore to FNO2Phe occurs very fast (within 11 ps), which is comparable to that of the first electron transfer step in photosystem I, from P700* to A(0). This genetically encoded, low-reduction potential unnatural amino acid (UAA) can significantly in-Trove our ability to investigate electron transfer mechanisms in complex reductases and facilitate the design of miniature proteins that mimic their functions.