Large conformational changes in the Escherichia coli tryptophan synthase beta(2) subunit upon pyridoxal 5'-phosphate binding.
To understand the basis for the lower activity of the tryptophan synthase beta(2) subunit in comparison to the alpha(2)beta(2) complex, we determined the crystal structures of apo-beta(2) and holo-beta(2) from Escherichia coli at 3.0 and 2.9 A resolutions, respectively. To our knowledge, this is the first report of both beta(2) subunit structures with and without pyridoxal-5'-phosphate. The apo-type molecule retained a dimeric form in solution, as in the case of the holo-beta(2) subunit. The subunit structures of both the apo-beta(2) and the holo-beta(2) forms consisted of two domains, namely the N domain and the C domain. Although there were significant structural differences between the apo- and holo-structures, they could be easily superimposed with a 22 degrees rigid body rotation of the C domain. The pyridoxal-5'-phosphate-bound holo-form had multiple interactions between the two domains and a long loop (residues 260-310), which were missing in the apo-form. Comparison of the structures of holo-Ecbeta(2) and Stbeta(2) in the alpha(2)beta(2) complex from Salmonella typhimurium (Stalpha(2)beta(2)) identified the cause of the lower enzymatic activity of holo-Ecbeta(2) in comparison with Stalpha(2)beta(2). The substrate (indole) gate residues, Tyr279 and Phe280, block entry of the substrate into the beta(2) subunit, although the indole can directly access the active site as a result of a wider cleft between the N and C domains in the holo-Ecbeta(2) subunit. In addition, the structure around betaAsp305 of the holo-Ecbeta(2) subunit was similar to the open state of Stalpha(2)beta(2) with low activity, resulting in lower activity of holo-Ecbeta(2).
Nishio
Kazuya
K
Institute for Protein Research, Osaka University, Japan.
Ogasahara
Kyoko
K
Morimoto
Yukio
Y
Tsukihara
Tomitake
T
Lee
Soo Jae
SJ
Yutani
Katsuhide
K
D000495:Allosteric Site; D000595:Amino Acid Sequence; D001051:Apoenzymes / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011732:Pyridoxal Phosphate / Q000737:chemistry* / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology; D016415:Sequence Alignment; D014367:Tryptophan Synthase / Q000737:chemistry* / Q000378:metabolism
D001051:Apoenzymes; D020035:Holoenzymes; D021122:Protein Subunits; D011732:Pyridoxal Phosphate; D014367:Tryptophan Synthase