Large conformational changes in the Escherichia coli tryptophan synthase β₂ subunit upon pyridoxal 5′‐phosphate binding

Abstract

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).

The Mtm1p carrier and pyridoxal 5′-phosphate cofactor trafficking in yeast mitochondria

“…The lower maximal ALAS activity observed for the Mtm1p-deficient mitochondria may indicate that the ALAS apoenzyme is unstable in the absence of PLP. Cofactor binding is known to significantly increase the stability of PLP-dependent enzymes, with as much as 30 kcal/mol stabilization of the PLP complex reported for cytosolic aspartate aminotransferase [45], and PLP binding has been reported to induce large conformational changes in the β-subunit of tryptophan synthase [46]. There is also evidence, at least in mammalian cells, for a mitochondrial protease that selectively degrades the ALAS apoprotein [47].…”

Crystal structures of open and closed forms of d‐serine deaminase from Salmonella typhimurium – implications on substrate specificity and catalysis

“…2B) [23], serine racemase from Rattus norvegicus and Homo sapiens [30], and OASS from S. typhimurium [31]. In tryptophan synthase β from E. coli [32] and l ‐serine dehydratase from Rattus norvegicus (rat liver LSD) [9], a similar domain movement is observed between apo and holo forms of the enzymes. Considering that SeMetDSD crystal was grown in the presence of the inhibitor (isoserine), it is reasonable to assume that SeMetDSD represents the ligand bound closed form of the enzyme while the WtDSD represents the unliganded open form of the enzyme.…”

Directed Evolution Improves the Enzymatic Synthesis of L-5-Hydroxytryptophan by an Engineered Tryptophan Synthase

“…Mutation analysis Chimera 1.13 was used to analyse the substrate channel space of tryptophan synthase (PDB: 2dh6), and it was found that the amino acid residues of tryptophan synthase at positions 231 and 382 were located in the loop formed by the substrate channel [24]. The amino acid residue at site 231 was mutated from valine to alanine, and the residue at site 382 was mutated from lysine to glycine, which may change the folding direction of the loop, thus changing the size and shape of channel entrance and promoting mutated enzymatic activity (Fig.…”