The TIM‐barrel fold: a versatile framework for efficient enzymes

Abstract

Recent studies on triosephosphate isomerase (TIM)-barrel enzymes highlight the remarkable versatility of the TIMbarrel scaffold. At least 15 distinct enzyme families use this framework to generate the appropriate active site geometry, always at the C-terminal end of the eight parallel L L-strands of the barrel. Sequence and structure comparisons now suggest that many of the TIM-barrel enzymes are evolutionarily related. Common structural properties of TIM-barrel enzymes are discussed. ß

Mimicking enzyme evolution by generating new (βα) 8 -barrels from (βα) 4 -half-barrels

“…For construction of hisFA with linker, the C-terminal half of hisA (hisA-C) was amplified by PCR using the plasmid SKϩ͞III P-P as the template, the oligonucleotide 5Ј-ATA GGA TCC GGT GTG GAG CCC GTG-3Ј with a BamHI site (in bold) as the 5Ј primer, and the oligonucleotide [2] 5Ј-ATA GCG GCC GCG CGA GCA TAT CTC TTC ATC AC-3Ј with a NotI site (in bold) as the 3Ј primer. The amplified hisA-C was cloned into pET24a(ϩ) by using BamHI and NotI, yielding the plasmid pET24a(ϩ)-hisA-C. Then, hisF-N was amplified by PCR using the plasmid SKϩ͞III P-P as the template, the oligonucleotide [3] 5Ј-AGC CAT ATG CTC GCT AAA AGA ATA ATC GCG-3Ј with a NdeI site (in bold) as the 5Ј primer, and the oligonucleotide 5Ј-GCC GGA TCC ACT CCC AAA AGT TTG-3Ј with a BamHI site (in bold) as the 3Ј primer. The amplified hisF-N was cloned into pET24a(ϩ)-hisA-C by using NdeI and BamHI, yielding the plasmid pET24a(ϩ)-hisFlinkA.…”

“…The linker was removed by PCR with pET24a(ϩ)-hisFlinkA as the template as follows. The N-terminal half of hisF was amplified by using the oligonucleotide [3] as the 5Ј primer and 5Ј-GAA CAC GGG CTC CAC ACT CCC AAA AGT TTG-3Ј as the 3Ј primer. The amplification product was used in a second PCR as a 5Ј primer together with the oligonucleotide [2] as the 3Ј primer.…”

“…The fold of the canonical (␤␣) 8 -barrel consists of a central barrel of eight parallel ␤-strands and eight external ␣-helices. Connecting loops are located at the N-terminal (␣-␤ loops) and Cterminal (␤-␣ loops) face of the barrel (3,4). Although (␤␣) 8 barrel enzymes catalyze a broad range of chemically diverse reactions, their active sites always are located at the C-terminal face of the barrel (5).…”

Establishing wild-type levels of catalytic activity on natural and artificial (βα) 8 -barrel protein scaffolds

“…The (␤␣) 8 -(or TIM) barrel is the most frequent and most versatile fold among naturally occurring enzymes (7,8). The canonical TIM-barrel is composed of 8 modular units, each of which consists of a ␤-strand and a ␣-helix that are connected by a ␤␣-loop; individual units are linked by ␣␤-loops.…”

Inactivation of Mandelate Racemase by 3-Hydroxypyruvate Reveals a Potential Mechanistic Link between Enzyme Superfamilies

“…44,45 Although the mechanism-based inhibition of these two NAL superfamily members by 3-HP also proceeds via initial Schiff-base formation followed by deprotonation, 44,45 the apparent second-order rate constant for the inactivation of MR exceeds that observed for inactivation of DHDPS (0.56 M -1 s -1 ) by ∼138-fold. This observation is in accord with the active site of MR being optimized to promote enolate formation.Many of the enzymes sharing the highly prevalent (α/β)8-barrel (or TIM barrel) folding topology 53,[57][58][59][60][61][62] are believed to have arisen via divergent evolution from a common ancestor or a limited number of progenitor enzymes. 53,60,[63][64][65][66][67] Many aldolases share this fold with members of the enolase and NAL superfamilies, as well as sharing a similar mechanistic theme, i.e., the formation of a "masked" carbanionic intermediate (Scheme 3).…”