Structural, Kinetic, and Mechanistic Analysis of an Asymmetric 4-Oxalocrotonate Tautomerase Trimer

Abstract

A 4-oxalocrotonate tautomerase (4-OT) trimer has been isolated from Burkholderia lata and a kinetic, mechanistic, and structural analysis has been performed. The enzyme is the third described oligomer state for 4-OT along with a homo-and heterohexamer. The 4-OT trimer is part of a small subset of sequences (133 sequences) within the 4-OT subgroup of the tautomerase superfamily (TSF). The TSF has two distinct features: members are composed of a single β-α-β unit (homo-and heterohexamer) or two consecutively joined β-α-β units (trimer) and generally have a catalytic amino-terminal proline. The enzyme, designated fused 4-OT, functions as a 4-OT where the active site groups (Pro-1, Arg-39, Arg-76, Phe-115, Arg-127) mirror those in the canonical 4-OT from Pseudomonas putida mt-2. Inactivation by 2-oxo-3-pentynoate suggests that Pro-1 of fused 4-OT has a low pK a enabling the prolyl nitrogen to function as a general base. A remarkable feature of the fused 4-OT is the absence of P3 rotational symmetry in the structure (1.5 Å resolution). The asymmetric arrangement of the trimer is not due to the fusion of the two β-α-β building blocks because an engineered "unfused" variant that breaks the covalent bond between the two units (to generate a heterohexamer) assumes the same asymmetric oligomerization state. It

Gene Fusion and Directed Evolution to Break Structural Symmetry and Boost Catalysis by an Oligomeric C−C Bond‐Forming Enzyme

“…Consequently, the Pro‐1 residues and active sites in subunits A and C are positioned adjacent to each other across one of the trimeric interfaces, while at another interface the Pro‐1 residue of chain B is positioned adjacent to Pro‐68 of chain A (Figure 3B). Intriguingly, a similar uncommon asymmetric trimeric architecture has recently been observed for certain naturally fused homotrimeric 4‐OT homologs [19, 20] . Analysis of the subunit interfaces indeed suggests that for 4‐OT(F11) the assembly of an asymmetric trimer is energetically more favorable than that of a symmetric trimer (Table S2) [20] .…”

Structural Basis for the Asymmetry of a 4-Oxalocrotonate Tautomerase Trimer

“…A majority of the sequences in this subset (133 sequences) fall into two clusters identified as the “Linker 2” and “Fused 4-OT” clusters (47 and 86 sequences, respectively) in the SSN. , Structural analysis of one protein (designated “linker 2”) in the Linker 2 cluster showed that it is a symmetrical trimer (Figure A) that consists of three αβ interfaces (where α and β refer to the N- and C-terminal regions, respectively, of the adjoining β–α–β units). Each interface has a single catalytic Pro-1.…”

“…Within the 4-OT subgroup, there is a subset of sequences double the length of that of the short 4-OTs. , These 4-OTs form a separate subgroup that still connects to the short 4-OTs in a sequence similarity network (SSN) . More importantly, they might share features with possible progenitors for the other four subgroups in the TSF, which are of the double length represented by the fused 4-OTs.…”

“…Symmetric and asymmetric trimers characterized in the fused 4-OT subset of sequences in the 4-OT subgroup . (A) Linker 2 showing the typical TSF trimer consisting of three αβ interfaces, each containing a catalytic Pro-1.…”

Gene Fusion and Directed Evolution to Break Structural Symmetry and Boost Catalysis by an Oligomeric C−C Bond‐Forming Enzyme

“…Intriguingly, a similar uncommon asymmetric trimeric architecture has recently been observed for certain naturally fused homotrimeric 4-OT homologs. [19,20] Analysis of the subunit interfaces indeed suggests that for 4-OT(F11) the assembly of an asymmetric trimer is energetically more favorable than that of a symmetric trimer (Table S2). [20] Hence, both natural and laboratory evolution of tandem fused 4-OT enzymes may thus result in unusual asymmetric trimers.…”