Kinetic and Structural Analysis of Two Linkers in the Tautomerase Superfamily: Analysis and Implications

Abstract

The tautomerase superfamily (TSF) is a collection of enzymes and proteins that share a simple β–α–β structural scaffold. Most members are constructed from a single-core β–α–β motif or two consecutively fused β–α–β motifs in which the N-terminal proline (Pro-1) plays a key and unusual role as a catalytic residue. The cumulative evidence suggests that a gene fusion event took place in the evolution of the TSF followed by duplication (of the newly fused gene) to result in the diversification of activity that is seen today. Analysis of the sequence similarity network (SSN) for the TSF identified several linking proteins (“linkers”) whose similarity links subgroups of these contemporary proteins that might hold clues about structure–function relationship changes accompanying the emergence of new activities. A previously uncharacterized pair of linkers (designated N1 and N2) was identified in the SSN that connected the 4-oxalocrotonate tautomerase (4-OT) and cis-3-chloroacrylic acid dehalogenase (cis-CaaD) subgroups. N1, in the cis-CaaD subgroup, has the full complement of active site residues for cis-CaaD activity, whereas N2, in the 4-OT subgroup, lacks a key arginine (Arg-39) for canonical 4-OT activity. Kinetic characterization and nuclear magnetic resonance analysis show that N1 has activities observed for other characterized members of the cis-CaaD subgroup with varying degrees of efficiencies. N2 is a modest 4-OT but shows enhanced hydratase activity using allene and acetylene compounds, which might be due to the presence of Arg-8 along with Arg-11. Crystallographic analysis provides a structural context for these observations.

Comparative Metabolic Pathways Analysis and Subtractive Genomics Profiling to Prioritize Potential Drug Targets Against Streptococcus pneumoniae

“…The 4-oxalocrotonate tautomerase enzyme is involved in benzonate and xylene degradation pathways which is unique to S. pneumonia . The benzonate and xylene degradation pathways degrade the aromatic compounds and amino acids into essential hydrocarbons to fulfill its carbon and energy requirements ( Medellin et al, 2020 ; Baas et al, 2021 ). On the other hand, the sensor histidine kinase is involved in the Two-Component System (TCS) of S. pneumonia and regulates certain physiological conditions.…”

Kinetic, Inhibition, and Structural Characterization of a Malonate Semialdehyde Decarboxylase-like Protein from Calothrix sp. PCC 6303: A Gateway to the non-Pro1 Tautomerase Superfamily Members

“…13 N2 is an enzyme from Gammaproteobacteria bacterium SG8_31 (UniProt accession A0A0S8FF56) and is in the 4-oxalocrotonate tautomerase (4-OT) subgroup, as described elsewhere. 13 The DEAE Sephadex, Phenyl-Sepharose 6 Fast Flow, and Sephadex G-75 resins were obtained from GE Healthcare Biosciences (Pittsburgh, PA). The Econo-Column chromatography columns were obtained from BioRad (Hercules, CA).…”

“…24 MSA can be generated from propiolate using the enzyme designated N2 (see Scheme 3). 13 N2 is from G. bacterium SG8_31 (UniProt accession A0A0S8FF56) and is in the 4-oxalocrotonate tautomerase (4-OT) subgroup, as described elsewhere. 13 Accordingly, N2 (410 μL of 6.4 mg/mL) was added to propiolic acid to make a final solution of 1 mL (21 mg/mL, 300 mM) in 100 mM dibasic Na 2 HPO 4 buffer made pH 8 with 1 M NaOH.…”

“…The 1 H NMR protocol for monitoring the reactions of 437 and JJ3 with 2hydroxymuconate followed that described elsewhere, as modified below. 13 Accordingly, an amount of 2-HM (1.2 mg) in 30 μL of dimethyl sulfoxide-d 6 (DMSO-d 6 ) was added to the 20 mM Na 3 PO 4 buffer (final total volume of 600 μL). The final pH of the solution was ∼8.0.…”

“…MSA was generated by the N2-catalyzed hydration of propiolate (Scheme 3), as previously described. 13 The kinetic parameters are summarized in Table 2.…”