The Birth of Genomic Enzymology: Discovery of the Mechanistically Diverse Enolase Superfamily

Abstract

Enzymes are categorized into superfamilies by sequence, structural, and mechanistic similarities. The evolutionary implications can be profound. Until the mid-1990s, the approach was fragmented largely due to limited sequence and structural data. However, in 1996, Babbitt et al. published a paper in Biochemistry that demonstrated the potential power of mechanistically diverse superfamilies to identify common ancestry, predict function, and, in some cases, predict specificity. This Perspective describes the findings of the original work and reviews the current understanding of structure and mechanism in the founding family members. The outcomes of the genomic enzymology approach have reached far beyond the functional assignment of members of the enolase superfamily, inspiring the study of superfamilies and the adoption of sequence similarity networks and genome context and yielding fundamental insights into enzyme evolution.

Figure 1.. The (β/α)₇β-barrel fold and active site of the enolase superfamily.

A. Ribbon diagram of enolase (PDB 1ONE) with metal-binding residues (gold stick) and metal (Mg²⁺; blue tint sphere) which are conserved and identifying features of the enolase superfamily. Catalytic residues in enolase at conserved positions in the active site (grey stick). Strands of the (β/α)₇β-barrel fold are highlighted in blue with strands numbered S1-S8 and N-terminal excursion from the barrel made transparent B. Comparison of the active-site residues in MR (blue; PDB 1MNS), MLE (pink; PDB 1BKH) and enolase (grey) with conserved metal position shown for MR (grey sphere).

Figure 2.. Comparative active sites in the MR subgroup.

Comparison of the active-site residues in MR (blue; PDB 1MNS), and A. GalD (gold) with conserved metal position shown for GalD (gold sphere) and D-lyxose (gold-stick) and B. GlcD (wheat; PDB 1ECQ) with metal position shown for GlcD (grey sphere) and 4-deoxyglucarate (wheat stick). C. Overlay of GalD (gold) with RspA (yellow; PDB 4IL2) with metal position (gold sphere) and D-lyxose (gold stick) shown for GalD.

Figure 3.. Conservative and diverged active sites in the MR subgroup.

A. Comparison of the conserved active-site residues in rTSγ (magenta; PDB 4A35) and FucD (PDB 2HXT; brown) with conserved metal position (silver sphere) and D-erythronohydroxamate (brown-stick) shown for FucD. Residues labelled according to the sequences in Babbitt et al. B. The active sites of MR (blue; PDB 1MNS) and ManD (purple; PDB 2QJJ) with metal position shown for ManD (grey sphere) and 2-keto-3-deoxy-D-gluconate (yellow stick) from PDB 2QJN. Note that the coordinates of ManD were used from 2QJJ as the electron density was resolved for the long loop from β-strand 2 bearing Tyr159; the position of 2-keto-3-deoxy-(D)-gluconate was obtained by superposition of ManD from PDB 2QJN.

Figure 4.. Conservative and diverged active sites in the MLE subgroup.

A. Comparison of the conserved active-site residues in MLEI (pink; PDB 1BKH) and A. MLEII (PDB 2CHR; green) with Mn²⁺ metal position shown for MLEII ((silver sphere); B. OSBS (purple; PDB 1SJB) with o-succinylbenzoic acid and Mg²⁺ shown as silver sphere and C. Beta-MAL (grey; PDB 1KKR) with (2S,3S)-3-methylaspartic acid. In B, C, and D and Mg²⁺ is shown as silver sphere.

Figure 5.. Active site of enolase complexed with substrate/product.

Enolase (PDB 1ONE) shown as ribbon with catalytic residues and phosphoenolpyruvate (grey stick) and Mg²⁺ (silver sphere); 2-phosphoglycerate (2-PGA) not shown.

Scheme 1.

Substrates, Intermediates, and Products for MR, MLE I, and Enolase

Scheme 2.

The GalD-catalyzed Reaction

Scheme 3.

The GlucD-catalyzed Reactions

Scheme 4.

Reactions catalyzed by RspA (Alt D/Man D)

Scheme 5.

Reactions catalyzed by FucD and ManD

Scheme 6.

The MLE II- and NAAAR-catalyzed Reactions

Scheme 7.

The OSBS-catalyzed Reaction

Scheme 8.

NAAAR-catalyzed racemization of M-acylamino acids

Scheme 9.

The MAL-catalyzed Reaction

Scheme 10.

Misassigned and Corrected Reactions for CPEPS