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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.

SUBMITTER: Allen KN

PROVIDER: S-EPMC10251251 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature

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

Allen Karen N KN Whitman Christian P CP

Biochemistry 20211019 46

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 <i>Biochemistry</i> that demonstrated the potential power of mechanistically diverse superfamilies to identify common ancestry, predict function, and, in some cases, predict specificity. This Perspec ...[more]

PMID: 34664940

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Project description:The l-rhamnonate dehydratase (RhamD) function was assigned to a previously uncharacterized family in the mechanistically diverse enolase superfamily that is encoded by the genome of Escherichia coli K-12. We screened a library of acid sugars to discover that the enzyme displays a promiscuous substrate specificity: l-rhamnonate (6-deoxy- l-mannonate) has the "best" kinetic constants, with l-mannonate, l-lyxonate, and d-gulonate dehydrated less efficiently. Crystal structures of the RhamDs from both E. coli K-12 and Salmonella typhimurium LT2 (95% sequence identity) were obtained in the presence of Mg (2+); the structure of the RhamD from S. typhimurium was also obtained in the presence of 3-deoxy- l-rhamnonate (obtained by reduction of the product with NaBH 4). Like other members of the enolase superfamily, RhamD contains an N-terminal alpha + beta capping domain and a C-terminal (beta/alpha) 7beta-barrel (modified TIM-barrel) catalytic domain with the active site located at the interface between the two domains. In contrast to other members, the specificity-determining "20s loop" in the capping domain is extended in length and the "50s loop" is truncated. The ligands for the Mg (2+) are Asp 226, Glu 252 and Glu 280 located at the ends of the third, fourth and fifth beta-strands, respectively. The active site of RhamD contains a His 329-Asp 302 dyad at the ends of the seventh and sixth beta-strands, respectively, with His 329 positioned to function as the general base responsible for abstraction of the C2 proton of l-rhamnonate to form a Mg (2+)-stabilized enediolate intermediate. However, the active site does not contain other acid/base catalysts that have been implicated in the reactions catalyzed by other members of the MR subgroup of the enolase superfamily. Based on the structure of the liganded complex, His 329 also is expected to function as the general acid that both facilitates departure of the 3-OH group in a syn-dehydration reaction and delivers a proton to carbon-3 to replace the 3-OH group with retention of configuration.

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

Publications

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

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