Residues Required for Activity in <i>Escherichia coli o</i>-Succinylbenzoate Synthase (OSBS) Are Not Conserved in All OSBS Enzymes

Zhu, Wan Wen; Wang, Chenxi; Jipp, Jacob; Ferguson, L.T.; Lucas, Stephanie N.; Hicks, Michael A.; Glasner, Margaret E.

doi:10.1021/bi300753j

Cited by 14 publications

(83 citation statements)

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“…Like most other members of the enolase superfamily, the five enzymes from the NSAR/OSBS subfamily are multimers (21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40). The three previously characterized NSAR/OSBS subfamily enzymes are (53). Subfamilies (shown as wedges) were defined by grouping sequences whose pairwise amino acid sequence identity is >40%.…”

Section: Resultsmentioning

confidence: 99%

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Loss of quaternary structure is associated with rapid sequence divergence in the OSBS family

Odokonyero

Sakai

Patskovsky³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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The rate of protein evolution is determined by a combination of selective pressure on protein function and biophysical constraints on protein folding and structure. Determining the relative contributions of these properties is an unsolved problem in molecular evolution with broad implications for protein engineering and function prediction. As a case study, we examined the structural divergence of the rapidly evolving o-succinylbenzoate synthase (OSBS) family, which catalyzes a step in menaquinone synthesis in diverse microorganisms and plants. On average, the OSBS family is much more divergent than other protein families from the same set of species, with the most divergent family members sharing <15% sequence identity. Comparing 11 representative structures revealed that loss of quaternary structure and large deletions or insertions are associated with the family's rapid evolution. Neither of these properties has been investigated in previous studies to identify factors that affect the rate of protein evolution. Intriguingly, one subfamily retained a multimeric quaternary structure and has small insertions and deletions compared with related enzymes that catalyze diverse reactions. Many proteins in this subfamily catalyze both OSBS and N-succinylamino acid racemization (NSAR). Retention of ancestral structural characteristics in the NSAR/OSBS subfamily suggests that the rate of protein evolution is not proportional to the capacity to evolve new protein functions. Instead, structural features that are conserved among proteins with diverse functions might contribute to the evolution of new functions.enolase superfamily | protein structure | protein structure-function relationships I nvestigating the causes and effects of protein sequence divergence is the key to identifying properties that enable proteins to evolve new functions. Previous studies found that constraints imposed by biophysical properties such as protein folding and stability, translational accuracy, and interactions with other proteins make a large contribution to the rate of protein evolution (1-11). However, the relative contributions of biophysical properties versus functional constraints is an open question (2). Given that the rate of protein evolution varies over several orders of magnitude, the evolutionary rate of each protein is probably determined by a unique blend of biophysical and functional constraints (12-15). Thus, the evolutionary simulations and statistical analyses of large protein datasets that comprise the primary focus of this field need to be supplemented with case studies.Here, we present the extraordinarily diverse o-succinylbenzoate synthase (OSBS) family as such a case study. The OSBS family belongs to the enolase superfamily, a group of evolutionarily related protein families that have a common fold but catalyze diverse reactions (16). The rate of sequence divergence in the OSBS family is much faster than other families in the enolase superfamily. For example, the average pairwise amino acid sequence identity of OSBSs fr...

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Section: Resultsmentioning

confidence: 99%

“…To date, our data do not support this idea, although experiments have been limited to a small number of active-site residues. Mutations of active-site residues have similar effects in E. coli OSBS, T. fusca OSBS, and P. putida mandelate racemase (MR), reducing k cat /K M by ∼10-to 500-fold (52)(53)(54)(55).…”

Section: Discussionmentioning

confidence: 99%

Loss of quaternary structure is associated with rapid sequence divergence in the OSBS family

Odokonyero

Sakai

Patskovsky³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…40,41 These observations reinforce the well-known difficulties in the functional annotation of closely related homologues. 42 …”

Section: Discussionmentioning

confidence: 99%

“…Characterization can assist in the identification of new homologues in the MSAD family, and potentially, their functional annotation, which is a fundamental problem in biochemistry. 42 …”

Section: Discussionmentioning

confidence: 99%

Kinetic, Mutational, and Structural Analysis of Malonate Semialdehyde Decarboxylase from Coryneform Bacterium Strain FG41: Mechanistic Implications for the Decarboxylase and Hydratase Activities

Guo¹,

Serrano²,

Poelarends

et al. 2013

Biochemistry

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Malonate semialdehyde decarboxylase from Pseudomonas pavonaceae 170 (designated Pp MSAD) is in a bacterial catabolic pathway for the nematicide 1,3-dichloropropene. MSAD has two known activities: it catalyzes the metal-ion independent decarboxylation of malonate semialdehyde to produce acetaldehyde and carbon dioxide, as well as a low-level hydration of 2-oxo-3-pentynoate to yield acetopyruvate. The latter activity is not known to be biologically relevant. Previous studies identified Pro-1, Asp-37, and a pair of arginines (Arg-73 and Arg-75) as critical residues in these activities. MSAD from Coryneform bacterium strain FG41 (designated FG41 MSAD) shares 38% pairwise sequence identity with the Pseudomonas enzyme including Pro-1 and Asp-37. However, Gln-73 replaces Arg-73, and the second arginine is shifted to Arg-76 by the insertion of a glycine. In order to determine how these changes relate to the activities of FG41 MSAD, the gene was cloned and the enzyme expressed and characterized. The enzyme has a comparable decarboxylase activity, but a significantly reduced hydratase activity. Mutagenesis along with crystal structures of the native enzyme (2.0 Å resolution) and the enzyme modified by a 3-oxopropanoate moiety (resulting from the incubation of enzyme and 3-bromopropiolate) (2.2 Å resolution) provided a structural basis. The roles of Pro-1 and Asp-37 are likely the same as those proposed for MSAD. However, the side chains of Thr-72, Gln-73, and Tyr-123 replace those of Arg-73 and Arg-75 in the mechanism and play a role in binding and catalysis. The structures also show that Arg-76 is likely too distant to play a direct role in the mechanism. FG41 MSAD is the second functionally annotated homologue in the MSAD family of the tautomerase superfamily and could represent a new subfamily.

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“…6–8 Members of two subfamilies, E. coli OSBS from the γ-Proteobacteria 1 subfamily and Amycolatopsis NSAR/OSBS from the NSAR/OSBS subfamily, were previously characterized. 9–14 T. fusca OSBS belongs to the Actinobacteria OSBS subfamily. Like the γ-Proteobacteria 1 subfamily, genome context indicates that OSBS is the biological function of all members of the Actinobacteria subfamily.…”

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Divergent Evolution of Ligand Binding in the o-Succinylbenzoate Synthase Family

et al. 2013

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Thermobifida fusca o-succinylbenzoate synthase (OSBS), a member of the enolase superfamily that catalyzes a step in menaquinone biosynthesis, shares 22% and 28% amino acid sequence identity with two previously characterized OSBS enzymes from Escherichia coli and Amycolatopsis sp. T-1-60, respectively. These values are considerably lower than typical sequence identities among homologous proteins that have the same function. To determine how such divergent enzymes catalyze the same reaction, we solved the structure of T. fusca OSBS and identified amino acids that are important for ligand binding. We discovered significant differences in structure and conformational flexibility between T. fusca OSBS and other members of the enolase superfamily. In particular, the 20s loop, a flexible loop in the active site that permits ligand binding and release in most enolase superfamily proteins, has a four-amino acid deletion and is well ordered in T. fusca OSBS. Instead, flexibility of a different region allows the substrate to enter from the other side of the active site. T. fusca OSBS was more tolerant of mutations at residues that were critical for activity in E. coli OSBS. Also, replacing active site amino acids found in one protein with the amino acids that occur at the same place in the other protein reduces catalytic efficiency. Thus, the extraordinary divergence between these proteins does not appear to reflect a higher tolerance of mutations. Instead, large deletions outside the active site were accompanied by alteration of active site size and electrostatic interactions, resulting in small but significant differences in ligand binding.

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Residues Required for Activity in Escherichia coli o-Succinylbenzoate Synthase (OSBS) Are Not Conserved in All OSBS Enzymes

Cited by 14 publications

References 50 publications

Loss of quaternary structure is associated with rapid sequence divergence in the OSBS family

Loss of quaternary structure is associated with rapid sequence divergence in the OSBS family

Kinetic, Mutational, and Structural Analysis of Malonate Semialdehyde Decarboxylase from Coryneform Bacterium Strain FG41: Mechanistic Implications for the Decarboxylase and Hydratase Activities

Divergent Evolution of Ligand Binding in the o-Succinylbenzoate Synthase Family

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