Evolution of enzymatic activity in the enolase superfamily: structural studies of the promiscuous o-succinylbenzoate synthase from Amycolatopsis.

Abstract

Divergent evolution of enzyme function is commonly explained by a gene duplication event followed by mutational changes that allow the protein encoded by the copy to acquire a new function. An alternate hypothesis is that this process is facilitated when the progenitor enzyme acquires a second function while maintaining the original activity. This phenomenon has been suggested to occur in the o-succinylbenzoate synthase (OSBS) from a species of Amycolatopsis that catalyzes not only the physiological syn-dehydration reaction of 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate but also an accidental racemization of N-acylamino acids [Palmer, D. R., Garrett, J. B., Sharma, V., Meganathan, R., Babbitt, P. C., and Gerlt, J. A. (1999) Biochemistry 38, 4252-4258]. To understand the molecular basis of this promiscuity, three-dimensional structures of liganded complexes of this enzyme have been determined, including the product of the OSBS reaction and three N-acylamino acid substrates for the N-acylamino acid racemase (NAAAR) reaction, N-acetylmethionine, N-succinylmethionine, and N-succinylphenylglycine, to 2.2, 2.3, 2.1, and 1.9 A resolution, respectively. These structures show how the active-site cavity can accommodate both the hydrophobic substrate for the OSBS reaction and the substrates for the accidental NAAAR reaction. As expected, the N-acylamino acid is sandwiched between lysines 163 and 263, which function as the catalytic bases for the abstraction of the alpha-proton in the (R)- and (S)-racemization reactions, respectively [Taylor Ringia, E. A., Garrett, J. B, Thoden, J. B., Holden, H. M., Rayment, I., and Gerlt, J. A. (2004) Biochemistry 42, 224-229]. Importantly, the protein forms specific favorable interactions with the hydrophobic amino acid side chain, alpha-carbon, carboxylate, and the polar components of the N-acyl linkage. Accommodation of the components of the N-acyl linkage appears to be the reason that this enzyme is capable of a racemization reaction on these substrates, whereas the orthologous OSBS from Escherichia coli lacks this functionality.

• Authors: James B Thoden, Erika A Taylor Ringia, James B Garrett, John A Gerlt, Hazel M Holden, Ivan Rayment
• Journal: Biochemistry (Biochemistry) Vol. 43 Issue 19 Pg. 5716-27 (May 18 2004) ISSN: 0006-2960 [Print] United States
• PMID: 15134446 (Publication Type: Comparative Study, Journal Article, Research Support, U.S. Gov't, Non-P.H.S., Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Acyl Coenzyme A
• Escherichia coli Proteins
• Multienzyme Complexes
• 4-(2'-carboxyphenyl)-4-oxobutyryl-coenzyme A ester
• N-acetylmethionine
• Methionine
• Phosphopyruvate Hydratase
• Succinate-CoA Ligases
• O-succinylbenzoate - CoA ligase

Topics

• Actinomycetales (enzymology)
• Acyl Coenzyme A (chemistry, metabolism)
• Acylation
• Binding Sites
• Crystallization
• Crystallography, X-Ray
• Enzyme Activation
• Escherichia coli Proteins (chemistry, metabolism)
• Evolution, Molecular
• Methionine (analogs & derivatives, chemistry, metabolism)
• Multienzyme Complexes (chemistry, metabolism)
• Phosphopyruvate Hydratase (chemistry, metabolism)
• Protein Binding
• Structural Homology, Protein
• Substrate Specificity
• Succinate-CoA Ligases (chemistry, metabolism)