Evolution of enzymatic activity in the enolase superfamily: structural studies of the promiscuous o-succinylbenzoate synthase from Amycolatopsis.
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.
Thoden
James B
JB
Department of Chemistry, University of Illinois, Urbana, Illinois 61801, USA.
Taylor Ringia
Erika A
EA
Garrett
James B
JB
Gerlt
John A
JA
Holden
Hazel M
HM
Rayment
Ivan
I
D000192:Actinomycetales / Q000201:enzymology*; D000214:Acyl Coenzyme A / Q000737:chemistry / Q000378:metabolism; D000215:Acylation; D001665:Binding Sites; D003460:Crystallization; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D019143:Evolution, Molecular; D008715:Methionine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism; D010751:Phosphopyruvate Hydratase / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D013389:Succinate-CoA Ligases / Q000737:chemistry* / Q000378:metabolism
D000214:Acyl Coenzyme A; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; C055511:4-(2'-carboxyphenyl)-4-oxobutyryl-coenzyme A ester; C006379:N-acetylmethionine; D008715:Methionine; D010751:Phosphopyruvate Hydratase; D013389:Succinate-CoA Ligases; C022693:O-succinylbenzoate - CoA ligase