Comparison of Alicyclobacillus acidocaldarius OSBS to its promiscuous NSAR/OSBS relatives

Denis Odokonyero, Andrew W. McMillan, Udupi A. Ramagopal, Rafael Toro, Dat P. Truong, Mingzhao Zhu, Mariana S. Lopez, Belema Somiari, Meghann Herman, Asma Aziz, Jeffrey B. Bonanno, Kenneth G. Hull, Stephen K. Burley, Daniel Romo, Steven C. Almo, Margaret E. Glasner

Research output: Contribution to journalArticle

Abstract

Studying the evolution of catalytically promiscuous enzymes like those from the N-succinylamino acid racemase/o-succinylbenzoate synthase (NSAR/OSBS) subfamily can reveal mechanisms by which new functions evolve. Some enzymes in this subfamily only have OSBS activity, while others catalyze OSBS and NSAR reactions. We characterized several NSAR/OSBS subfamily enzymes as a step toward determining the structural basis for evolving NSAR activity. Three enzymes were promiscuous, like most other characterized NSAR/OSBS subfamily enzymes. However, Alicyclobacillus acidocaldarius OSBS (AaOSBS) efficiently catalyzes OSBS activity but lacks detectable NSAR activity. Competitive inhibition and molecular modeling show that AaOSBS binds N-succinylphenylglycine with moderate affinity in a site that overlaps its normal substrate. Based on possible steric conflicts identified by molecular modeling and sequence conservation within the NSAR/OSBS subfamily, we identified one mutation, Y299I, which increased NSAR activity from undetectable to 1.2 x 102 M-1s-1 without affecting OSBS activity. This mutation does not appear to affect binding affinity, but instead affects kcat, by reorienting the substrate or modifying conformation changes to allow both catalytic lysines to access the proton that is moved during the reaction. This is the first site known to affect reaction specificity in the NSAR/OSBS subfamily. However, this gain of activity was obliterated by a second mutation, M18F. Epistatic interference by M18F was unexpected because a phenylalanine at this position is important in another NSAR/OSBS enzyme. Together, modest NSAR activity of Y299I AaOSBS and epistasis between sites 18 and 299 indicate that additional sites influenced the evolution of NSAR reaction specificity in the NSAR/OSBS subfamily.

Original languageEnglish (US)
JournalBiochemistry
DOIs
StateAccepted/In press - Jan 26 2018

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Alicyclobacillus
Racemases and Epimerases
Acids
Enzymes
Molecular modeling
Mutation
Substrates
o-succinylbenzoic acid synthase
Phenylalanine
Lysine
Conformations
Protons
Conservation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Odokonyero, D., McMillan, A. W., Ramagopal, U. A., Toro, R., Truong, D. P., Zhu, M., ... Glasner, M. E. (Accepted/In press). Comparison of Alicyclobacillus acidocaldarius OSBS to its promiscuous NSAR/OSBS relatives. Biochemistry. https://doi.org/10.1021/acs.biochem.8b00088

Comparison of Alicyclobacillus acidocaldarius OSBS to its promiscuous NSAR/OSBS relatives. / Odokonyero, Denis; McMillan, Andrew W.; Ramagopal, Udupi A.; Toro, Rafael; Truong, Dat P.; Zhu, Mingzhao; Lopez, Mariana S.; Somiari, Belema; Herman, Meghann; Aziz, Asma; Bonanno, Jeffrey B.; Hull, Kenneth G.; Burley, Stephen K.; Romo, Daniel; Almo, Steven C.; Glasner, Margaret E.

In: Biochemistry, 26.01.2018.

Research output: Contribution to journalArticle

Odokonyero, D, McMillan, AW, Ramagopal, UA, Toro, R, Truong, DP, Zhu, M, Lopez, MS, Somiari, B, Herman, M, Aziz, A, Bonanno, JB, Hull, KG, Burley, SK, Romo, D, Almo, SC & Glasner, ME 2018, 'Comparison of Alicyclobacillus acidocaldarius OSBS to its promiscuous NSAR/OSBS relatives', Biochemistry. https://doi.org/10.1021/acs.biochem.8b00088
Odokonyero, Denis ; McMillan, Andrew W. ; Ramagopal, Udupi A. ; Toro, Rafael ; Truong, Dat P. ; Zhu, Mingzhao ; Lopez, Mariana S. ; Somiari, Belema ; Herman, Meghann ; Aziz, Asma ; Bonanno, Jeffrey B. ; Hull, Kenneth G. ; Burley, Stephen K. ; Romo, Daniel ; Almo, Steven C. ; Glasner, Margaret E. / Comparison of Alicyclobacillus acidocaldarius OSBS to its promiscuous NSAR/OSBS relatives. In: Biochemistry. 2018.
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