Omega Aminotransferase from P. aeruginosa

  Margo Goldfarb '20 and Miriam Hyman '21


Contents:


I. Introduction

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The Pseudomonas aeruginosa omega-aminotransferase is a class III aminotransferase that has a substrate specificity for (S)-a-Methylbenzylamine (MBA), beta-alanine, 4 aminobutyrate and pyruvate. Aminotransferases catalyze the transfer of an amino group from an amino to a keto acid using pyridoxal phosphate (PLP) as a cofactor. During transamination, aminotransferases bind to PLP to form a Schiff base with a lysine in their active site resulting in an internal aldimine. PLP subsequently forms an external aldimine with the amino donor. Hydrolysis of the resulting ketamine leads to keto product, but leaves PMP which must be regenerated to PLP. In a dehydration reaction, the keto donor forms a Schiff base with PMP. Lysine dehydrogenates and then displaces the transaminated ketodonor to regenerate the internal aldimine PLP from the external aldimine.

Scheme 1. General aminotransferase reaction mechanism using the PLP cofactor. Reaction mechanism Lyskowski, A., Gruber, C., Steinkellner, G., Schürmann, M., Schwab, H., Gruber, K., & Steiner, K. (2014). Crystal structure of an (R)-selective omega-transaminase from Aspergillus terreus. PLoS One, 9(1), e87350.

The majority of aminotransferase classes catalyze the transamination of alpha amino acids (classes I,II, IV and V). However, class III aminotransferases catalyze transamination of substrates with amines at omega positions, and are known as omega-aminotransferases. This reaction is more challenging to catalyze, but many amino transferase reactions with industrial applications lack an alpha carboxyl group, so there is much interest in class III omega-aminotransferases which are able to do these reactions.


II. General Structure



Pseudomonas aeruginosa omega-aminotransferase is a with two catalytic dimers. It has a molecular weight of 200 kDa, and each monomer has a molecular weight of 50 kDa. Two at the interface of the two dimers stabilize the tetramer. Each calcium ion is coordinated by the carboxyl groups of Asp180 of the two adjacent subunits as well as four water molecules . Four evenly spaced , one for each subunit, sit in pockets formed at the interface of the noncatalytic dimers. Each of the chloride ions coordinates with Phe173, Phe322, Ser323, and Met172 .

Omega aminotransferase fits into the class of type I PLP fold enzymes which consist of a . The small domain comprises N- and C-termini of the polypeptide chain. The folds into an alpha/beta/alpha sandwich made up of a seven-stranded beta sheet. The is made up of two beta sheets; a four-stranded N-terminal beta sheet (the last sheet is donated by C-terminus) and an antiparallel beta sheet which is pinned between three alpha helices on one side and the large domain. This interface between the large and small domains composes the active site of the enzyme. The enzyme contains four cofactor binding domains, at the bottom of each active site. All bound

While PLP is the natural cofactor for P. aeruginosa omega-aminotransferase, during crystallization of the enzyme PLP crystalized outside of the active site. However, PLP crystallized in the active site when it was bound to the suicide inhibitor gabalucine, resulting in PXG. PXG was subsequently used as a proxy for PLP in the analysis of active site interactions with the cofactor.


III. PLP binding

When Pseudomonas aeruginosa omega-aminotransferase binds , it induces a conformational change in its active site. The (up to residue 36), which is disordered in the apoenzyme (unbound) structure, becomes ordered in the holoenzyme (bound) structure and occupies the position of the unwound helix.

The holoenzyme structure features bound cofactor, Schiff-base PLP–Lys288, bound at the bottom of the active site. PLP binds at the interface of the catalytic dimer, between the two domains of a , where the Lysine is in between beta strands 9 and 10. The phosphate group of PLP makes hydrogen bonds to Gly120, Thr327, Thr327, and Ser121 . The carboxyl group of Asp259 makes a hydrogen bond to the pyridine-ring N atom of PLP . Asp259 is stabilized by interactions with the imidazole ring of His154 . The pyridine ring of PLP is sandwiched between the side chains of Tyr153 and Val261, which lie perpendicular to the cofactor ring .

Cocrystallization with gabaculine, the irreversible GABA aminotransferase inhibitor, allowed for the determination of the inhibitor complex structure. The inhibitor-bound complex provides valuable information about the active-site, the determinant of the substrate specificity. Gabaculine is covalently bound to C4 of PLP as the mCPP complex in all four monomers. It binds to the carboxyl group of mCPP and makes hydrogen bonds to the side-chain O atom of PA Gln421 and the side-chain N atoms of PA Trp61 and PA Arg414. These three residues form a rigid .


IV. Substrate Specificity

Class III omega-aminotransferases all catalyze transaminations at omega carbon positions, but they differ in substrate specificity. Pseudomonas aeruginosa omega-aminotransferase has a narrow substrate specificity, accepting only beta alanine, 4-aminobutyrate and MBA as amino donors, and catalyzes amino transfer to pyruvate.

Scheme 2. Amino and Keto donors donors

Sayer et al. (2013) used the suicide inhibitor of PLP to interpret the mechanisms behinds Pseudomonas aeruginosa omega-aminotransferase’s substrate selectivity. Unlike more flexible aminotransferases, the enzyme has a at a fixed distance from the cofactor, formed by the side chain O atom of Gln421, and the side chain N atoms of Arg414 and Trp61. The rigid structure of the omega aminotransferase’s active site limits its substrate specificity, but makes it very active towards beta-alanine.

When the native substrate, beta-alanine, is modeled in the carboxylate binding site, the amino end is ideally positioned for transamination. This is further favored by the positioning of , which is involved in proton abstraction. When smaller amino donors are bound at the carboxylate site, the amino group is too far from PLP to form a Schiff base. Larger amino donors are not able to occupy the active site as the side-chain of sterically blocks any amino acid beyond the beta-carbon.

Understanding the mechanisms for substrate selectivity has significant industrial applications. Omega aminotransferases are promising tools for synthesizing medically-relevant enantiopure compounds due to their broad substrate specificity, high enantioselectivity, and high turnover number as well as no need for cofactor regeneration.

Omega aminotransferases have been engineered to efficiently produce optically pure amines and beta amino acids (Cho et al., 2008; Shin et al., 2015). Further research has explored the potential for omega aminotransferases to catalyze enantiopure ketone synthesis, however, more work is needed to establish omega aminotransferases as an efficient means to producing this class of compounds (Han et al., 2019).


V. References

Cho, B. K., Park, H. Y., Seo, J. H., Kim, J., Kang, T. J., Lee, B. S., & Kim, B. G. (2008). Redesigning the substrate specificity of omega-aminotransferase for the kinetic resolution of aliphatic chiral amines. Biotechnology and bioengineering, 99(2), 275-284.

Han, S. W., & Shin, J. S. (2019). Activity Improvements of an Engineered omega-transaminase for Ketones Are Positively Correlated with Those for Cognate Amines. Biotechnology and Bioprocess Engineering, 24(1), 176-182.

Shin, G., Mathew, S., & Yun, H. (2015). Kinetic resolution of amines by (R)-selective omega-transaminase from Mycobacterium vanbaalenii. Journal of Industrial and Engineering Chemistry, 23, 128-133.

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