(569c) Characterization of an ?-Amino Ester Hydrolase from Xanthomonas Campestris Pv. Campestris suitable for Cephalosporin Synthesis but Not Amoxicillin Synthesis

β-lactam antibiotics, especially those derived from penicillins and cephalosporins, are continually the most prescribed antibiotics in the world [1]. Currently, penicillin G acylase (PGA) is the most used enzyme for the biocatalytic synthesis of β-lactam antibiotic and is employed industrially as an efficient alternative to chemical synthesis routes. α-amino ester hydrolases (AEH) have recently gained interest as alternative β-lactam antibiotic acylases to PGA for their enhanced reaction rates and stereospecificity, but AEHs remain largely uncharacterized. To date, only AEHs from Xanthomonas citri and Acetobacter turbidans have reported crystal structures and substrate specificities [2,3]. There is interest in expanding the number of characterized AEHs and quantifying their synthetic abilities across a wide range of β-lactam antibiotics. Prior work in our group on AEH from Xanthomonas campestris pv. campestris focused primarily on characterizing activity toward commercially less interesting ampicillin [4].

This work characterizes activity, structure, and thermostability of AEH from X. campestris pv. campestris as well as two promising quadruple variants [5]. Enzymatic synthesis couples an activated acyl side chain such as phenylglycine methyl ester (PGME) or p-hydroxyphenylglycine methyl ester (HPGME) with a β-lactam nucleus such as 6-aminopenicillanic acid (6-APA) or 7-aminodesacetoxycephalosporanic acid (7-ADCA). Different combinations of these moieties produce unique antibiotics (cephalexin from 7-ADCA and PGME, amoxicillin from HPGME and 6-APA, and cefadroxil from HPGME and 7-ADCA). AEH catalyzes β-lactam antibiotic synthesis as well as acyl donor hydrolysis and β-lactam antibiotic hydrolysis, leading to the formation of unwanted byproducts. Synthesis selectivity is dependent on 6-APA or 7-ADCA concentration and quantified with the parameters α, β_o, and γ used in the reaction scheme developed by Youshko and Svedas [6]. AEH melting temperature was measured with differential scanning fluorimetry (DSF) while molecular weight and oligomeric state of AEH in solution were verified using size exclusion chromatography/multi-angle laser light scattering (SEC-MALLS) and analytical ultracentrifugation (AUC).

AEH was found to be more selective for cephalexin and cefadroxil synthesis than for amoxicillin synthesis. Maximum selectivity, quantified by 1/γ, was found to be between 0.37–0.5 for amoxicillin, 1.41–1.78 for cefadroxil, and 5.6–7.64 for cephalexin. AEH hydrolysis activity had an order of magnitude higher affinity (K_M) and reactivity (k_cat) toward PGME over HPGME. These trends indicate AEH’s suitability for cephalosporin synthesis over amoxicillin synthesis primarily due to the higher nucleophilic reactivity of 7-ADCA relative to 6-APA.

AEH has found to have two melting points, one at 25°C and another at 40°C. This suggests that the low thermostability of AEH could be due to separate domains unfolding within the AEH monomer. It was also found that 1M NaCl stabilizes AEH in solution and increases the first melting point to >40°C, however, this high salt concentration eliminates AEH activity. SEC-MALLS demonstrates that the molecular weight of AEH in solution is approximately that of the monomer, suggesting the lack of tetrameric AEH that is found in the crystal structure. The presence of AEH as an active monomer in solution is further confirmed with AUC [7].

References:

[1] H. Gelband et al., Wound Healing Southern Africa 8.2 (2015) 30-34.

[2] J.J. Polderman-Tijmes et al., Appl. Environ. Microbiol. 68 (2002) 211–218.

[3] J.J. Polderman-Tijmes et al., J. Biol. Chem. 277 (2002) 28474–28482.

[4] J.K. Blum, A.S. Bommarius, J. Mol. Catal. B: Enzym. 67 (2010) 21-28

[5] J.K. Blum et al., J. Biotechnol. 160 (2012) 214-221

[6] M.I. Youshko, and V.K. Śvedas, Biochemistry (Moscow) 65 (2000) 1367-1375.

[7] C. Lagerman, A. Bommarius, Characterization of an α-amino ester hydrolase from X. campestris pv. campestris for biocatalytic production of β-Lactam antibiotics, To be submitted.