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. 2010 Oct;67(1-2):21-28.
doi: 10.1016/j.molcatb.2010.06.014.

Amino ester hydrolase from Xanthomonas campestris pv. campestris, ATCC 33913 for enzymatic synthesis of ampicillin

Janna K Blum  1 , Andreas S Bommarius
Affiliations

Amino ester hydrolase from Xanthomonas campestris pv. campestris, ATCC 33913 for enzymatic synthesis of ampicillin

Janna K Blum et al. J Mol Catal B Enzym. 2010 Oct.

Abstract

α-Amino ester hydrolases (AEH) are a small class of proteins, which are highly specific for hydrolysis or synthesis of α-amino containing amides and esters including β-lactam antibiotics such as ampicillin, amoxicillin, and cephalexin. A BLAST search revealed the sequence of a putative glutaryl 7-aminocephalosporanic acid (GL-7-ACA) acylase 93% identical to a known AEH from Xanthomonas citri. The gene, termed gaa, was cloned from the genomic DNA of Xanthomonas campestris pv. campestris sp. strain ATCC 33913 and the corresponding protein was expressed into Escherichia coli. The purified protein was able to perform both hydrolysis and synthesis of a variety of α-amino β-lactam antibiotics including (R)-ampicillin and cephalexin, with optimal ampicillin hydrolytic activity at 25 °C and pH 6.8, with kinetic parameters of k(cat) of 72.5 s(-1) and K(M) of 1.1 mM. The synthesis parameters α, β(o), and γ for ampicillin, determined here first for this class of proteins, are α = 0.25, β(o) = 42.8 M(-1), and γ = 0.23, and demonstrate the excellent synthetic potential of these enzymes. An extensive study of site-directed mutations around the binding pocket of X. campestris pv. campestris AEH strongly suggests that mutation of almost any first-shell amino acid residues around the active site leads to inactive enzyme, including Y82, Y175, D207, D208, W209, Y222, and E309, in addition to those residues forming the catalytic triad, S174, H340, and D307.

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Figures

Fig. 1
Fig. 1
Protein gel of the expressed AEH protein from X. campestris pv. campestris. Lane 1: pETXccH protein lysate, Lane 2: pETXccH unbound fraction, Lane 3: pETXccH Wash 1, Lane 4: pETXccH Wash 2, Lane 5: pETXccH eluent. The desired protein band is approximately 68 kDa.
Fig. 2
Fig. 2
(A) The temperature profile for the hydrolysis of ampicillin using the AEH from X. campestris pv. campestris. An Arrhenius fit of the temperature activation energy (Ea = 57 kJ mol−1, A = 7.3E+11 s−1, R2 = 0.95) and deactivation constants (Ed = −82 kJ mol−1, A = 3.4E−14 s−1, R2 = 1.0). Only the first four deactivation points were considered in the fit, initial activity at >35 °C is difficult to quantify since the deactivation occurs in <1 min. (B) CD thermal scan result fit to a two-state deactivation model, resulting in a calculated Tm = 32.4 °C. (C) Residual activity of AEH after incubation at reported temperature for 30 min, protein was immediately quenched on ice prior to determining residual activity at 25 °C as described in Section 2. (D) Residual activity at 25 °C of AEH after incubation at 30 °C at reported time.
Fig. 3
Fig. 3
A. PyMol representation of the active-site residues from the AEH from A. turbidans shown with (R)-PG bound, pdb 2b4k [22] aligned with the RhcocE. The proteins were aligned using PyMol software using the alpha carbons of the catalytic triad (Xcc SER-174, Xcc HIS-340, and Xcc ASP-307) to an RMS of 0.6 Å. The residue number reflects the numbering for X. campestris pv. campestris (XCC) and for the RhcocE. B. RosettaBackrub model [33] for mutation Asp207Asn, the top 10 structures are shown (blue) the wild-type structure is shown in green. The position of the Asp208 side chain is repositioned away from the α-amino of (R)-phenylglcyine, which impacts the polar interactions between (R)-PG and Asp208. (For interpretation of the references to colors in this figure legend, the reader is referred to the web version of this article.)
Fig. 4
Fig. 4
(A) Kinetically controlled synthesis of (R)-ampicillin with AEH from X. campestris pv. campestris starting with 20 mM 6-APA and 60 mM R-PGME (B) Kinetically controlled synthesis of (R)-ampicillin employing AEH from X. campestris pv. campestris starting with 20 mM 6-APA and 60 mM S-PGME (C) Synthesis of (R/S)-ampicillin starting with 20 mM 6-APA and 90 mM racemic PGME. (D) Plot of the change in enantioselectivity of the reaction over time, when starting with racemic PGME.
Fig. 5
Fig. 5
Plot of [RPG] versus [AMP] results for six synthesis reactions at various [6APA]o :[RPGME]o ratios and concentrations (20:20 mM, 33:20 mM, 45:20 mM, 60:20 mM, 20:33 mM; 20:45 mM; 20:60 mM), fit to the model equation (Eq. (1)) with parameters α= 0.25, βo = 42.8 M−1, and γ = 0.23. The experimental results are shown by the data points, while the model fits are represented by the lines.
Scheme 1
Scheme 1
Synthesis reaction of Ampicillin using AEH.
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