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

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 kcat of 72.5 s−1 and KM of 1.1 mM. The synthesis parameters α, βo, and γ for ampicillin, determined here first for this class of proteins, are α = 0.25, βo = 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.

Stabilizing biocatalysts

“…39 Similarly, a-amino ester hydrolases have been shown to have great potential for industrial synthesis of b-lactam antibiotics when compared to the industrially available penicillin G acylase. 41 However, these hydrolases are thermally instable as they are easily degraded into smaller peptides. Their rapid degradation at just slightly above ambient temperatures requires further stabilization of the enzyme (Fig.…”

3D Structure Modeling of Alpha-Amino Acid Ester Hydrolase from Xanthomonas rubrilineans

“…Unlike penicillin G acylase (PA), which consists of two different subunits, XrAEH is a homotetramer of four identical subunits with the active site located inside each subunit. According to X-ray diffraction analysis data [4-7], the presence of three types of key amino acid residues is a characteristic feature of α-amino ester hydrolase: …”

“…Some data is available on AEH isolated from bacteria Acetobacter turbidans ATCC 9325 (ActAEH) [4, 5] and Xanthomonas citri IF0 3835 (XcAEH) [6], X. campestris pv. campestris ATCC 33913 [7], and a number of other sources. The Protein Data Bank (PDB) contains three-dimensional structures of only two enzymes that exhibit the highest activity in antibiotics synthesis - ActAEH and XcAEH.…”

Scope and limitations of optical pure hydantoins as chiral auxiliaries in asymmetric Mannich reactions

“…It is well known that β-amino esters are appropriate substrates for the preparation of β-lactams, which are the central constituents of a large family of antibiotics [13][14][15][16][17][18] . In our work, β-amino esters 4a and 4g-4k were treated with lithium hexamethyldisilazide(LiHMDS) at -20 °C in anhydrous tetrahydrofuran(THF) to afford the target β-lactams 5a and 5g-5k in excellent yields(Scheme 3).…”