title: 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 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.
authors.affiliation: School of Chemical and Biomolecular Engineering, Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, 315 Ferst Drive, Atlanta, GA 30332-0363, USA.
authors.forename: Janna K
authors.initials: JK
authors.lastname: Blum
authors.forename: Andreas S
authors.initials: AS
authors.lastname: Bommarius
references.citation: Biomacromolecules. 2001 Spring;2(1):95-104
references.pmid: 11749160
references.citation: Angew Chem Int Ed Engl. 2006 Feb 13;45(8):1236-41
references.pmid: 16411254
references.citation: Appl Environ Microbiol. 2002 Jan;68(1):211-8
references.pmid: 11772629
references.citation: Appl Microbiol Biotechnol. 2003 Jun;61(5-6):385-92
references.pmid: 12679848
references.citation: J Biol Chem. 2003 Jun 20;278(25):23076-84
references.pmid: 12684501
references.citation: Appl Microbiol Biotechnol. 2003 Jan;60(5):507-14
references.pmid: 12536249
references.citation: Biochem J. 1974 Mar;137(3):497-503
references.pmid: 4424889
references.citation: Curr Opin Struct Biol. 2009 Oct;19(5):596-604
references.pmid: 19765975
references.citation: Nucleic Acids Res. 2010 Jul;38(Web Server issue):W569-75
references.pmid: 20462859
references.citation: Eur J Biochem. 2003 Sep;270(18):3675-83
references.pmid: 12950251
references.citation: J Biotechnol. 2008 Jan 1;133(1):18-26
references.pmid: 17933411
references.citation: FEBS Lett. 2006 Feb 20;580(5):1465-71
references.pmid: 16469317
references.citation: Proc Natl Acad Sci U S A. 2009 Jun 16;106 Suppl 1:9995-10000
references.pmid: 19528653
references.citation: Biochim Biophys Acta. 2002 Sep 23;1599(1-2):134-40
references.pmid: 12479414
references.citation: Biotechnol J. 2006 May;1(5):531-6
references.pmid: 16892288
references.citation: J Biol Chem. 2002 Aug 9;277(32):28474-82
references.pmid: 12011065
references.citation: Protein Eng Des Sel. 2004 Jul;17(7):571-9
references.pmid: 15333773
references.citation: J Mol Biol. 2004 Jul 16;340(4):783-95
references.pmid: 15223320
references.citation: Biotechnol Bioeng. 2002 Jun 5;78(5):589-93
references.pmid: 12115129
references.citation: Nat Struct Biol. 2002 Jan;9(1):17-21
references.pmid: 11742345
references.citation: BMC Biotechnol. 2007 Nov 14;7:77
references.pmid: 18001472
references.citation: J Biol Chem. 2002 Nov 1;277(44):42121-7
references.pmid: 12198140
references.citation: J Biol Chem. 2006 Mar 3;281(9):5804-10
references.pmid: 16377627
references.citation: Biotechnol Bioeng. 1993 Sep 20;42(7):800-6
references.pmid: 18613126
references.citation: Angew Chem Int Ed Engl. 2005 Jul 4;44(27):4192-6
references.pmid: 15929154
references.citation: Mol Plant Pathol. 2004 Jan 1;5(1):1-15
references.pmid: 20565577
references.citation: Nat Protoc. 2007;2(4):891-903
references.pmid: 17446890