Enzymatic biosynthesis of cephalexin

Rhee, Dong Kwon; Lee, S. B.; Rhee, Jae-Sung; Ryu, Dewey D. Y.; Hospodka, J.

doi:10.1002/bit.260220610

Cited by 33 publications

(13 citation statements)

References 14 publications

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“…One of the first AEHs that was isolated and characterized is the enzyme from Xanthomonas citri (5)(6)(7)(8)(9)(10)(11). This enzyme was found to be a homotetramer with subunits of 72 kDa (6).…”

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confidence: 99%

The Sequence and Crystal Structure of the α-Amino Acid Ester Hydrolase from Xanthomonas citri Define a New Family of β-Lactam Antibiotic Acylases

Barends

Polderman-Tijmes

Jekel

et al. 2003

Journal of Biological Chemistry

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␣-Amino acid ester hydrolases (AEHs) catalyze the hydrolysis and synthesis of esters and amides with an ␣-amino group. As such, they can synthesize ␤-lactam antibiotics from acyl compounds and ␤-lactam nuclei obtained from the hydrolysis of natural antibiotics. This article describes the gene sequence and the 1.9-Å resolution crystal structure of the AEH from Xanthomonas citri. The enzyme consists of an ␣/␤-hydrolase fold domain, a helical cap domain, and a jellyroll ␤-domain. Structural homology was observed to the Rhodococcus cocaine esterase, indicating that both enzymes belong to the same class of bacterial hydrolases. Docking of a ␤-lactam antibiotic in the active site explains the substrate specificity, specifically the necessity of an ␣-amino group on the substrate, and explains the low specificity toward the ␤-lactam nucleus.␤-Lactam antibiotics form a large family of widely applied antibacterials. Most of them are derived from a handful of naturally occurring antibiotics like penicillin G, penicillin V, and cephalosporin C by replacing their acyl groups with synthetic ones. Initially, this was achieved by chemical means but at present, enzymatic methods are preferred (1). A well known enzyme used for these conversions is penicillin acylase (EC 3.5.1.11) from Escherichia coli. This enzyme is used both for the production of the ␤-lactam nucleus 6-aminopenicillanic acid (6-APA) 1 by cleaving off phenylacetic acid from penicillin G and for the coupling of new acyl groups to 6-APA or other ␤-lactam nuclei. Penicillin acylase is, however strongly inhibited by its product phenylacetic acid (2), which must therefore be removed before coupling of a new acyl group to the ␤-lactam nucleus can take place. In addition, ␤-lactam nuclei are not very stable at the alkaline pH optimum of penicillin acylase.By contrast, ␣-amino acid ester hydrolases (AEHs) do not have these disadvantages. These enzymes catalyze the hydrolysis and synthesis of esters and amides of ␣-amino acids exclusively, and do not attack the amide bond of a ␤-lactam. They can be used to acylate a ␤-lactam using an ester as acyl donor, as shown in Fig. 1. Because the AEHs require an ␣-amino group on the substrate, they are not inhibited by phenylacetic acid (3). Together with their ability to accept various ␤-lactam nuclei without cleaving them, this makes them suitable for generating widely used antibiotics such as ampicillin, amoxicillin, and the cephalosporins cephadroxil and cephalexin. The slightly acidic pH optimum of AEH, which is beneficial for ␤-lactam stability, is another advantage of AEHs for biocatalytic applications, as is their stereospecificity toward the acyl donor (4).One of the first AEHs that was isolated and characterized is the enzyme from Xanthomonas citri (5-11). This enzyme was found to be a homotetramer with subunits of 72 kDa (6). Kinetic studies indicated the occurrence of an acyl-enzyme intermediate in the hydrolysis and acylation reactions of ␤-lactam antibiotics (3,7,12).This article reports the gene and 1.9-Å resolution ...

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confidence: 99%

The Sequence and Crystal Structure of the α-Amino Acid Ester Hydrolase from Xanthomonas citri Define a New Family of β-Lactam Antibiotic Acylases

Barends

Polderman-Tijmes

Jekel

et al. 2003

Journal of Biological Chemistry

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“…In Xanthomonas spp., the resistance for cephalexin is associated with the presence of a gene coding for a beta-lactamase (Weng et al, 1999). Also, the commercial synthesis of cephalexin is currently done using bacteria belonging to the genus Xanthomonas (Rhee, Lee, Rhee, Ryu, & Hospodka, 1980). Cycloheximide is fungicidal and inhibited all the fungal saprophytes in our samples.…”

Section: Discussionmentioning

confidence: 97%

Development of a semi-selective medium for isolation of Xanthomonas campestris pv. musacearum from banana plants

Tripathi

Tushemereirwe³

et al. 2006

Eur J Plant Pathol

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Banana Xanthomonas wilt, caused by Xanthomonas campestris pv. musacearum, is a new threat to banana cultivation in eastern Africa. The causal bacterium grows slowly in culture and is easily overgrown by contaminants. A selective culture medium for isolation of X. c. pv. musacearum will facilitate disease study. A medium that suppressed saprophytic growth and possessed diagnostic characters for the pathogen was developed. Various carbon sources were tested with two isolates of X. c. pv. musacearum, and sucrose was selected as main carbon source. The susceptibility of X. c. pv. musacearum and other bacterial strains was tested with 29 different antibiotics. Cephalexin and cycloheximide had no effect on X. c. pv. musacearum but cephalexin inhibited most of the saprophytes and cycloheximide inhibited the fungal contaminants. Based on these studies, we have developed a semi-selective medium YTSA-CC containing yeast extract (1%), tryptone (1%), sucrose (1%), agar (1.5%), cephalexin (50 mg l -1 ) and cycloheximide (150 mg l -1 ), pH 7.0. The pathogen X. c. pv. musacearum was easily identified as yellowish, mucoid and circular colonies on YTSA-CC medium. This simple semi-selective medium was effective for isolation of X. c. pv. musacearum from infected banana tissues and soil, and it should be a valuable tool in ecological and epidemiological studies.

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“…The PGA catalyzed synthesis of cephalexin (CEX) from 7-ADCA and D-phenylglycine methyl ester (PGM) is an example of kinetically controlled synthesis [18][19][20][21][22]. The enzymatic synthesis of -lactam antibiotics as described above is accompanied by two side reactions, (1) hydrolysis of the activated acyl donor (PGM) and (2) hydrolysis of the resultant antibiotics (CEX), leading to loss of the activated acyl donor and limiting the product yield.…”

Section: Preprintsmentioning

confidence: 99%

Impact of polybasic alcohols on biocompatibility and selectivity of Penicillin G Acylase for kinetically controlled synthesis

Shi

Zhu-an

Shen

2015

Preprint

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The enzyme catalyzed synthesis of cephalexin (CEX) from 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) and D-phenylglycine methyl ester (PGM) by Penicillin G acylase (PGA) is a model for kinetically controlled synthesis. The parallel hydrolysis of PGM, the activated acyl donor, is the principle competing pathway in this reaction, limiting the synthetic yield and reaction efficiency. To improve the performance of PGA catalyzed CEX synthesis, the biocompatibility and selectivity of various co-solvents were investigated. Polybasic alcohols such as ethylene glycol, glycerol and PEG400 did not cause deleterious changes to the enzyme, whereas monobasic alcohols, such as butyl alcohol, disrupted the PGA activity. Compared with the reaction in aqueous medium, the use of ethylene glycol as a co-solvent was found to have good selectivity in order to facilitate CEX synthesis and significantly minimize PGM hydrolysis. The pH of ethylene glycol medium was also optimized. The mechanism of the enhanced effect of polybasic alcohols as co-solvents on both biocompatibility and selectivity of enzymatic kinetically controlled synthesis is suggested.

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Enzymatic biosynthesis of cephalexin

Cited by 33 publications

References 14 publications

The Sequence and Crystal Structure of the α-Amino Acid Ester Hydrolase from Xanthomonas citri Define a New Family of β-Lactam Antibiotic Acylases

The Sequence and Crystal Structure of the α-Amino Acid Ester Hydrolase from Xanthomonas citri Define a New Family of β-Lactam Antibiotic Acylases

Development of a semi-selective medium for isolation of Xanthomonas campestris pv. musacearum from banana plants

Impact of polybasic alcohols on biocompatibility and selectivity of Penicillin G Acylase for kinetically controlled synthesis

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