Active Site Residues of Cephalosporin Acylase Are Critical Not Only for Enzymatic Catalysis but Also for Post-translational Modification

Kim, Sanggu; Kim, Youngsoo

doi:10.1074/jbc.m109603200

Cited by 30 publications

(28 citation statements)

References 18 publications

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“…Amino acid changes affecting glutaryl side chain binding, such as S170C, Y205I or -S, R229K or -S, and F349T, simultaneously lost the activities for both the second cleavage and substrate hydrolysis partially or completely (25). Glutarate is a competitive inhibitor for the hydrolysis of Gl-7-ACA.…”

Section: Tablementioning

confidence: 99%

“…From studies with the P. diminuta CA, it was known that Glu 159 is important for hydrolysis of the spacer from the immature ␣Ј-subunit. Replacing Glu by Leu, Met, or Asn prevented the second cleavage reaction, without affecting the release of the ␤-subunit by the first autoproteolytic reaction (25). We created the P. sp.…”

Section: Lc-ms Identification Of the Second Cleavagementioning

confidence: 99%

“…Based on these observations, it was proposed that the second cleavage is carried out by inter-molecular interaction using the same active site for substrate hydrolysis and released a 9-aa spacer for CA from Pseudomonas sp. GK16 or an 11-aa spacer for CA from Pseudomonas diminuta (21,22,25). However, the second cleavage of S170C was not performed when incubated with the mature enzyme (28).…”

mentioning

confidence: 99%

“…However, the second cleavage of S170C was not performed when incubated with the mature enzyme (28). Although the lengths of spacers reported are different, Glu 159 was reported to be crucial for the second cleavage of both CA precursors from Pseudomonas GK16 and P. diminuta (25,27,28). Based on these observations, mutants containing an integral ␣Ј-subunit were crystallized to deduce the intra-molecular mechanism for the second cleavage.…”

mentioning

confidence: 99%

“…It has been debated for years whether the second cleavage is inter-or intra-molecular, which amino acid residue catalyzes the reaction, and what determines the site of cleavage (25,(27)(28)(29). Matured CA from Pseudomonas sp.…”

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confidence: 99%

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The N-terminal Nucleophile Serine of Cephalosporin Acylase Executes the Second Autoproteolytic Cleavage and Acylpeptide Hydrolysis

Yin

Deng

Zhao

et al. 2011

Journal of Biological Chemistry

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Cephalosporin acylase (CA) precursor is translated as a single polypeptide chain and folds into a self-activating pre-protein. Activation requires two peptide bond cleavages that excise an internal spacer to form the mature ␣␤ heterodimer. Using Q-TOF LC-MS, we located the second cleavage site between Glu 159 and Gly 160 , and detected the corresponding 10-aa spacer 160 GDPPDLADQG 169 of CA mutants. The site of the second cleavage depended on Glu 159 : moving Glu into the spacer or removing 5-10 residues from the spacer sequence resulted in shorter spacers with the cleavage at the carboxylic side of Glu. The mutant E159D was cleaved more slowly than the wild-type, as were mutants G160A and G160L. This allowed kinetic measurements showing that the second cleavage reaction was a firstorder, intra-molecular process. Glutaryl-7-aminocephalosporanic acid is the classic substrate of CA, in which the N-terminal Ser 170 of the ␤-subunit, is the nucleophile. Glu and Asp resemble glutaryl, suggesting that CA might also remove N-terminal Glu or Asp from peptides. This was indeed the case, suggesting that the N-terminal nucleophile also performed the second proteolytic cleavage. We also found that CA is an acylpeptide hydrolase rather than a previously expected acylamino acid acylase. It only exhibited exopeptidase activity for the hydrolysis of an externally added peptide, supporting the intra-molecular interaction. We propose that the final CA activation is an intramolecular process performed by an N-terminal nucleophile, during which large conformational changes in the ␣-subunit C-terminal region are required to bridge the gap between Glu 159 and Ser 170 .

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Section: Tablementioning

confidence: 99%

Section: Lc-ms Identification Of the Second Cleavagementioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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The N-terminal Nucleophile Serine of Cephalosporin Acylase Executes the Second Autoproteolytic Cleavage and Acylpeptide Hydrolysis

Yin

Deng

Zhao

et al. 2011

Journal of Biological Chemistry

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Mutational Analysis of a Key Residue in the Substrate Specificity of a Cephalosporin Acylase

et al. 2004

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beta-Lactam acylases are crucial for the synthesis of semisynthetic cephalosporins and penicillins. Unfortunately, there are no cephalosporin acylases known that can efficiently hydrolyse the amino-adipic side chain of Cephalosporin C. In a previous directed evolution experiment, residue Asn266 of the glutaryl acylase from Pseudomonas SY-77 was identified as being important for substrate specificity. In order to explore the function of this residue in substrate specificity, we performed a complete mutational analysis of position 266. Codons for all amino acids were introduced in the gene, 16 proteins that could be functionally expressed in Escherichia coli were purified to homogeneity and their catalytic parameters were determined. The mutant enzymes displayed a broad spectrum of affinities and activities, pointing to the flexibility of the enzyme at this position. Mutants in which Asn266 was changed into Phe, Gln, Trp and Tyr displayed up to twofold better catalytic efficiency (k(cat)/K(m))than the wild-type enzyme when adipyl-7-aminodesacetoxycephalosporanic acid (adipyl-7-ADCA) was used as substrate, due to a decreased K(m). Only mutants SY-77(N266H) and SY-77(N266M) showed an improvement of both catalytic parameters, resulting in 10- and 15-times higher catalytic efficiency with adipyl-7-ADCA, respectively. Remarkably, the catalytic activity (k(cat)) of SY-77(N266M) when using adipyl-7-ADCA as substrate was as high as when glutaryl-7-aminocephalosporanic acid (glutaryl-7-ACA) was used, and approaches commercially interesting activity. SY-77(N266Q), SY-77(N266H) and SY-77(N266M) mutants showed a modest improvement in hydrolysing Cephalosporin C. Since these mutants also have a good catalytic efficiency when adipyl-7-ADCA is used and are still active towards glutaryl-7-ACA, they can be regarded as broad substrate acylases. These results demonstrate that the combination of directed evolution for the identification of important positions, together with saturation mutagenesis for finding the optimal amino acid, is a very effective method for finding improved biocatalysts.

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Glutaryl-7-aminocephalosporanic-acid acylase

2009

Class 3 Hydrolases

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Active Site Residues of Cephalosporin Acylase Are Critical Not Only for Enzymatic Catalysis but Also for Post-translational Modification

Cited by 30 publications

References 18 publications

The N-terminal Nucleophile Serine of Cephalosporin Acylase Executes the Second Autoproteolytic Cleavage and Acylpeptide Hydrolysis

The N-terminal Nucleophile Serine of Cephalosporin Acylase Executes the Second Autoproteolytic Cleavage and Acylpeptide Hydrolysis

Mutational Analysis of a Key Residue in the Substrate Specificity of a Cephalosporin Acylase

Glutaryl-7-aminocephalosporanic-acid acylase

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