Crystal Structures of Glutaryl 7-Aminocephalosporanic Acid Acylase:  Insight into Autoproteolytic Activation

Kim, Jin-Kwang; Yang, In Seok; Rhee, Sangkee; Dauter, Zbigniew; Lee, Young Sik; Park, Sung Soo; Kim, Kyung Hyun

doi:10.1021/bi027181x

Cited by 46 publications

(62 citation statements)

References 34 publications

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“…Instead, two mechanisms, an intra-molecular interaction catalyzed by Glu 159 and an inter-molecular interaction catalyzed by Ntn, have been proposed for the reaction (21,25,28).…”

Section: Discussionmentioning

confidence: 99%

“…GK16 (21,28), 10 aa (GDPPDLADQG) from Pseudomonas sp. 130 (6), and 11 aa (EGDPPDLADQG) from P. diminuta (29).…”

Section: Lc-ms Identification Of the Second Cleavagementioning

confidence: 99%

“…The first proteolytic step cleaves the CA precursor between Gly 169 and Ser 170 to generate the mature ␤-subunit with the catalytic N-terminal Ser 170 (or numbered S␤1 in the mature enzyme), and the ␣Ј-subunit with the spacer still attached (21). To identify the C terminus of the mature CA ␣-subunit (after the spacer has been removed), CA from Pseudomonas sp.…”

Section: Lc-ms Identification Of the Second Cleavagementioning

confidence: 99%

“…Matured CA from Pseudomonas sp. GK16 was considered to be an (␣␤) 2 heterotetramer complex before releasing a 9-aa spacer (21,30,31). Mutant S170C kept its capability for the first cleavage, which releases the ␤-subunit, but completely lost the activities for both second cleavage to generate the mature ␣-subunit, and substrate (Gl-7-ACA) hydrolysis.…”

mentioning

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%

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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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“…Instead, two mechanisms, an intra-molecular interaction catalyzed by Glu 159 and an inter-molecular interaction catalyzed by Ntn, have been proposed for the reaction (21,25,28).…”

Section: Discussionmentioning

confidence: 99%

“…GK16 (21,28), 10 aa (GDPPDLADQG) from Pseudomonas sp. 130 (6), and 11 aa (EGDPPDLADQG) from P. diminuta (29).…”

Section: Lc-ms Identification Of the Second Cleavagementioning

confidence: 99%

Section: Lc-ms Identification Of the Second Cleavagementioning

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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Biocatalytic Synthesis of β‐lactam Antibiotics

Deaguero

Blum

Bommarius

2010

Encyclopedia of Industrial Biotechnology

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The penicillins, cephalosporins, carbapenems, and monobactams, make up the β‐lactam family of antibiotics. There are currently more than 25 antibiotics in this family that are approved by the United States Food and Drug Administration and over 50 companies that manufacture them. Their four‐membered β‐lactam ring, which is the part of the antibiotic that is responsible for their bactericidal capabilities, is sensitive to acids, bases, and heat. Therefore, the original chemical synthesis processes utilized to make these compounds required subzero temperatures, hazardous chemicals for protecting groups, and large volumes of organic solvents, rendering them both environmentally harmful and economically taxing. The original syntheses are increasingly being replaced with biocatalytic syntheses which are carried out at ambient temperatures, in aqueous medium, and without the use of auxiliary chemicals, rendering them environmentally benign and economically advantageous processes. The most important enzymes utilized for these processes are penicillin G acylases, penicillin V acylases, cephalosporin acylases, and expandases.

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Protein Engineering of d‐Succinylase from Cupriavidus sp. for d‐Amino Acid Synthesis and the Structural Implications

et al. 2021

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d‐Amino acids are important chiral building blocks for pharmaceuticals and agrochemicals. Previously, we have used d‐Succinylase (DSA) from Cupriavidus sp. P4‐10‐C and N‐succinyl amino acid racemase (NSAR, EC.4.2.1.113) from Geobacillus stearothermophilus NCA1503 to produce d‐amino acids via the dynamic kinetic resolution of N‐succinyl‐dl‐amino acids. However, the use of this bioconversion system remains challenging for industrial application due to the insufficient enantioselectivity of DSA toward N‐succinyl‐d‐amino acids. Therefore, we screened DSA mutants for improved enantioselectivity by directed evolution. Several mutants showed improved enantioseletivity compared to wild‐type DSA. L182E mutant had superior enantioselectivity, and the thermal stability was also remarkably improved by this single mutation. We solved the crystal structure of the L182E mutant in complex with succinic acids at a resolution 2.0 Å. The mutated residues in all generated mutants that showed improved enantioselectivity (including the substituted Glu182 in the L182E mutant) are found very close to the active site. The solved crystal structure also provides some rationale to explain the higher thermostability of the L182E mutant compared to wild‐type DSA. d‐phenylalanine and d‐tryptophan were produced in high conversion (approximately 90%) with 98.8% ee and 99.6% ee, respectively, using coupled L182E DSA and NSAR with the one‐pot enzymatic method. These data suggested that L182E DSA may be a useful biocatalyst for industrial d‐amino acids production.

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Crystal Structures of Glutaryl 7-Aminocephalosporanic Acid Acylase: Insight into Autoproteolytic Activation

Cited by 46 publications

References 34 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

Biocatalytic Synthesis of β‐lactam Antibiotics

Protein Engineering of d‐Succinylase from Cupriavidus sp. for d‐Amino Acid Synthesis and the Structural Implications

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