Glu121‐Lys319 salt bridge between catalytic and N‐terminal domains is pivotal for the activity and stability of <i>Escherichia coli</i> aminopeptidase N

Gumpena, R.; Kishor, Chandan; Ganji, Roopa Jones; Jain, Nishant; Addlagatta, A.

doi:10.1002/pro.2060

Cited by 15 publications

(11 citation statements)

References 28 publications

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“…To estimate the binding ability of 40 peptides and their influence on the stability of Naa50, thermal shift (T m ) assays were performed (35) (Fig. 4).…”

Section: Resultsmentioning

confidence: 99%

Human Naa50 Protein Displays Broad Substrate Specificity for Amino-terminal Acetylation

Reddi¹,

Saddanapu²,

Chinthapalli

et al. 2016

Journal of Biological Chemistry

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Amino-terminal acetylation is a critical co-translational modification of the newly synthesized proteins in a eukaryotic cell carried out by six amino-terminal acetyltransferases (NATs). All NATs contain at least one catalytic subunit, and some contain one or two additional auxiliary subunits. For example, NatE is a complex of Naa10, Naa50, and Naa15 (auxiliary). In the present study, the crystal structure of human Naa50 suggested the presence of CoA and acetylated tetrapeptide (AcMMXX) that have co-purified with the protein. Biochemical and thermal stability studies on the tetrapeptide library with variations in the first and second positions confirm our results from the crystal structure that a peptide with Met-Met in the first two positions is the best substrate for this enzyme. In addition, Naa50 acetylated all MXAA peptides except for MPAA. Transcriptome analysis of 10 genes that make up six NATs in humans from eight different cell lines suggests that components of NatE are transcribed in all cell lines, whereas others are variable. Because Naa10 is reported to acetylate all amino termini that are devoid of methionine and Naa50 acetylates all other peptides that are followed by methionine, we believe that NatE complex can be a major contributor for amino-terminal acetylation at the ribosome exit tunnel.Co-translational protein modifications are important biochemical events. Prominent among them are amino-terminal modifications such as methionine cleavage, acetylation, and myristoylation (1-3). Initiator methionine in eukaryotic proteins is removed co-translationally by the enzyme methionine aminopeptidase (MetAP) 3 when the second amino acid is small and uncharged (1). About 70% of the matured proteins do not retain their amino-terminal methionine (4). Similarly, about 90% of all newly synthesized cytosolic proteins undergo aminoterminal acetylation (5). Protein amino-terminal acetylation is carried out by six amino-terminal acetyltransferases (NATs) named sequentially NatA to NatF, classified based on substrate preference (6 -8). NatA acetylates the peptides with serine, alanine, threonine, cysteine, and valine on the amino termini that are formed after MetAP action at the ribosome exit tunnel (5, 9), whereas NatB acts on methionine followed by acidic residues in the second position (10, 11). NatC, NatE, and NatF act on methionine that is followed by hydrophobic amino acids like leucine, phenylalanine, isoleucine, and tryptophan (8, 10, 12, 13). Additionally, NatF also acetylates methionine that is followed specifically by a lysine (14). NatD acetylates the amino termini of Ser-Gly of histones H2A and H4 (15). Each of the NATs (NatA-NatF) is a complex of one or two of the enzymes labeled serially as Naa10, Naa20, Naa30, Naa40, Naa50, and Naa60 attached to one or two of the auxiliary proteins named Naa15, Naa25, Naa35, and Naa38 (7, 9). Some NATs are known to bind to the ribosome at the exit tunnel through their auxiliary subunits (11, 16). Protein amino-terminal acetylation has potential effects on cell ...

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“…To estimate the binding ability of 40 peptides and their influence on the stability of Naa50, thermal shift (T m ) assays were performed (35) (Fig. 4).…”

Section: Resultsmentioning

confidence: 99%

Human Naa50 Protein Displays Broad Substrate Specificity for Amino-terminal Acetylation

Reddi¹,

Saddanapu²,

Chinthapalli

et al. 2016

Journal of Biological Chemistry

Self Cite

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“…3, all the eight five-point mutants had the same CD spectral and fluorescence spectroscopy with those of wild-type enzyme, indicating that the mutations had little influence on the secondary and tertiary structure of enzyme. Salt bridge has important effects on catalytic efficiency of enzymes [18], [19]. The thermostable NADPH:FMN oxidoreductase from B. subtilis was stabilized by four salt bridges formed by the side chains of residues Lys 109 and Asp 137 [18].…”

Section: Resultsmentioning

confidence: 99%

“…The thermostable NADPH:FMN oxidoreductase from B. subtilis was stabilized by four salt bridges formed by the side chains of residues Lys 109 and Asp 137 [18]. The Glu 121-Lys 319 salt bridge between catalytic and N-terminal domains is important for the catalytic efficiency and stability of E. coli aminopeptidase N [19]. In this work, based on the 3-D structure model of the wild-type and the mutant M145I-214A-229T-247T-317I (Fig.…”

Section: Resultsmentioning

confidence: 99%

Structure-Guided Systems-Level Engineering of Oxidation-Prone Methionine Residues in Catalytic Domain of an Alkaline α-Amylase from Alkalimonas amylolytica for Significant Improvement of Both Oxidative Stability and Catalytic Efficiency

et al. 2013

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High oxidative stability and catalytic efficiency are required for the alkaline α-amylases to keep the enzymatic performance under the harsh conditions in detergent industries. In this work, we attempted to significantly improve both the oxidative stability and catalytic efficiency of an alkaline α-amylase from Alkalimonas amylolytica by engineering the five oxidation-prone methionine residues around the catalytic domain via a systematic approach. Specifically, based on the tertiary structure analysis, five methionines (Met 145, Met 214, Met 229, Met 247 and Met 317) were individually substituted with oxidation-resistant threonine, isoleucine and alaline, respectively. Among the created 15 mutants, 7 mutants M145A, M145I, M214A, M229A, M229T, M247T and M317I showed significantly enhanced oxidative stability or catalytic efficiency. In previous work, we found that the replacement of M247 with leucine could significantly improve the oxidative stability. Thus, these 8 positive mutants (M145A, M145I, M214A, M229A, M229T, M247T, M247L and M317I) were used to conduct the second round of combinational mutations. Among the constructed 85 mutants (25 two-point mutants, 36 three-point mutants, 16 four-point mutants and 8 five-point mutants), the mutant M145I-214A-229T-247T-317I showed a 5.4-fold increase in oxidative stability and a 3.0-fold increase in catalytic efficiency. Interestingly, the specific activity, alkaline stability and thermal stability of this mutant were also increased. The increase of salt bridge and hydrogen bonds around the catalytic domain contributed to the significantly improved catalytic efficiency and stability, as revealed by the three-dimensional structure model of wild-type alkaline α-amylase and its mutant M145I-214A-229T-247T-317I. With the significantly improved oxidative stability and catalytic efficiency, the mutant M145I-214A-229T-247T-317I has a great potential as a detergent additive, and this structure-guided systems engineering strategy may be useful for the protein engineering of the other microbial enzymes to fulfill industrial requirements.

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“…Finally, the TFP6-encoding gene from the strain CF-1 is located close to a gene encoding for the putative puromycin-sensitive aminopeptidase PSA (or endopeptidase N from the M1 class peptidase) [26]. This enzyme is considered the major aminopeptidase in E. coli involved in cytosolic protein degradation, which enables the use of amino acids as nutrients [27].…”

Section: General Thiol-reducing System (Tfp1-tfp2-tfp6-tfp11-tfp13)mentioning

confidence: 99%

Deciphering the Role of Multiple Thioredoxin Fold Proteins of Leptospirillum sp. in Oxidative Stress Tolerance

González

Álamos

Rivero

et al. 2020

IJMS

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Thioredoxin fold proteins (TFPs) form a family of diverse proteins involved in thiol/disulfide exchange in cells from all domains of life. Leptospirillum spp. are bioleaching bacteria naturally exposed to extreme conditions like acidic pH and high concentrations of metals that can contribute to the generation of reactive oxygen species (ROS) and consequently the induction of thiol oxidative damage. Bioinformatic studies have predicted 13 genes that encode for TFP proteins in Leptospirillum spp. We analyzed the participation of individual tfp genes from Leptospirillum sp. CF-1 in the response to oxidative conditions. Genomic context analysis predicted the involvement of these genes in the general thiol-reducing system, cofactor biosynthesis, carbon fixation, cytochrome c biogenesis, signal transduction, and pilus and fimbria assembly. All tfp genes identified were transcriptionally active, although they responded differentially to ferric sulfate and diamide stress. Some of these genes confer oxidative protection to a thioredoxin-deficient Escherichia coli strain by restoring the wild-type phenotype under oxidative stress conditions. These findings contribute to our understanding of the diversity and complexity of thiol/disulfide systems, and of adaptations that emerge in acidophilic microorganisms that allow them to thrive in highly oxidative environments. These findings also give new insights into the physiology of these microorganisms during industrial bioleaching operations.

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Glu121‐Lys319 salt bridge between catalytic and N‐terminal domains is pivotal for the activity and stability of Escherichia coli aminopeptidase N

Cited by 15 publications

References 28 publications

Human Naa50 Protein Displays Broad Substrate Specificity for Amino-terminal Acetylation

Human Naa50 Protein Displays Broad Substrate Specificity for Amino-terminal Acetylation

Structure-Guided Systems-Level Engineering of Oxidation-Prone Methionine Residues in Catalytic Domain of an Alkaline α-Amylase from Alkalimonas amylolytica for Significant Improvement of Both Oxidative Stability and Catalytic Efficiency

Deciphering the Role of Multiple Thioredoxin Fold Proteins of Leptospirillum sp. in Oxidative Stress Tolerance

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