Nonnatural amino acid incorporation into the methionine 214 position of the metzincin Pseudomonas aeruginosa alkaline protease

Walasek, Paula; Honek, John F.

doi:10.1186/1471-2091-6-21

Cited by 19 publications

(15 citation statements)

References 59 publications

(60 reference statements)

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“…Similar studies on MMP-2 showed unaltered activity for the serine and leucine mutants but complete loss of activity and enhanced susceptibility to proteolytic degradation for the cysteine mutant (11). In serralysins, substitution of difluoromethionine for methionine revealed no significant differences in activity or in the result of differential scanning calorimetry in Pseudomonas aeruginosa alkaline protease (12). In contrast, studies on Erwinia chrysanthemi PrtC revealed lower levels of protein expression and diminished catalytic efficiency toward resorufin-casein for the leucine (85% of the wild type), isoleucine (50%), and alanine (23%) mutants and subtle changes in the crystal structure at the active site of the first two mutants and a cysteine mutant.…”

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confidence: 63%

On the Relevance of the Met-turn Methionine in Metzincins

Tallant

García‐Castellanos²,

Baumann³

et al. 2010

Journal of Biological Chemistry

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The metzincins are a clan of metallopeptidases consisting of families that share a series of structural elements. Among them is the Met-turn, a tight 1,4-turn found directly below the zincbinding site, which is structurally and spatially conserved and invariantly shows a methionine at position 3 in all metzincins identified. The reason for this conservation has been a matter of debate since its discovery. We have studied this structural element in Methanosarcina acetivorans ulilysin, the structural prototype of the pappalysin family, by generating 10 mutants that replaced methionine with proteogenic amino acids. We compared recombinant overexpression yields, autolytic and tryptic activation, proteolytic activity, thermal stability, and three-dimensional structure with those of the wild type. All forms were soluble and could be purified, although with varying yields, and three variants underwent autolysis, could be activated by trypsin, and displayed significant proteolytic activity. All variants were analyzed for the thermal stability of their zymogens. None of the mutants analyzed proved more stable or active than the wild type. Both bulky and small side chains, as well as hydrophilic ones, showed diminished thermal stability. Two mutants, leucine and cysteine, crystallized and showed three-dimensional structures that were indistinguishable from the wild type. These studies reveal that the Met-turn acts as a plug that snugly inserts laterally into a core structure created by the protein segment engaged in zinc binding and thus contributes to its structural integrity, which is indispensable for function. Replacement of the methionine with residues that deviate in size, side-chain conformation, and chemical properties impairs the plug-core interaction and prejudices molecular stability and activity.Evolutionary conservation of structural elements in proteins usually results from stringent steric requirements for function. Specific structures enable particular chemical groups to be placed appropriately in three-dimensional space for reaction, transport, regulation, and scaffolding (1). One such case of conserved structural elements is the Met-turn, found in the catalytic domains of all structurally characterized families of the metzincin clan of zinc-dependent metallopeptidases. These include astacins, ADAMs 4 /adamalysins, serralysins, matrix metalloproteinases (MMPs), leishmanolysins, snapalysins, and pappalysins (2-5). The catalytic domains span between ϳ130 and ϳ260 residues and fold into globular moieties, which are divided by an active site cleft into an upper N-terminal and a lower C-terminal subdomain when viewed in the standard orientation (Fig. 1A) (5). The N-terminal subdomain comprises a ␤-sheet and two helices, the backing helix and the active-site helix, as the minimum common core of repetitive secondary structure elements (5). The latter helix includes the first stretch of a zinc-binding consensus sequence, HEXX-HXXG/NXXH/D (amino acid one-letter code), which includes three protein ligands of the ca...

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confidence: 63%

On the Relevance of the Met-turn Methionine in Metzincins

Tallant

García‐Castellanos²,

Baumann³

et al. 2010

Journal of Biological Chemistry

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“…However, the acylated tRNA ( 125 I-TyrtRNA CUA Gly-dCA) was prepared without any modifications to avoid the tedious procedures of isolation from the cellular mixture. Using this acylated tRNA, 125 I-Tyr-tRNA CUA Gly-dCA, the suppression efficiency was 30% [38,39]. Subsequently, they employed runoff transcription [40] to generate the semisynthetic nonsense suppressor tRNA for in vitro incorporation of L-3-[ 125 I] iodotyrosine ( 125 I-Tyr) into the 16-mer polypeptide (Met-Gly-Leu-Tyr-Leu-Gly-LeuPhe-Stop-Gly-Leu-Tyr-Leu-Gly-Leu-Phe-Stop-Stop) [41].…”

Section: Exploiting Biosynthetic Machinery: Cotranslational Approachmentioning

confidence: 99%

“…By using RSI, selenomethionine [120], difluoromethionine [125], trifluoromethionine [126], norleucine [123,127], homopropargylglycine (Hpg) [128], azidohomoalanine (Aha) [129], homoallylglycine (Hag) [130], and methoxinine [127] have been incorporated using methionyl-tRNA synthetase (MetRS); per-thiaproline, 3 [131,132]; trifluorovaline (TfV), 2-amino-3-methyl-4-pentenoic acid have been incorporated using isoleucyl-tRNA synthetase (IleRS) [133][134][135]; o-fluorophenylalanine, m-fluorophenylalanine, pFF [136], 2-pyridylalanine, 3-pyridylalanine, and 4-pyridylalanine [137] have been incorporated using PheRS; 4-aminotryptophan (4AW), 5-aminotryptophan (5AW), 4-fluorotryptophan (4FW), 6-fluorotryptophan (6FW), 5-hydroxytryptophan (5HW), 7AzW have been incorporated using TyrRS [138][139][140][141]; and trifluoroleucine has been incorporated using leucyl-tRNA synthetase (LeuRS) [142,143] in E. coli (Table 12.3). Budisa and coworkers have explored RSI for the integration of Hpg and norleucine into the superoxide dismutase (SOD) protein in yeast [123].…”

Section: Endogenous Aarsmentioning

confidence: 99%

Posttranslational Modification of Proteins Incorporating Nonnatural Amino Acids

Yang

Montclare

2012

Functional Polymers by Post‐Polymerization Modification

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“…Thus, it may be possible to alter the catalytic properties of these redox proteins by perturbing the electronic features of the methionine thioether group. We have previously demonstrated that the fluorinated Met analogues L-difluoromethionine (S-difluoromethyl-L-homocysteine; L-DFM) and L-trifluoromethionine (S-trifluoromethyl-L-homocysteine; L-TFM) can be biosynthetically incorporated into recombinant proteins without diminishing catalytic activity [10][11][12][13][14]. Ab initio modeling experiments with these compounds suggest that the strongly electron-withdrawing effect of the fluorine atoms on the methyl group should significantly decrease the nucleophilicity of the sulfur atom, which could alter some aspects of the interaction between this atom and a metal ligand [5].…”

Section: Introductionmentioning

confidence: 99%