Solubility of artificial proteins with random sequences

AbstractA library of artificial random proteins of 141 amino acid residues of which 95 are random and which includes the 20 kinds of amino acids was prepared. Out of the 25 identified random proteins, 5 were soluble in the cell lysate, indicating that about 20% of the random proteins expressed in Escherichia coli are expected to be soluble. The soluble random proteins RP3–42 and RP3–45 and insoluble RP3–70 were purified. The solubility of the purified form is the same as that in the cell lysate

Three-dimensional Structure of Nylon Hydrolase and Mechanism of Nylon-6 Hydrolysis

This research was originally published in the Journal of Biological Chemistry. Seiji Negoro, Naoki Shibata, Yusuke Tanaka, Kengo Yasuhira, Hiroshi Shibata, Haruka Hashimoto, Young-Ho Lee, Shohei Oshima, Ryuji Santa, Shohei Oshima, Kozo Mochiji, Yuji Goto, Takahisa Ikegami, Keisuke Nagai, Dai-ichiro Kato, Masahiro Takeo and Yoshiki Higuchi. Three-dimensional Structure of Nylon Hydrolase and Mechanism of Nylon-6 Hydrolysis. J. Biol. Chem. 2012; 287, 5079-5090. © the American Society for Biochemistry and Molecular Biolog

Degradation Potential of the Nonylphenol Monooxygenase of Sphingomonas sp. NP5 for Bisphenols and Their Structural Analogs

The nonylphenol-degrading bacterium Sphingomonas sp. strain NP5 has a very unique monooxygenase that can attack a wide range of 4-alkylphenols with a branched side chain. Due to the structural similarity, it can also attack bisphenolic compounds, which are very important materials for the synthesis of plastics and resins, but many of them are known to or suspected to have endocrine disrupting effects to fish and animals. In this study, to clarify the substrate specificity of the enzyme (NmoA) for bisphenolic compounds, degradation tests using the cell suspension of Pseudomonas putida harboring the nonylphenol monooxygenase gene (nmoA) were conducted. The cell suspension degraded several bisphenols including bisphenol F, bisphenol S, 4,4′-dihydroxybenzophenone, 4,4′-dihydroxydiphenylether, and 4,4′-thiodiphenol, indicating that this monooxygenase has a broad substrate specificity for compounds with a bisphenolic structure

Nylon-Oligomer Hydrolase Promoting Cleavage Reactions in Unnatural Amide Compounds

International audienceThe active site of 6-aminohexanoate-dimer hydrolase, a nylon-6 byproduct-degrading enzyme with a β-lactamase fold, possesses a Ser112/Lys115/Tyr215 catalytic triad similar to the one of penicillin-recognizing family of serine-reactive hydrolases but includes a unique Tyr170 residue. By using a reactive quantum mechanics/molecular mechanics (QM/MM) approach, we work out its catalytic mechanism and related functional/structural specificities. At variance with other peptidases, we show that the involvement of Tyr170 in the enzyme–substrate interactions is responsible for a structural variation in the substrate-binding state. The acylation via a tetrahedral intermediate is the rate-limiting step, with a free-energy barrier of ∼21 kcal/mol, driven by the catalytic triad Ser112, Lys115, and Tyr215, acting as a nucleophile, general base, and general acid, respectively. The functional interaction of Tyr170 with this triad leads to an efficient disruption of the tetrahedral intermediate, promoting a conformational change of the substrate favorable for proton donation from the general acid

Unraveling the degradation of artificial amide bonds in nylon oligomer hydrolase: from induced-fit to acylation processes

International audienceTo elucidate how the nylon oligomer hydrolase (NylB) acquires its peculiar degradation activity towards non-biological amide bonds, we inspect the underlying enzymatic processes going from the induced-fit upon substrate binding to acylation. Specifically, we investigate the mutational effects of two mutants, Y170F and D181G, indicated in former experiments as crucial systems because of their specific amino acid residues. To this aim, by resorting on first-principles molecular dynamics complemented with metadynamics we provide a detailed insight into the underlying acylation mechanism. Our results show that while in the wild type (WT) the Tyr170 residue points the NH group towards the proton-acceptor site of an artificial amide bond, hence ready to react, in the Y170F this does not occur. The reason is ascribed to the absence of Tyr170 in the mutant, replaced by phenylalanine, unable to form hydrogen bonds with the amide bond, thus resulting in an increase of the activation barrier of more than 10 kcal/mol. Nonetheless, despite the lack of hydrogen bonding between the Y170F and the mutant, also in this case the highest free energy barrier for the induced-fit is similar to that of WT. This seems to suggest that, in the induced-fit process, kinetics is little affected by the mutation. On the basis of additional structural homology analyses on enzymes of the same family, we suggest that natural selection is responsible for the development of the peculiar hydrolytic activity of Arthrobacter sp. KI72