Biochemistry

Journal: Biochemistry

Abbreviation

Biochem

Publisher

American Chemical Society

ISSN

0006-2960
1520-4995

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Publications 1 - 10 of 19

Peptide Targeting of an Intracellular Receptor of the Secretory Pathway
Tirla, Alina; Rivera Fuentes, Pablo (2019)
Biochemistry
Kinetic and Structural Characterization of the Self-Labeling Protein Tags HaloTag7, SNAP-tag, and CLIP-tag
Wilhelm, Jonas; Kühn, Stefanie; Tarnawski, Miroslaw; et al. (2021)
Biochemistry
The self-labeling protein tags (SLPs) HaloTag7, SNAP-tag, and CLIP-tag allow the covalent labeling of fusion proteins with synthetic molecules for applications in bioimaging and biotechnology. To guide the selection of an SLP-substrate pair and provide guidelines for the design of substrates, we report a systematic and comparative study of the labeling kinetics and substrate specificities of HaloTag7, SNAP-tag, and CLIP-tag. HaloTag7 reaches almost diffusion-limited labeling rate constants with certain rhodamine substrates, which are more than 2 orders of magnitude higher than those of SNAP-tag for the corresponding substrates. SNAP-tag labeling rate constants, however, are less affected by the structure of the label than those of HaloTag7, which vary over 6 orders of magnitude for commonly employed substrates. Determining the crystal structures of HaloTag7 and SNAP-tag labeled with fluorescent substrates allowed us to rationalize their substrate preferences. We also demonstrate how these insights can be exploited to design substrates with improved labeling kinetics.
Hydrogen-Bonding Interactions at the DNA Terminus Promote Extension from Methylguanine Lesions by Human Extender DNA Polymerase zeta
Räz, Michael H.; Sturla, Shana J.; Gahlon, Hailey (2018)
Biochemistry
The Acyl-CoA Specificity of Human Lysine Acetyltransferase KAT2A
Anmangandla, Ananya; Ren, Yuxiang; Fu, Qin; et al. (2022)
Biochemistry
Protein post-translational modification sserve to regulate a broad range of cellular functions including signal transduction, transcription, and metabolism. Protein lysine residue sundergo many post-translational acylations and are regulated by a range of enzymes, such as histone acetyl transferases (HATs)and histone deacetylases (HDACs).KAT2A,well characterized as a lysine acetyltransferase for both histone and nonhistone substrates, has been reported to tolerate addition alacyl-CoA substrates, such as succinyl-CoA, and shows nonacetyl transferase activity in specific biologicalcontexts.In thiswork, we investigatethe acyl-CoA substrate preference of KAT2A and attempt to determine whether and to what extent addition alacyl-CoA substrates may be utilized by KAT2A in a cellula rcontext. We show that while KAT2A can bind and utilize malonyl-CoA, its activity with succinyl-CoA or glutaryl-CoA is very weak, and acetylation is still the most efficient activity for KAT2A in vitro and in cells.
Cooperative Assembly of Hsp70 Subdomain Clusters
Wright, Maya A.; Aprile, Francesco A.; Bellaiche, Mathias M.J.; et al. (2018)
Biochemistry
Conformational Switching of the Nuclear Exosome during Ribosome Biogenesis
Ban, Nenad; Kummer, Eva; Ban, Nenad (2018)
Biochemistry
Intrinsically Disordered Regions in the Transcription Factor MYC:MAX Modulate DNA Binding via Intramolecular Interactions
Schütz, Stefan; Bergsdorf, Christian; Hänni-Holzinger, Sandra; et al. (2024)
Biochemistry
The basic helix-loop-helix leucine zipper (bHLH-LZ) transcription factor (TF) MYC is in large part an intrinsically disordered oncoprotein. In complex with its obligate heterodimerization partner MAX, MYC preferentially binds E-Box DNA sequences (CANNTG). At promoters containing these sequence motifs, MYC controls fundamental cellular processes such as cell cycle progression, metabolism, and apoptosis. A vast network of proteins in turn regulates MYC function via intermolecular interactions. In this work, we establish another layer of MYC regulation by intramolecular interactions. We used nuclear magnetic resonance (NMR) spectroscopy to identify and map multiple binding sites for the C-terminal MYC:MAX DNA-binding domain (DBD) on the intrinsically disordered regions (IDRs) in the MYC N-terminus. We find that these binding events in trans are driven by electrostatic attraction, that they have distinct affinities, and that they are competitive with DNA binding. Thereby, we observe the strongest effects for the N-terminal MYC box 0 (Mb0), a conserved motif involved in MYC transactivation and target gene induction. We prepared recombinant full-length MYC:MAX complex and demonstrate that the interactions identified in this work are also relevant in cis, i.e., as intramolecular interactions. These findings are supported by surface plasmon resonance (SPR) experiments, which revealed that intramolecular IDR:DBD interactions in MYC decelerate the association of MYC:MAX complexes to DNA. Our work offers new insights into how bHLH-LZ TFs are regulated by intramolecular interactions, which open up new possibilities for drug discovery.
(Reverse) Evolution of a Promiscuous Isochorismate Pyruvate Lyase into an Efficient Chorismate Mutase
Künzler, Dominik E.; Bressan, Luca; Jäger, Linda; et al. (2025)
Biochemistry
PchB is an isochorismate pyruvate lyase (IPL) involved in siderophore biosynthesis in Pseudomonas aeruginosa . Besides catalyzing the [1,5]-sigmatropic rearrangement of isochorismate, PchB also has weak chorismate mutase (CM) activity, promoting the [3,3]-sigmatropic transformation of chorismate. It has been suggested that the secondary metabolism enzyme PchB evolved from a primary metabolism CM precursor. Here, we employed directed evolution to convert PchB (back) into an efficient CM. A total of seven residues around the active site differing between PchB and a prototypical CM from Escherichia coli were randomized, and the resulting gene library was subjected to selection for CM activity. After growth selection in an auxotrophic strain, a catalyst with 10-fold increased CM activity emerged. The improved enzyme was again randomized at three active site positions and subjected to selection, leading to a PchB variant with a k cat/K m of 96,000 M-1 s-1, which is 40 times higher than that of the parent enzyme and well within the range of dedicated natural CMs. The facile conversion of an IPL into a CM by directed evolution coincides with the fact that both reactions proceed through mechanistically interesting pericyclic processes, reaction types otherwise rarely used by enzymes. When probing typical established CMs for catalytic promiscuity, we discovered spurious IPL activity for the secreted CM from Mycobacterium tuberculosis . Our results hint at active site features, particularly a Val at the bottom of the substrate-binding pocket that may have served as a steppingstone for the evolution of IPL activity in a primordial CM.
Crystal Structure and Mechanistic Molecular Modeling Studies of Mycobacterium tuberculosis Diterpene Cyclase Rv3377c
Zhang, Yue; Prach, Lisa M.; O’Brien, Terrence E.; et al. (2020)
Biochemistry
Terpenes make up the largest class of natural products, with extensive chemical and structural diversity. Diterpenes, mostly isolated from plants and rarely prokaryotes, exhibit a variety of important biological activities and valuable applications, including providing antitumor and antibiotic pharmaceuticals. These natural products are constructed by terpene synthases, a class of enzymes that catalyze one of the most complex chemical reactions in biology: converting simple acyclic oligo-isoprenyl diphosphate substrates to complex polycyclic products via carbocation intermediates. Here we obtained the second ever crystal structure of a class II diterpene synthase from bacteria, tuberculosinol pyrophosphate synthase (i.e., Halimadienyl diphosphate synthase, MtHPS, or Rv3377c) from Mycobacterium tuberculosis (Mtb). This enzyme transforms (E,E,E)-geranylgeranyl diphosphate into tuberculosinol pyrophosphate (Halimadienyl diphosphate). Rv3377c is part of the Mtb diterpene pathway along with Rv3378c, which converts tuberculosinol pyrophosphate to 1-tuberculosinyl adenosine (1-TbAd). This pathway was shown to exist only in virulent Mycobacterium species, but not in closely related avirulent species, and was proposed to be involved in phagolysosome maturation arrest. To gain further insight into the reaction pathway and the mechanistically relevant enzyme substrate binding orientation, electronic structure calculation and docking studies of reaction intermediates were carried out. Results reveal a plausible binding mode of the substrate that can provide the information to guide future drug design and anti-infective therapies of this biosynthetic pathway.
Concurrent cooperativity and substrate inhibition in the epoxidation of carbamazepine by cytochrome P450 3A4 active site mutants inspired by molecular dynamics simulations
Müller, Christian S.; Knehans, Tim; Davydov, Dmitri R.; et al. (2015)
Biochemistry
Cytochrome P450 3A4 (CYP3A4) is the major human P450 responsible for the metabolism of carbamazepine (CBZ). To explore the mechanisms of interactions of CYP3A4 with this anticonvulsive drug, we carried out multiple molecular dynamics (MD) simulations, starting with the complex of CYP3A4 manually docked with CBZ. On the basis of these simulations, we engineered CYP3A4 mutants I369F, I369L, A370V, and A370L, in which the productive binding orientation was expected to be stabilized, thus leading to increased turnover of CBZ to the 10,11-epoxide product. In addition, we generated CYP3A4 mutant S119A as a control construct with putative destabilization of the productive binding pose. Evaluation of the kinetics profiles of CBZ epoxidation demonstrate that CYP3A4-containing bacterial membranes (bactosomes) as well as purified CYP3A4 (wild-type and mutants I369L/F) exhibit substrate inhibition in reconstituted systems. In contrast, mutants S119A and A370V/L exhibit S-shaped profiles that are indicative of homotropic cooperativity. MD simulations with two to four CBZ molecules provide evidence that the substrate-binding pocket of CYP3A4 can accommodate more than one molecule of CBZ. Analysis of the kinetics profiles of CBZ metabolism with a model that combines the formalism of the Hill equation with an allowance for substrate inhibition demonstrates that the mechanism of interactions of CBZ with CYP3A4 involves multiple substrate-binding events (most likely three). Despite the retention of the multisite binding mechanism in the mutants, functional manifestations reveal an exquisite sensitivity to even minor structural changes in the binding pocket that are introduced by conservative substitutions such as I369F, I369L, and A370V.

Publications 1 - 10 of 19

Journal: Biochemistry

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