Reaction Mechanism of Histone Demethylation in αKG-dependent Non-Heme Iron Enzymes.

Histone demethylases (KDMs) catalyze histone lysine demethylation, an important epigenetic process that controls gene expression in eukaryotes, and represent important cancer drug targets for cancer treatment. Demethylation of histone is comprised of sequential reaction steps including oxygen activation, decarboxylation, and demethylation. The initial oxygen binding and activation steps have been studied. However, the information on the complete catalytic reaction cycle is limited, which has impeded the structure-based design of inhibitors targeting KDMs. Here we report the mechanism of the complete reaction steps catalyzed by a representative nonheme iron αKG-dependent KDM, PHF8 using QM/MM approaches. The atomic-level understanding on the complete reaction mechanism of PHF8 would shed light on the structure-based design of selective inhibitors targeting KDMs to intervene in cancer epigenetics.

Mechanism of Phosphatidylglycerol Activation Catalyzed by Prolipoprotein Diacylglyceryl Transferase.

Lipoproteins are essential for bacterial survival. Bacterial lipoprotein biosynthesis is accomplished by sequential modification by three enzymes in the inner membrane, all of which are emerging antimicrobial targets. The X-ray crystal structure of prolipoprotein diacylglyceryl transferase (Lgt) and apolipoprotein N-acyl transferase (Lnt) has been reported. However, the mechanisms of the post-translational modification catalyzed by these enzymes have not been understood. Here, we studied the mechanism of the transacylation reaction catalyzed by Lgt, the first enzyme for lipoprotein modification using molecular docking, molecular dynamics, and quantum mechanics/molecular mechanics (QM/MM) calculations. Our results suggest that Arg143, Arg239, and Glu202 play a critical role in stabilizing the glycerol-1-phosphate head group and activating the glycerol C3-O ester bond of the phosphatidylglycerol (PG) substrate. With PG binding, the opening of the L6-7 loop mediated by the highly conserved Arg236 residue as a gatekeeper is observed, which facilitates the release of the modified lipoprotein product, as well as the entry of another PG substrate. Further QM/MM studies revealed that His103 acts as a catalytic base to abstract a proton from the cysteine residue of the preproliprotein, initiating the diacylglyceryl transfer from PG to preprolipoprotein. This is the first study on the mechanism of lipoprotein modification catalyzed by a post-translocational processing enzyme. The transacylation mechanism of Lgt would shed light on the development of novel antimicrobial therapies targeting the challenging enzymes involved in the post-translocational modification pathway of lipoproteins.

Artificial cysteine-lipases with high activity and altered catalytic mechanism created by laboratory evolution.

Engineering artificial enzymes with high activity and catalytic mechanism different from naturally occurring enzymes is a challenge in protein design. For example, many attempts have been made to obtain active hydrolases by introducing a Ser → Cys exchange at the respective catalytic triads, but this generally induced a breakdown of activity. We now report that this long-standing dogma no longer pertains, provided additional mutations are introduced by directed evolution. By employing Candida antarctica lipase B (CALB) as the model enzyme with the Ser-His-Asp catalytic triad, a highly active cysteine-lipase having a Cys-His-Asp catalytic triad and additional mutations W104V/A281Y/A282Y/V149G can be evolved, showing a 40-fold higher catalytic efficiency than wild-type CALB in the hydrolysis of 4-nitrophenyl benzoate, and tolerating bulky substrates. Crystal structures, kinetics, MD simulations and QM/MM calculations reveal dynamic features and explain all results, including the preference of a two-step mechanism involving the zwitterionic pair Cys105-/His224+ rather than a concerted process.

Synthesis of carbon-14-labelled peptides.

Carbon-14 (14 C)-labelled active pharmaceutical ingredients (APIs) and investigational medicinal products (IMPs) are required for phase 0/I to phase III mass balance and micro-dosing clinical trials. In some cases, this may involve the synthesis of 14 C-labelled peptides, and the analysis can be performed by accelerated mass spectrometry (AMS). The 14 C-peptide is typically prepared by the solid-phase peptide synthesis (SPPS) approach using custom-made glassware for the key coupling steps. Further modification of the purified 14 C-peptide can then be performed.

Radionuclide antibody-conjugates, a targeted therapy towards cancer.

Targeted alpha therapy (TAT) is an investigational procedure which utilises monoclonal antibodies (mAbs), peptide conjugates and/or other chemical compounds. These bio-vectors are able to transport a dose of alpha particles to destroy cancer cells. Radionuclide antibody-conjugates (RACs), labelled with beta emitters, have already been used in humans. More recently, TAT has been introduced to treat oncological diseases mainly leukaemia and lymphoma. Encouraging results have also been obtained in solid neoplasms with the administration of anti-tenascin. This chimeric antibody labelled with astatine-211 was delivered in patients with recurrent brain tumours into a surgically created cavity. Conversely, a clinical trial using a standard TAT approach to treat patients with metastatic melanoma, observed the shrinkage of the solid tumour mass. This response in melanoma may lead to an alternative mechanism for TAT, called tumour-antivascular- alpha-therapy (TAVAT), and forms the basis of a novel approach to the treatment of cancer disease states. In this paper, we will concentrate mainly on the application of TAT using antibodies. In particular, an investigation into the major general features connected with the use of alpha emitters in cancer therapy will be discussed. The prospective role of TAT with RACs will also be outlined briefly, especially focussing on the most important therapeutic strategies to date based on antibodies radiolabelled with beta emitters.