Mechanism of pyranopterin ring formation in molybdenum cofactor biosynthesis

The molybdenum cofactor (Moco) is essential for all kingdoms of life, plays central roles in various biological processes, and must be biosynthesized de novo. During Moco biosynthesis, the characteristic pyranopterin ring is constructed by a complex rearrangement of guanosine 5′-triphosphate (GTP) into cyclic pyranopterin (cPMP) through the action of two enzymes, MoaA and MoaC (molybdenum cofactor biosynthesis protein A and C, respectively). Conventionally, MoaA was considered to catalyze the majority of this transformation, with MoaC playing little or no role in the pyranopterin formation. Recently, this view was challenged by the isolation of 3′,8-cyclo-7,8-dihydro-guanosine 5′-triphosphate (3′,8-cH2GTP) as the product of in vitro MoaA reactions. To elucidate the mechanism of formation of Moco pyranopterin backbone, we performed biochemical characterization of 3′,8-cH2GTP and functional and X-ray crystallographic characterizations of MoaC. These studies revealed that 3′,8-cH2GTP is the only product of MoaA that can be converted to cPMP by MoaC. Our structural studies captured the specific binding of 3′,8-cH2GTP in the active site of MoaC. These observations provided strong evidence that the physiological function of MoaA is the conversion of GTP to 3′,8-cH2GTP (GTP 3′,8-cyclase), and that of MoaC is to catalyze the rearrangement of 3′,8-cH2GTP into cPMP (cPMP synthase). Furthermore, our structure-guided studies suggest that MoaC catalysis involves the dynamic motions of enzyme active-site loops as a way to control the timing of interaction between the reaction intermediates and catalytically essential amino acid residues. Thus, these results reveal the previously unidentified mechanism behind Moco biosynthesis and provide mechanistic and structural insights into how enzymes catalyze complex rearrangement reactions.

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MOLYBDENUM COFACTOR BIOSYNTHESIS IN Neurospora crassa: BIOCHEMICAL CHARACTERIZATION OF PLEIOTROPIC MOLYBDOENZYME MUTANTS nit-7, nit-8, nit-9A, B and C

Photochemistry and Photobiology ◽

10.1111/j.1751-1097.1995.tb09242.x ◽

1995 ◽

Vol 61 (1) ◽

pp. 54-60 ◽

Cited By ~ 5

Author(s):

Immanuel S. Heck ◽

Helga Ninnemann

Keyword(s):

Neurospora Crassa ◽

Biochemical Characterization ◽

Molybdenum Cofactor ◽

Cofactor Biosynthesis ◽

Molybdenum Cofactor Biosynthesis

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Conformation-Specific Inhibitory Anti-MMP-7 Monoclonal Antibody Sensitizes Pancreatic Ductal Adenocarcinoma Cells to Chemotherapeutic Cell Kill

Cancers ◽

10.3390/cancers13071679 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1679

Author(s):

Vishnu Mohan ◽

Jean P. Gaffney ◽

Inna Solomonov ◽

Maxim Levin ◽

Mordehay Klepfish ◽

...

Keyword(s):

Monoclonal Antibody ◽

Active Site ◽

Drug Targets ◽

Fas Ligand ◽

Specific Activity ◽

Matrix Metalloproteases ◽

Ductal Adenocarcinoma ◽

Enzyme Active Site ◽

Induced Apoptosis

Matrix metalloproteases (MMPs) undergo post-translational modifications including pro-domain shedding. The activated forms of these enzymes are effective drug targets, but generating potent biological inhibitors against them remains challenging. We report the generation of anti-MMP-7 inhibitory monoclonal antibody (GSM-192), using an alternating immunization strategy with an active site mimicry antigen and the activated enzyme. Our protocol yielded highly selective anti-MMP-7 monoclonal antibody, which specifically inhibits MMP-7′s enzyme activity with high affinity (IC50 = 132 ± 10 nM). The atomic model of the MMP-7-GSM-192 Fab complex exhibited antibody binding to unique epitopes at the rim of the enzyme active site, sterically preventing entry of substrates into the catalytic cleft. In human PDAC biopsies, tissue staining with GSM-192 showed characteristic spatial distribution of activated MMP-7. Treatment with GSM-192 in vitro induced apoptosis via stabilization of cell surface Fas ligand and retarded cell migration. Co-treatment with GSM-192 and chemotherapeutics, gemcitabine and oxaliplatin elicited a synergistic effect. Our data illustrate the advantage of precisely targeting catalytic MMP-7 mediated disease specific activity.

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Systematic analysis and integrative discovery of active-site subpocket-specific dehydroquinate synthase inhibitors combating antibiotic-resistant Staphylococcus aureus infection

Journal of Bioinformatics and Computational Biology ◽

10.1142/s0219720018500270 ◽

2018 ◽

Vol 16 (06) ◽

pp. 1850027

Author(s):

Quanfeng Liu ◽

Liping Li ◽

Fei Xu

Keyword(s):

Staphylococcus Aureus ◽

Active Site ◽

Shikimate Pathway ◽

In Vitro Susceptibility ◽

Systematic Analysis ◽

Antibiotic Resistant ◽

Enzyme Active Site ◽

Specific Inhibitors ◽

Dehydroquinate Synthase

Shikimate pathway plays an essential role in the biosynthesis of aromatic amino acids in various plants and bacteria, which consists of seven key enzymes and they are all attractive targets for antibacterial agent development due to their absence in humans. The Staphylococcus aureus dehydroquinate synthase (SaDHQS) is involved in the second step of shikimate pathway, which catalyzes the NAD[Formula: see text]-dependent conversion of 3-deoxy-D-arabino-heptulosonate-7-phosphate to dehydroquinate via multiple steps. The enzyme active site can be characterized by two spatially separated subpockets 1 and 2, which represent the reaction center of substrate adduct with NAD[Formula: see text] nicotinamide moiety and the assistant binding site of NAD[Formula: see text] adenine moiety, respectively. In silico virtual screening is performed against a biogenic compound library to discover SaDHQS subpocket-specific inhibitors, which were then tested against both antibiotic-sensitive and antibiotic-resistant S. aureus strains by using in vitro susceptibility test. The activity profile of hit compounds has no considerable difference between the antibiotic-sensitive and -resistant strains. The subpocket 1-specific inhibitors exhibit a generally higher activity than subpocket 2-specific inhibitors, and they also hold a strong selectivity between their cognate and noncognate subpockets. Dynamics and energetics analyses reveal that the SaDHQS active site prefers to interact with amphipathic and polar inhibitors by forming multiple hydrogen bonds and van der Waals packing at the complex interfaces of the two subpockets with their cognate inhibitors.

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Molecular genetic analysis of the moa operon of Escherichia coli K-12 required for molybdenum cofactor biosynthesis

Molecular Microbiology ◽

10.1111/j.1365-2958.1993.tb01652.x ◽

1993 ◽

Vol 8 (6) ◽

pp. 1071-1081 ◽

Cited By ~ 70

Author(s):

S. L. Rivers ◽

E. McNairn ◽

F. Blasco ◽

G. Giordano ◽

D. H. Boxer

Keyword(s):

Escherichia Coli ◽

Genetic Analysis ◽

Molecular Genetic ◽

Molecular Genetic Analysis ◽

Molybdenum Cofactor ◽

Cofactor Biosynthesis ◽

Molybdenum Cofactor Biosynthesis ◽

K 12

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A specific amino acid context in EGFR and HER2 phosphorylation sites enables selective binding to the active site of Src homology phosphatase 2 (SHP2)

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011422 ◽

2020 ◽

Vol 295 (11) ◽

pp. 3563-3575 ◽

Cited By ~ 2

Author(s):

Zachary Hartman ◽

Werner J. Geldenhuys ◽

Yehenew M. Agazie

Keyword(s):

Amino Acid ◽

Active Site ◽

Specific Binding ◽

Tyrosine Phosphatase ◽

Strong Inhibitor ◽

Selective Binding ◽

Specific Amino Acid ◽

Src Homology ◽

Her2 Signaling

The Src homology phosphatase 2 (SHP2) is a cytoplasmic enzyme that mediates signaling induced by multiple receptor tyrosine kinases, including signaling by the epidermal growth factor receptor (EGFR) family (EGFR1–4 or the human homologs HER1–4). In EGFR (HER1) and EGFR2 (HER2) signaling, SHP2 increases the half-life of activated Ras by blocking recruitment of Ras GTPase-activating protein (RasGAP) to the plasma membrane through dephosphorylation of docking sites on the receptors. However, it is unclear how SHP2 selectively recognizes RasGAP-binding sites on EGFR and HER2. In this report, we show that SHP2-targeted pTyr residues exist in a specific amino acid context that allows selective binding. More specifically, we show that acidic residues N-terminal to the substrate pTyr in EGFR and HER2 mediate specific binding by the SHP2 active site, leading to blockade of RasGAP binding and optimal signaling by the two receptors. Molecular modeling studies revealed that a peptide derived from the region of pTyr992-EGFR packs well and makes stronger interactions with the SHP2 active site than with the SHP1 active site, suggesting a built-in mechanism that enables selective substrate recognition by SHP2. A phosphorylated form of this peptide inhibits SHP2 activity in vitro and EGFR and HER2 signaling in cells, suggesting inhibition of SHP2 protein tyrosine phosphatase activity by this peptide. Although we do not expect this peptide to be a strong inhibitor by itself, we foresee that the insights into SHP2 selectivity described here will be useful in future development of active-site small molecule-based inhibitors.

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A thylakoid membrane-bound and redox-active rubredoxin (RBD1) functions in de novo assembly and repair of photosystem II

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1903314116 ◽

2019 ◽

Vol 116 (33) ◽

pp. 16631-16640 ◽

Cited By ~ 4

Author(s):

José G. García-Cerdán ◽

Ariel L. Furst ◽

Kent L. McDonald ◽

Danja Schünemann ◽

Matthew B. Francis ◽

...

Keyword(s):

Photosystem Ii ◽

Thylakoid Membrane ◽

De Novo Assembly ◽

Biochemical Characterization ◽

De Novo ◽

Midpoint Potential ◽

Photosynthetic Organisms ◽

Redox Active ◽

Photooxidative Damage

Photosystem II (PSII) undergoes frequent photooxidative damage that, if not repaired, impairs photosynthetic activity and growth. How photosynthetic organisms protect vulnerable PSII intermediate complexes during de novo assembly and repair remains poorly understood. Here, we report the genetic and biochemical characterization of chloroplast-located rubredoxin 1 (RBD1), a PSII assembly factor containing a redox-active rubredoxin domain and a single C-terminal transmembrane α-helix (TMH) domain. RBD1 is an integral thylakoid membrane protein that is enriched in stroma lamellae fractions with the rubredoxin domain exposed on the stromal side. RBD1 also interacts with PSII intermediate complexes containing cytochrome b559. Complementation of the Chlamydomonas reinhardtii (hereafter Chlamydomonas) RBD1-deficient 2pac mutant with constructs encoding RBD1 protein truncations and site-directed mutations demonstrated that the TMH domain is essential for de novo PSII assembly, whereas the rubredoxin domain is involved in PSII repair. The rubredoxin domain exhibits a redox midpoint potential of +114 mV and is proficient in 1-electron transfers to a surrogate cytochrome c in vitro. Reduction of oxidized RBD1 is NADPH dependent and can be mediated by ferredoxin-NADP+ reductase (FNR) in vitro. We propose that RBD1 participates, together with the cytochrome b559, in the protection of PSII intermediate complexes from photooxidative damage during de novo assembly and repair. This role of RBD1 is consistent with its evolutionary conservation among photosynthetic organisms and the fact that it is essential in photosynthetic eukaryotes.

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