Genome replication in flavivirus requires (−) strand RNA synthesis, (+) strand RNA synthesis, and 5′-RNA capping and methylation. To carry out viral genome replication, flavivirus assembles a replication complex, consisting of both viral and host proteins, on the cytoplasmic side of the endoplasmic reticulum (ER) membrane. Two major components of the replication complex are the viral non-structural (NS) proteins NS3 and NS5. Together they possess all the enzymatic activities required for genome replication, yet how these activities are coordinated during genome replication is not clear. We provide an overview of the flaviviral genome replication process, the membrane-bound replication complex, and recent crystal structures of full-length NS5. We propose a model of how NS3 and NS5 coordinate their activities in the individual steps of (−) RNA synthesis, (+) RNA synthesis, and 5′-RNA capping and methylation.
Flavivirus nonstructural protein 5 (NS5) consists of methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) domains, which catalyze 5’-RNA capping/methylation and RNA synthesis, respectively, during viral genome replication. Although the crystal structure of flavivirus NS5 is known, no data about the quaternary organization of the functional enzyme are available. We report the crystal structure of dengue virus full-length NS5, where eight molecules of NS5 are arranged as four independent dimers in the crystallographic asymmetric unit. The relative orientation of each monomer within the dimer, as well as the orientations of the MTase and RdRp domains within each monomer, is conserved, suggesting that these structural arrangements represent the biologically relevant conformation and assembly of this multi-functional enzyme. Essential interactions between MTase and RdRp domains are maintained in the NS5 dimer via inter-molecular interactions, providing evidence that flavivirus NS5 can adopt multiple conformations while preserving necessary interactions between the MTase and RdRp domains. Furthermore, many NS5 residues that reduce viral replication are located at either the inter-domain interface within a monomer or at the inter-molecular interface within the dimer. Hence the X-ray structure of NS5 presented here suggests that MTase and RdRp activities could be coordinated as a dimer during viral genome replication.
The structural underpinnings of enzyme substrate specificity are investigated in a pair of copper amine oxidases (CAOs) from Hansenula polymorpha (HPAO-1 and HPAO-2). The X-ray crystal structure (to 2.0 Å resolution) and steady state kinetic data of the second copper amine oxidase (HPAO-2) are presented for comparison to HPAO-1. Despite 34 % sequence identity and superimposable active site residues implicated in catalysis, the enzymes vary considerably in their substrate entry channel. The previously studied CAO, HPAO-1, has a narrow substrate channel. In contrast HPAO-2 has a wide funnel-shaped substrate channel, which also contains a side-chamber. In addition, there are a number of amino acid changes within the channels of HPAO-2 and HPAO-1 that may sterically impact the ability of substrates to form covalent Schiff base catalytic intermediates and to initiate chemistry. These differences can partially explain the greatly different substrate specificities as characterized by k cat /K m value differences: in HPAO-1, the k cat /K m for methylamine is 330-fold greater than for benzylamine, whereas in HPAO-2 it is benzylamine that is the better substrate by 750-fold. In HPAO-2 an inflated D k cat /K m (methylamine) in relation to D k cat / K m (benzylamine) indicates that proton abstraction has been impeded more than substrate release. In HPAO-1, D k cat /K m (S) changes little with the slow substrate, and indicates a similar increase in the energy barriers that control both substrate binding and subsequent catalysis. In neither case is k cat / K m for the second substrate, O 2, significantly altered. These results reinforce the modular nature of the active sites of CAOs and show that multiple factors contribute to substrate specificity and catalytic efficiency. In HPAO-1, the enzyme with the smaller substrate binding pocket, both initial substrate binding and proton loss are affected by an increase in substrate size, while in HPAO-2, the enzyme with the larger substrate binding pocket, the rate of proton loss is differentially affected when a phenyl substituent in substrate is reduced to the size of a methyl group. † This work was supported by NIH grants, GM39296 to J.P.K., GM66569 to C.M.W. and Chemistry-Biology Interface Training Grant GM-008700 to B.J.J, and Minnesota Medical Foundation grant 3714-9221-06, Office of the Dean of the Graduate School of the University of Minnesota grant 21087, and a Minnesota Partnership for Biotechnology and Medical Genomics grant SPAP-05-0013-P-FY06 to C.M.W. ‡ Co-ordinates and structure factors have been deposited in the Protein Data Bank as entry 3loy.*Address correspondence to: Carrie M. Wilmot, Tel: (612) 624-2406, Fax: (612) 624-5121, wilmo004@umn.edu and Judith P. Klinman, Tel: (510) 642-2668, Fax (510) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptCopper amine oxidases (CAOs) 1 are virtually ubiquitous in aerobic organisms, and catalyze the oxidative deamination of primary amines in the following overall reaction:While usually...
Copper amine oxidases (CAOs) are a ubiquitous group of enzymes that catalyze the conversion of primary amines to aldehydes coupled to the reduction of O2 to H2O2. These enzymes utilize a wide range of substrates from methylamine to polypeptides. Changes in CAO activity are correlated with a variety of human diseases, including diabetes mellitus, Alzheimer’s disease, and inflammatory disorders. CAOs contain a cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ), that is required for catalytic activity and synthesized through the post-translational modification of a tyrosine residue within the CAO polypeptide. TPQ generation is a self-processing event only requiring the addition of oxygen and Cu(II) to the apoCAO. Thus, the CAO active site supports two very different reactions: TPQ synthesis, and the two electron oxidation of primary amines. Crystal structures are available from bacterial through to human sources, and have given insight into substrate preference, stereospecificity, and structural changes during biogenesis and catalysis. In particular both these processes have been studied in crystallo through the addition of native substrates. These latter studies enable intermediates during physiological turnover to be directly visualized, and demonstrate the power of this relatively recent development in protein crystallography.
Background: Copper amine oxidases activate O 2 either at the copper center or aminoquinol cofactor. Results: Catalytic intermediates from the oxidative half-reaction are structurally and spectroscopically characterized. Conclusion: The mechanism of O 2 activation may depend on accessibility dictated by two conformers of the quinone cofactor. Significance: Structural changes that inform on catalytic mechanism have been revealed in the ubiquitous copper amine oxidases.
Ehrlichia chaffeensis, the causative agent of human monocytotropic ehrlichiosis, secretes several effector proteins that bind host DNA to modulate host gene expression. The tandem repeat protein 120 (TRP120), one of the largest effector proteins, has four nearly identical tandem repeat (TR) regions that each consists of 80 amino acids. In addition to playing a role in ehrlichial binding and internalization, TRP120 translocates to the host nucleus where it is thought to function as a transcription factor that modulates gene expression. However, sequence analysis of TRP120 does not identify the presence of DNA-binding or trans-activation domains typical of classical eukaryotic transcription factors. Thus, the mechanism by which TRP120 binds DNA and modulates gene expression remains elusive. Herein, we expressed the TR regions of the TRP120 protein, and characterized its solution structure and ability to bind DNA. TRP120, expressed as either a one or two TR repeat, is a monomer in solution, and is mostly disordered as determined by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. Using NMR spectroscopy, we further show that the 1 TR construct selectively binds GC-rich DNA. Although low pH was required for TRP120 TR-DNA interaction, acidic pH alone does not induce any significant structural changes in the TR region. This suggests that TRP120 folds into an ordered structure upon forming a protein-DNA complex, and thus folding of TRP120 TR is coupled with DNA binding.
PqqB is an enzyme involved in the biosynthesis of pyrroloquinoline quinone (PQQ) and a distal member of the metallo-β-lactamase (MBL) superfamily. PqqB lacks two residues in the conserved signature motif HxHxDH that makes up the key metal-chelating elements that can bind up to two metal ions at the active site of MBLs and other members of its superfamily. Here we report crystal structures of PqqB bound to Mn2+, Mg2+, Cu2+ and Zn2+. These structures demonstrate that PqqB can still bind metal ions at the canonical MBL active site. The fact that PqqB can adapt its side chains to chelate a wide spectrum of metal ions with different coordination features on a uniform main chain scaffold demonstrates its metal binding plasticity. This plasticity may provide insights into the structural basis of promiscuous activities found in ensembles of metal complexes within this superfamily. Furthermore, PqqB belongs to a small subclass of MBLs that contain an additional CxCxxC motif that binds a structural Zn2+. Our data support a key role for this motif in dimerization.
PDB References: apoHPAO-1, 3sx1; Co II -apoHPAO-1, 3sxx; Cu I -apoHPAO-1, 3t0u.Copper amine oxidases (CAOs) catalyze the oxidative deamination of primary amines to their corresponding aldehydes, with the concomitant reduction of O 2 to H 2 O 2 . Catalysis requires two cofactors: a mononuclear copper center and the cofactor 2,4,5-trihydroxyphenylalanine quinone (TPQ). TPQ is synthesized through the post-translational modification of an endogenous tyrosine residue and requires only oxygen and copper to proceed. TPQ biogenesis in CAO can be supported by alternate metals, albeit at decreased rates. A variety of factors are thought to contribute to the degree to which a metal can support TPQ biogenesis, including Lewis acidity, redox potential and electrostatic stabilization capability. The crystal structure has been solved of one of two characterized CAOs from the yeast Hansenula polymorpha (HPAO-1) in its metal-free (apo) form, which contains an unmodified precursor tyrosine residue instead of fully processed TPQ (HPAO-1 was denoted HPAO in the literature prior to 2010). Structures of apoHPAO-1 in complex with Cu I and Co II have also been solved, providing structural insight into metal binding prior to biogenesis.
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