scholarly journals Three Conserved Regions in Baculovirus Sulfhydryl Oxidase P33 Are Critical for Enzymatic Activity and Function

2017 ◽  
Vol 91 (23) ◽  
Author(s):  
Wenhua Kuang ◽  
Huanyu Zhang ◽  
Manli Wang ◽  
Ning-Yi Zhou ◽  
Fei Deng ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Active Site ◽  
Salt Bridge ◽  
Sulfhydryl Oxidase ◽  
Dimer Interface ◽  
Conserved Regions ◽  
Occlusion Bodies ◽  
Budded Virus ◽  
And Function ◽  
Substrate Proteins

ABSTRACT Baculoviruses encode a conserved sulfhydryl oxidase, P33, which is necessary for budded virus (BV) production and multinucleocapsid occlusion-derived virus (ODV) formation. Here, the structural and functional relationship of P33 was revealed by X-ray crystallography, site-directed mutagenesis, and functional analysis. Based on crystallographic characterization and structural analysis, a series of P33 mutants within three conserved regions, i.e., the active site, the dimer interface, and the R127-E183 salt bridge, were constructed. In vitro experiments showed that mutations within the active site and dimer interface severely impaired the sulfhydryl oxidase activity of P33, while the mutations in the salt bridge had a relatively minor influence. Recombinant viruses containing mutated P33 were constructed and assayed in vivo. Except for the active-site mutant AXXA, all other mutants produced infectious BVs, although certain mutants had a decreased BV production. The active-site mutant H114A, the dimer interface mutant H227D, and the salt bridge mutant R127A-E183A were further analyzed by electron microscopy and bioassays. The occlusion bodies (OBs) of mutants H114A and R127A-E183A had a ragged surface and contained mostly ODVs with a single nucleocapsid. The OBs of all three mutants contained lower numbers of ODVs and had a significantly reduced oral infectivity in comparison to control virus. Crystallographic analyses further revealed that all three regions may coordinate with one another to achieve optimal function of P33. Taken together, our data revealed that all the three conserved regions are involved in P33 activity and are crucial for virus morphogenesis and peroral infectivity. IMPORTANCE Sulfhydryl oxidase catalyzes disulfide bond formation of substrate proteins. P33, a baculovirus-encoded sulfhydryl oxidase, is different from other cellular and viral sulfhydryl oxidases, bearing unique features in tertiary and quaternary structure organizations. In this study, we found that three conserved regions, i.e., the active site, dimer interface, and the R127-E183 salt bridge, play important roles in the enzymatic activity and function of P33. Previous observations showed that deletion of p33 results in a total loss of budded virus (BV) production and in morphological changes in occlusion-derived virus (ODV). Our study revealed that certain P33 mutants lead to occlusion bodies (OBs) with a ragged surface, decreased embedded ODVs, and reduced oral infectivity. Interestingly, some P33 mutants with impaired ODV/OB still retained BV productivity, indicating that the impacts on BV and on ODV/OB are two distinctly different functions of P33, which are likely to be performed via different substrate proteins.

scholarly journals Residue R113 Is Essential for PhoP Dimerization and Function: a Residue Buried in the Asymmetric PhoP Dimer Interface Determined in the PhoPN Three-Dimensional Crystal Structure

2003 ◽  
Vol 185 (1) ◽  
pp. 262-273 ◽  
Author(s):  
Yinghua Chen ◽  
Catherine Birck ◽  
Jean-Pierre Samama ◽  
F. Marion Hulett
Keyword(s):  
Crystal Structure ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Salt Bridge ◽  
Pho Regulon ◽  
Dimer Interface ◽  
And Function ◽  
Negative Phenotype

ABSTRACT Bacillus subtilis PhoP is a member of the OmpR/PhoB family of response regulators that is directly required for transcriptional activation or repression of Pho regulon genes in conditions under which Pi is growth limiting. Characterization of the PhoP protein has established that phosphorylation of the protein is not essential for PhoP dimerization or DNA binding but is essential for transcriptional regulation of Pho regulon genes. DNA footprinting studies of PhoP-regulated promoters showed that there was cooperative binding between PhoP dimers at PhoP-activated promoters and/or extensive PhoP oligomerization 3′ of PhoP-binding consensus repeats in PhoP-repressed promoters. The crystal structure of PhoPN described in the accompanying paper revealed that the dimer interface between two PhoP monomers involves nonidentical surfaces such that each monomer in a dimer retains a second surface that is available for further oligomerization. A salt bridge between R113 on one monomer and D60 on another monomer was judged to be of major importance in the protein-protein interaction. We describe the consequences of mutation of the PhoP R113 codon to a glutamate or alanine codon and mutation of the PhoP D60 codon to a lysine codon. In vivo expression of either PhoPR113E, PhoPR113A, or PhoPD60K resulted in a Pho-negative phenotype. In vitro analysis showed that PhoPR113E was phosphorylated by PhoR (the cognate histidine kinase) but was unable to dimerize. Monomeric PhoPR113E∼P was deficient in DNA binding, contributing to the PhoPR113E in vivo Pho-negative phenotype. While previous studies emphasized that phosphorylation was essential for PhoP function, data reported here indicate that phosphorylation is not sufficient as PhoP dimerization or oligomerization is also essential. Our data support the physiological relevance of the residues of the asymmetric dimer interface in PhoP dimerization and function.

scholarly journals Structural basis of the dominant inheritance of hypermethioninemia associated with the Arg264His mutation in the MAT1A gene

2020 ◽  
Vol 76 (6) ◽  
pp. 594-607
Author(s):  
Jiraporn Panmanee ◽  
Svetlana V. Antonyuk ◽  
S. Samar Hasnain
Keyword(s):  
Active Site ◽  
Autosomal Recessive ◽  
Salt Bridge ◽  
Pathogenic Mutation ◽  
Structural Basis ◽  
Crystallographic Structure ◽  
Common Mutation ◽  
Dominant Inheritance ◽  
Gene Encoding ◽  
Dimer Interface

Methionine adenosyltransferase (MAT) deficiency, characterized by isolated persistent hypermethioninemia (IPH), is caused by mutations in the MAT1A gene encoding MATαl, one of the major hepatic enzymes. Most of the associated hypermethioninemic conditions are inherited as autosomal recessive traits; however, dominant inheritance of hypermethioninemia is caused by an Arg264His (R264H) mutation. This mutation has been confirmed in a screening programme of newborns as the most common mutation in babies with IPH. Arg264 makes an inter-subunit salt bridge located at the dimer interface where the active site assembles. Here, it is demonstrated that the R264H mutation results in greatly reduced MAT activity, while retaining its ability to dimerize, indicating that the lower activity arises from alteration at the active site. The first crystallographic structure of the apo form of the wild-type MATαl enzyme is provided, which shows a tetrameric assembly in which two compact dimers combine to form a catalytic tetramer. In contrast, the crystal structure of the MATαl R264H mutant reveals a weaker dimeric assembly, suggesting that the mutation lowers the affinity for dimer–dimer interaction. The formation of a hetero-oligomer with the regulatory MATβV1 subunit or incubation with a quinolone-based compound (SCR0911) results in the near-full recovery of the enzymatic activity of the pathogenic mutation R264H, opening a clear avenue for a therapeutic solution based on chemical interventions that help to correct the defect of the enzyme in its ability to metabolize methionine.

scholarly journals Fe(2)OG: an integrated HMM profile-based web server to predict and analyze putative non-haem iron(II)- and 2-oxoglutarate-dependent dioxygenase function in protein sequences

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Siddhartha Kundu
Keyword(s):  
Amino Acid ◽  
Water Molecule ◽  
Active Site ◽  
Ferrous Iron ◽  
Web Server ◽  
Protein Sequences ◽  
Diverse Group ◽  
And Function ◽  
Functionally Diverse ◽  
Haem Iron

Abstract Objective Non-haem iron(II)- and 2-oxoglutarate-dependent dioxygenases (i2OGdd), are a taxonomically and functionally diverse group of enzymes. The active site comprises ferrous iron in a hexa-coordinated distorted octahedron with the apoenzyme, 2-oxoglutarate and a displaceable water molecule. Current information on novel i2OGdd members is sparse and relies on computationally-derived annotation schema. The dissimilar amino acid composition and variable active site geometry thereof, results in differing reaction chemistries amongst i2OGdd members. An additional need of researchers is a curated list of sequences with putative i2OGdd function which can be probed further for empirical data. Results This work reports the implementation of $$Fe\left(2\right)OG$$ F e 2 O G , a web server with dual functionality and an extension of previous work on i2OGdd enzymes $$\left(Fe\left(2\right)OG\equiv \{H2OGpred,DB2OG\}\right)$$ F e 2 O G ≡ { H 2 O G p r e d , D B 2 O G } . $$Fe\left(2\right)OG$$ F e 2 O G , in this form is completely revised, updated (URL, scripts, repository) and will strengthen the knowledge base of investigators on i2OGdd biochemistry and function. $$Fe\left(2\right)OG$$ F e 2 O G , utilizes the superior predictive propensity of HMM-profiles of laboratory validated i2OGdd members to predict probable active site geometries in user-defined protein sequences. $$Fe\left(2\right)OG$$ F e 2 O G , also provides researchers with a pre-compiled list of analyzed and searchable i2OGdd-like sequences, many of which may be clinically relevant. $$Fe(2)OG$$ F e ( 2 ) O G , is freely available (http://204.152.217.16/Fe2OG.html) and supersedes all previous versions, i.e., H2OGpred, DB2OG.

Protein Immobilization in Metal–Organic Frameworks by Covalent Binding

2014 ◽  
Vol 67 (11) ◽  
pp. 1629 ◽  
Author(s):  
Xuan Wang ◽  
Trevor A. Makal ◽  
Hong-Cai Zhou
Keyword(s):  
Enzymatic Activity ◽  
Enzyme Immobilization ◽  
Working Conditions ◽  
Active Site ◽  
Covalent Binding ◽  
Metal Organic Frameworks ◽  
Protein Immobilization ◽  
Immobilization Of Enzymes ◽  
Metal Organic ◽  
Biological Catalysis

Metal–organic frameworks (MOFs), possessing a well defined system of pores, demonstrate extensive potential serving as a platform in biological catalysis. Successful immobilization of enzymes in a MOF system retains the enzymatic activity, renders the active site more accessible to the substrate, and promises recyclability for reuse, and solvent adaptability in a broad range of working conditions. This highlight describes enzyme immobilization on MOFs via covalent binding and its significance.

scholarly journals Acetylcholine Receptor Gating at Extracellular Transmembrane Domain Interface: the “Pre-M1” Linker

2007 ◽  
Vol 130 (6) ◽  
pp. 559-568 ◽  
Author(s):  
Prasad Purohit ◽  
Anthony Auerbach
Keyword(s):  
Acetylcholine Receptors ◽  
State Energy ◽  
Transmembrane Domain ◽  
Salt Bridge ◽  
Side Chain ◽  
Coupling Energy ◽  
And Function ◽  
Loop 2 ◽  
Α Subunits ◽  
Α1 Subunit

Charged residues in the β10–M1 linker region (“pre-M1”) are important in the expression and function of neuromuscular acetylcholine receptors (AChRs). The perturbation of a salt bridge between pre-M1 residue R209 and loop 2 residue E45 has been proposed as being a principle event in the AChR gating conformational “wave.” We examined the effects of mutations to all five residues in pre-M1 (positions M207–P211) plus E45 in loop 2 in the mouse α1-subunit. M207, Q208, and P211 mutants caused small (approximately threefold) changes in the gating equilibrium constant (Keq), but the changes for R209, L210, and E45 were larger. Of 19 different side chain substitutions at R209 on the wild-type background, only Q, K, and H generated functional channels, with the largest change in Keq (67-fold) from R209Q. Various R209 mutants were functional on different E45 backgrounds: H, Q, and K (E45A), H, A, N, and Q (E45R), and K, A, and N (E45L). Φ values for R209 (on the E45A background), L210, and E45 were 0.74, 0.35, and 0.80, respectively. Φ values for R209 on the wt and three other backgrounds could not be estimated because of scatter. The average coupling energy between 209/45 side chains (six different pairs) was only −0.33 kcal/mol (for both α subunits, combined). Pre-M1 residues are important for expression of functional channels and participate in gating, but the relatively modest changes in closed- vs. open-state energy caused mutations, the weak coupling energy between these residues and the functional activity of several unmatched-charge pairs are not consistent with the perturbation of a salt bridge between R209 and E45 playing the principle role in gating.

Oxidative protein folding in the mammalian endoplasmic reticulum

2004 ◽  
Vol 32 (5) ◽  
pp. 655-658 ◽  
Author(s):  
C.E. Jessop ◽  
S. Chakravarthi ◽  
R.H. Watkins ◽  
N.J. Bulleid
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Redox State ◽  
Structure And Function ◽  
Reduced Form ◽  
Secretory Proteins ◽  
Oxidative Protein Folding ◽  
Disulphide Bonds ◽  
And Function ◽  
Substrate Proteins

Native disulphide bonds are essential for the structure and function of many membrane and secretory proteins. Disulphide bonds are formed, reduced and isomerized in the endoplasmic reticulum of mammalian cells by a family of oxidoreductases, which includes protein disulphide isomerase (PDI), ERp57, ERp72, P5 and PDIR. This review will discuss how these enzymes are maintained in either an oxidized redox state that allows them to form disulphide bonds in substrate proteins or a reduced form that allows them to perform isomerization and reduction reactions, how these opposing pathways may co-exist within the same compartment and why so many oxidoreductases exist when PDI alone can perform all three of these functions.

scholarly journals Molecular biology of baculovirus and its use in biological control in Brazil

1999 ◽  
Vol 34 (10) ◽  
pp. 1733-1761 ◽  
Author(s):  
Maria Elita Batista de Castro ◽  
Marlinda Lobo de Souza ◽  
William Sihler ◽  
Júlio Carlyle Macedo Rodrigues ◽  
Bergmann Morais Ribeiro
Keyword(s):  
Molecular Biology ◽  
Recombinant Dna ◽  
Host Cells ◽  
Virus Propagation ◽  
Viral Particles ◽  
Occlusion Bodies ◽  
Budded Virus ◽  
Recombinant Dna Techniques ◽  
High Level ◽  
Eukaryotic Organisms

Baculoviruses are insect viruses found mainly in Lepidoptera. The family Baculoviridae is taxonomically divided in two genera, Nucleopolyhedrovirus and Granulovirus, which differ by occlusion body morphology. NPVs (Nucleopolyhedroviruses) have polyhedrical inclusion bodies (PIBs) containing multiple viral particles, while GVs (Granuloviruses) appear to be generally single particles occluded in oval shaped occlusion bodies. During the life cycle, two different viral progenies are produced: BV (Budded Virus) and PDV (Polyhedra Derived Virus), which are essential for the infectious process and virus propagation in host cells. Baculoviruses are being used for pest control and they are especially safe due to their specificity and invertebrate-restricted host range. Baculoviruses have been used as vectors for high level protein expression ofheterologous genes from prokaryotic and eukaryotic organisms. Also, recombinant DNA techniques have allowed the production of genetically modified viral insecticides. This study is a review on the taxonomy, structure, replication and molecular biology of baculoviruses, as well as their use as bioinsecticides in Brazil.

Characterization of anEscherichia coliSulfite Oxidase Homologue Reveals the Role of a Conserved Active Site Cysteine in Assembly and Function†

Biochemistry ◽  
2005 ◽  
Vol 44 (30) ◽  
pp. 10339-10348 ◽  
Author(s):  
Stephen J. Brokx ◽  
Richard A. Rothery ◽  
Guijin Zhang ◽  
Derek P. Ng ◽  
Joel H. Weiner
Keyword(s):  
Active Site ◽  
Active Site Cysteine ◽  
And Function

scholarly journals Reverse evolution leads to genotypic incompatibility despite functional and active site convergence

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Miriam Kaltenbach ◽  
Colin J Jackson ◽  
Eleanor C Campbell ◽  
Florian Hollfelder ◽  
Nobuhiko Tokuriki
Keyword(s):  
Active Site ◽  
Adaptive Landscape ◽  
Sequence Structure ◽  
Activity Increase ◽  
Enzyme Active Site ◽  
Fundamental Relationship ◽  
And Function ◽  
Alternative Set ◽  
Shed Light ◽  
New Mutations

Understanding the extent to which enzyme evolution is reversible can shed light on the fundamental relationship between protein sequence, structure, and function. Here, we perform an experimental test of evolutionary reversibility using directed evolution from a phosphotriesterase to an arylesterase, and back, and examine the underlying molecular basis. We find that wild-type phosphotriesterase function could be restored (>104-fold activity increase), but via an alternative set of mutations. The enzyme active site converged towards its original state, indicating evolutionary constraints imposed by catalytic requirements. We reveal that extensive epistasis prevents reversions and necessitates fixation of new mutations, leading to a functionally identical sequence. Many amino acid exchanges between the new and original enzyme are not tolerated, implying sequence incompatibility. Therefore, the evolution was phenotypically reversible but genotypically irreversible. Our study illustrates that the enzyme's adaptive landscape is highly rugged, and different functional sequences may constitute separate fitness peaks.

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