Molecular arrangements that accompany binding of rice xylanase inhibitor protein OsXIP and the Rhizopus oryzae GH11 xylanase RXyn2.

Plants have evolved xylanase inhibitor proteins as part of their defense mechanisms against phytopathogens. The rice xylanase inhibitor protein (OsXIP) is structurally similar to GH18 chitinase and homologous to wheat XIP-type inhibitor (XIP-I), which inhibits both GH10 and GH11 xylanases. Various inhibition and interaction analyses showed that OsXIP competitively inhibits the hydrolytic activity of GH11 xylanase RXyn2, but not the activity of GH10 xylanase RXyn1 from Rhizopus oryzae.

β1,6-Selective Enzymatic N-Acetylglucosamination Catalyzed by the Family GH84 N-Acetyl-β-D-glucosaminidase from Bacteroides thetaiotaomicron and its Glycosyl Acceptor Specificity.

The chemoenzymatic synthesis of oligosaccharides presents a highly attractive methodology with significant potential for diverse applications, particularly through using various glycosidases. In this study, the O-glycan core 6 disaccharide moiety, GlcNAcβ1-6GalNAc, was successfully synthesized via enzymatic glycosylation using an N-acetyl-β-D-glucosaminidase from Bacteroides thetaiotaomicron (BtOGA), a member of glycoside hydrolase family 84 (GH84), alongside an N-acetyl-D-glucosamine oxazoline derivative (GlcNAc-oxa) as the glycosyl donor. Furthermore, an investigation into glycosyl acceptor recognition in BtOGA-catalyzed enzymatic glycosylation indicated that the presence of an aromatic group at the anomeric position and an axial hydroxy group at the 4-position of the saccharide moiety is crucial for effective recognition of BtOGA as a glycosyl acceptor.

Bidirectional electro-enzymatic reaction of coenzyme F using benzyl viologen and F-dependent sulfite reductase.

Coenzyme F is recognized as a crucial electron carrier in methane-generating metabolism but, beyond this, has garnered significant attention for its role in diverse microbial physiologies and relevance in industrial, medical, and environmental applications. However, one limitation of current application of F is the necessity of chemical electron donors for its reduction. In this study, an electrochemical reaction system was designed to facilitate electron transfer between the electrode and F using F-dependent sulfite reductase (Fsr) as the catalyst and benzyl viologen (BV) as the redox mediator.

Nestin Forms a Flexible Cytoskeleton by Means of a Huge Tail Domain That Is Reversibly Stretched and Contracted by Weak Forces.

Nestin is a type VI intermediate filament protein and a well-known neural stem cell marker. It is also expressed in high-grade cancer cells, forming copolymerized filaments with vimentin. We previously showed that nestin inhibits the binding of vimentin's tail domain to actin filaments (AFs) by steric hindrance through its large nestin tail domain (NTD), thereby increasing three-dimensional cytoskeleton network mobility, enhancing cell flexibility, and promoting cancer progression.

Periplasmic chitooligosaccharide-binding protein requires a three-domain organization for substrate translocation.

Periplasmic solute-binding proteins (SBPs) specific for chitooligosaccharides, (GlcNAc) (n = 2, 3, 4, 5 and 6), are involved in the uptake of chitinous nutrients and the negative control of chitin signal transduction in Vibrios. Most translocation processes by SBPs across the inner membrane have been explained thus far by two-domain open/closed mechanism. Here we propose three-domain mechanism of the (GlcNAc) translocation based on experiments using a recombinant VcCBP, SBP specific for (GlcNAc) from Vibrio cholerae.

Protein expression and purification, molecular interaction, and X-ray crystallographic analysis of baculovirus protein PK2.

Phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α) by eIF2α kinases is a common mechanism to regulate the initiation of translation under stress conditions. The PK2 protein from baculovirus Autographica californica multiple nucleopolyhedrovirus (AcMNPV) binds and inhibits eIF2α kinases to ensure efficient virus propagation. The C-terminal region of PK2 shares a homology with the C-lobe of eIF2α kinases, but the N-terminal region of PK2 is unique to the orthologous proteins.

Crystal Structures of Csm2 and Csm3 in the Type III-A CRISPR-Cas Effector Complex.

Clustered regularly interspaced short palindromic repeat (CRISPR) loci and CRISPR-associated (Cas) genes encode CRISPR RNAs (crRNA) and Cas proteins, respectively, which play important roles in the adaptive immunity system (CRISPR-Cas system) in prokaryotes. The crRNA and Cas proteins form ribonucleoprotein effector complexes to capture and degrade invading genetic materials with base complementarity to the crRNA guide sequences. The Csm complex, a type III-A effector complex, comprises five Cas proteins (Csm1-Csm5) and a crRNA, which co-transcriptionally degrades invading DNA and RNA.

Molecular insights into replication initiation by Qβ replicase using ribosomal protein S1.

Ribosomal protein S1, consisting of six contiguous OB-folds, is the largest ribosomal protein and is essential for translation initiation in Escherichia coli. S1 is also one of the three essential host-derived subunits of Qβ replicase, together with EF-Tu and EF-Ts, for Qβ RNA replication in E. coli.

Structural basis for high substrate-binding affinity and enantioselectivity of 3-quinuclidinone reductase AtQR.

(R)-3-Quinuclidinol, a useful compound for the synthesis of various pharmaceuticals, can be enantioselectively produced from 3-quinuclidinone by 3-quinuclidinone reductase. Recently, a novel NADH-dependent 3-quinuclidionone reductase (AtQR) was isolated from Agrobacterium tumefaciens, and showed much higher substrate-binding affinity (>100 fold) than the reported 3-quinuclidionone reductase (RrQR) from Rhodotorula rubra. Here, we report the crystal structure of AtQR at 1.

Structural basis of stereospecific reduction by quinuclidinone reductase.

Chiral molecule (R)-3-quinuclidinol, a valuable compound for the production of various pharmaceuticals, is efficiently synthesized from 3-quinuclidinone by using NADPH-dependent 3-quinuclidinone reductase (RrQR) from Rhodotorula rubra. Here, we report the crystal structure of RrQR and the structure-based mutational analysis. The enzyme forms a tetramer, in which the core of each protomer exhibits the α/β Rossmann fold and contains one molecule of NADPH, whereas the characteristic substructures of a small lobe and a variable loop are localized around the substrate-binding site.

Translocation and rotation of tRNA during template-independent RNA polymerization by tRNA nucleotidyltransferase.

The 3'-terminal CCA (CCA-3' at positions 74-76) of tRNA is synthesized by CCA-adding enzyme using CTP and ATP as substrates, without a nucleic acid template. In Aquifex aeolicus, CC-adding and A-adding enzymes collaboratively synthesize the CCA-3'. The mechanism of CCA-3' synthesis by these two enzymes remained obscure.

Structure of the Ca(2+)-saturated C-terminal domain of scallop troponin C in complex with a troponin I fragment.

Troponin C (TnC) is the Ca(2+)-sensing subunit of troponin that triggers the contraction of striated muscles. In scallops, the striated muscles consume little ATP energy in sustaining strong contractile forces. The N-terminal domain of TnC works as the Ca(2+) sensor in vertebrates, whereas scallop TnC uses the C-terminal domain as the Ca(2+) sensor, suggesting that there are differences in the mechanism of the Ca(2+)-dependent regulation of muscles between invertebrates and vertebrates.

Expression, purification, crystallization and X-ray analysis of 3-quinuclidinone reductase from Agrobacterium tumefaciens.

(R)-3-Quinuclidinol is a useful chiral building block for the synthesis of various pharmaceuticals and can be produced from 3-quinuclidinone by asymmetric reduction. A novel 3-quinuclidinone reductase from Agrobacterium tumefaciens (AtQR) catalyzes the stereospecific reduction of 3-quinuclidinone to (R)-3-quinuclidinol with NADH as a cofactor. Recombinant AtQR was overexpressed in Escherichia coli, purified and crystallized with NADH using the sitting-drop vapour-diffusion method at 293 K.

Mechanism for template-independent terminal adenylation activity of Qβ replicase.

The genomic RNA of Qβ virus is replicated by Qβ replicase, a template-dependent RNA polymerase complex. Qβ replicase has an intrinsic template-independent RNA 3'-adenylation activity, which is required for efficient viral RNA amplification in the host cells. However, the mechanism of the template-independent 3'-adenylation of RNAs by Qβ replicase has remained elusive.

Molecular basis for RNA polymerization by Qβ replicase.

Core Qβ replicase comprises the Qβ virus-encoded RNA-dependent RNA polymerase (β-subunit) and the host Escherichia coli translational elongation factors EF-Tu and EF-Ts. The functions of the host proteins in the viral replicase are not clear. Structural analyses of RNA polymerization by core Qβ replicase reveal that at the initiation stage, the 3'-adenine of the template RNA provides a stable platform for de novo initiation.

Mechanism for the alteration of the substrate specificities of template-independent RNA polymerases.

PolyA polymerase (PAP) adds a polyA tail onto the 3'-end of RNAs without a nucleic acid template, using adenosine-5'-triphosphate (ATP) as a substrate. The mechanism for the substrate selection by eubacterial PAP remains obscure. Structural and biochemical studies of Escherichia coli PAP (EcPAP) revealed that the shape and size of the nucleobase-interacting pocket of EcPAP are maintained by an intra-molecular hydrogen-network, making it suitable for the accommodation of only ATP, using a single amino acid, Arg(197).

Assembly of Q{beta} viral RNA polymerase with host translational elongation factors EF-Tu and -Ts.

Replication and transcription of viral RNA genomes rely on host-donated proteins. Qbeta virus infects Escherichia coli and replicates and transcribes its own genomic RNA by Qbeta replicase. Qbeta replicase requires the virus-encoded RNA-dependent RNA polymerase (beta-subunit), and the host-donated translational elongation factors EF-Tu and -Ts, as active core subunits for its RNA polymerization activity.

Mechanism for the definition of elongation and termination by the class II CCA-adding enzyme.

The CCA-adding enzyme synthesizes the CCA sequence at the 3' end of tRNA without a nucleic acid template. The crystal structures of class II Thermotoga maritima CCA-adding enzyme and its complexes with CTP or ATP were determined. The structure-based replacement of both the catalytic heads and nucleobase-interacting neck domains of the phylogenetically closely related Aquifex aeolicus A-adding enzyme by the corresponding domains of the T.

Crystallization and preliminary X-ray analysis of the NADPH-dependent 3-quinuclidinone reductase from Rhodotorula rubra.

(R)-3-Quinuclidinol is a useful compound that is applicable to the synthesis of various pharmaceuticals. The NADPH-dependent carbonyl reductase 3-quinuclidinone reductase from Rhodotorula rubra catalyzes the stereospecific reduction of 3-quinuclidinone to (R)-3-quinuclidinol and is expected to be utilized in industrial production of this alcohol. 3-Quinuclidinone reductase from R.

Homodimeric structure and double-stranded RNA cleavage activity of the C-terminal RNase III domain of human dicer.

Human Dicer contains two RNase III domains (RNase IIIa and RNase IIIb) that are responsible for the production of short interfering RNAs and microRNAs. These small RNAs induce gene silencing known as RNA interference. Here, we report the crystal structure of the C-terminal RNase III domain (RNase IIIb) of human Dicer at 2.

Crystal structure of the PIN domain of human telomerase-associated protein EST1A.

Saccharomyces cerevisiae Est1p is a telomerase-associated protein essential for telomere length homeostasis. hEST1A is one of the three human Est1p homologues and is considered to be involved not only in regulation of telomere elongation or capping but also in nonsense-mediated degradation of RNA. hEST1A is composed of two conserved regions, Est1p homology and PIN (PilT N-terminus) domains.

Crystallization and preliminary X-ray analysis of the PIN domain of human EST1A.

Human EST1A (ever shorter telomeres 1A) is associated with most or all active telomerase in cell extracts and is involved either directly or indirectly in telomere elongation and telomere capping. The C-terminal region of EST1A contains the PIN (PilT N-terminus) domain, a putative nuclease domain. The PIN domain of human EST1A was expressed, purified and crystallized by the sitting-drop vapour-diffusion method.

Crystallization and preliminary X-ray analysis of the C-terminal RNase III domain of human Dicer.

Human Dicer protein contains two RNase III domains (RNase IIIa and RNase IIIb) which are involved in the production of short interfering RNAs (siRNAs). The C-terminal RNase III domain (RNase IIIb) of human Dicer was expressed, purified and crystallized by the sitting-drop vapour-diffusion method. The crystals belonged to space group C222(1), with unit-cell parameters a = 88.