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This work presents a thorough characterization of Helaina recombinant human lactoferrin (rhLF, Effera™) expressed in a yeast system at an industrial scale for the first time. Proteomic analysis confirmed that its amino acid sequence is identical to that of native human LF. N-linked glycans were detected at three known glycosylation sites, namely, Asparagines-156, -497, and -642 and they were predominantly oligomannose structures having five to nine mannoses. Helaina rhLF's protein secondary structure was nearly identical to that of human milk lactoferrin (hmLF), as revealed by microfluidic modulation spectroscopy. Results of small-angle X-ray scattering (SAXS) and analytical ultracentrifugation analyses confirmed that, like hmLF, Helaina rhLF displayed well-folded globular structures in solution. Reconstructed solvent envelopes of Helaina rhLF, obtained through the SAXS analysis, demonstrated a remarkable fit with the reported crystalline structure of iron-bound native hmLF. Differential scanning calorimetry investigations into the thermal stability of Helaina rhLF revealed two distinct denaturation temperatures at 68.7 ± 0.9 °C and 91.9 ± 0.5 °C, consistently mirroring denaturation temperatures observed for apo- and holo-hmLF. Overall, Helaina rhLF differed from hmLF in the N-glycans they possessed; nevertheless, the characterization results affirmed that Helaina rhLF was of high purity and exhibited globular structures closely akin to that of hmLF. [ABSTRACT FROM AUTHOR]

Numerous post-transcriptional modifications of transfer RNAs have vital roles in translation. The 2-methylthio-N.sup.6-isopentenyladenosine (ms.sup.2i.sup.6A) modification occurs at position 37 (A37) in transfer RNAs that contain adenine in position 36 of the anticodon, and serves to promote efficient A:U codon-anticodon base-pairing and to prevent unintended base pairing by near cognates, thus enhancing translational fidelity.sup.1-4. The ms.sup.2i.sup.6A modification is installed onto isopentenyladenosine (i.sup.6A) by MiaB, a radical S-adenosylmethionine (SAM) methylthiotransferase. As a radical SAM protein, MiaB contains one [Fe.sub.4S.sub.4].sub.RS cluster used in the reductive cleavage of SAM to form a 5E-deoxyadenosyl 5E-radical, which is responsible for removing the C.sup.2 hydrogen of the substrate.sup.5. MiaB also contains an auxiliary [Fe.sub.4S.sub.4].sub.aux cluster, which has been implicated.sup.6-9 in sulfur transfer to C.sup.2 of i.sup.6A37. How this transfer takes place is largely unknown. Here we present several structures of MiaB from Bacteroides uniformis. These structures are consistent with a two-step mechanism, in which one molecule of SAM is first used to methylate a bridging [micro]-sulfido ion of the auxiliary cluster. In the second step, a second SAM molecule is cleaved to a 5E-deoxyadenosyl 5E-radical, which abstracts the C.sup.2 hydrogen of the substrate but only after C.sup.2 has undergone rehybridization from sp.sup.2 to sp.sup.3. This work advances our understanding of how enzymes functionalize inert C-H bonds with sulfur. Crystal structures reveal the catalytic mechanism through which the radical S-adenosylmethionine enzyme MiaB adds a methylthio group onto tRNA., Author(s): Olga A. Esakova [sup.1] , Tyler L. Grove [sup.2] , Neela H. Yennawar [sup.3] , Arthur J. Arcinas [sup.4] [sup.6] , Bo Wang [sup.1] , Carsten Krebs [sup.1] [sup.4] [...]

Expansins are small extracellular proteins that promote turgor-driven extension of plant cell walls. EXPB1 (also called Zea m 1) is a member of the [beta]-expansin subfamily known in the allergen literature as group-1 grass pollen allergens. EXPB1 induces extension and stress relaxation of grass cell walls. To help elucidate expansin's mechanism of wall loosening, we determined the structure of EXPB1 by x-ray crystallography to 2.75-[Angstrom] resolution. EXPB1 consists of two domains closely packed and aligned so as to form a long, shallow groove with potential to bind a glycan backbone of [approximately equal to] 10 sugar residues. The structure of EXPB1 domain 1 resembles that of family-45 glycoside hydrolase (GH45), with conservation of most of the residues in the catalytic site. However, EXPB1 lacks a second aspartate that serves as the catalytic base required for hydrolytic activity in GH45 enzymes. Domain 2 of EXPB1 is an Ig-like [beta]-sandwich, with aromatic and polar residues that form a potential surface for polysaccharide binding in line with the glycan binding cleft of domain 1. EXPB1 binds to maize cell walls, most strongly to xylans, causing swelling of the cell wall. Tests for hydrolytic activity by EXPB1 with various wall polysaccharides proved negative. Moreover, GH45 enzymes and a GH45-related protein called 'swollenin' lacked wall extension activity comparable to that of expansins. We propose a model of expansin action in which EXPB1 facilitates the local movement and stress relaxation of arabinoxylancellulose networks within the wall by noncovalent rearrangement of its target. plant cell wall

Substrate binding of the human mitochondrial branched chain aminotransferase with substrate amino acids, isoleucine ketimine and pyridoxamine phosphate, does not involve domain or loop closure as seen in other proteins. Furthermore, cysteine residues 315 and 318 are important for the enzyme structure and form a thiol-thiolate hydrogen bond.

X-ray crystallographic study of the crystalline gamma-chymotrypsin in aqueous and nonaqueous solvents containing the inhibitor N-acetyl-D-tryptophan indicates that in the nonaqueous environment the indole ring of the inhibitor binds with the pocket of the enzyme for L-amino acids. Inhibitor binding is absent in the aqueous structure. This differential binding affinity of inhibitor in aqueous and nonaqueous solvents accounts for the enzymatic memory induced in the chymotrypsin by the inhibitor in nonaqueous solvents. Nonaqueous structure has three 2-propanol and five hexane, while only one 2-propanol is observed in the aqueous structure. Number of water molecules in both structures is increased by the addition of 2-propanol.

Analysis of crystal structures of gamma-chymotrypsin in hexane-soaked crystals and aqueous environment at 2.2 angstroms resolution reveals that the native structure has 97 water molecules while the hexane-soaked crystal has 130 water molecules and seven hexane molecules. The thermal factors for both water molecules and protein atoms in the native structure are higher than those in the hexane structure.

The kinetic and X-ray structure characterization of Streptococcus pneumoniae DHFR (spDHFR) was carried out to understand the mechanism of a drug-resistant dihydrofolate reductase (DHFR) from a pathogenic bacterial source. The insights gained for the specific amino acids in the active site coupled with detailed structural analysis could be applied to design inhibitors targeting drug-resistant pathogenic bacterial DHFRs.