Impact of cellulose properties on enzymatic degradation by bacterial GH48 enzymes: Structural and mechanistic insights from processive Bacillus licheniformis Cel48B cellulase.

Processive cellulases are highly efficient molecular engines involved in the cellulose breakdown process. However, the mechanism that processive bacterial enzymes utilize to recruit and retain cellulose strands in the catalytic site remains poorly understood. Here, integrated enzymatic assays, protein crystallography and computational approaches were combined to study the enzymatic properties of the processive BlCel48B cellulase from Bacillus licheniformis. Hydrolytic efficiency, substrate binding affinity, cleavage patterns, and the apparent processivity of bacterial BlCel48B are significantly impacted by the cellulose size and its surface morphology. BlCel48B crystallographic structure was solved with ligands spanning -5 to -2 and +1 to +2 subsites. Statistical coupling analysis and molecular dynamics show that co-evolved residues on active site are critical for stabilizing ligands in the catalytic tunnel. Our results provide mechanistic insights into BlCel48B molecular-level determinants of activity, substrate binding, and processivity on insoluble cellulose, thus shedding light on structure-activity correlations of GH48 family members in general.

Redesigning N-glycosylation sites in a GH3 ß-xylosidase improves the enzymatic efficiency.

BACKGROUND: ß-Xylosidases are glycoside hydrolases (GHs) that cleave xylooligosaccharides and/or xylobiose into shorter oligosaccharides and xylose. Aspergillus nidulans is an established genetic model and good source of carbohydrate-active enzymes (CAZymes). Most fungal enzymes are N-glycosylated, which influences their secretion, stability, activity, signalization, and protease protection. A greater understanding of the N-glycosylation process would contribute to better address the current bottlenecks in obtaining high secretion yields of fungal proteins for industrial applications. RESULTS: In this study, BxlB-a highly secreted GH3 ß-xylosidase from A. nidulans, presenting high activity and several N-glycosylation sites-was selected for N-glycosylation engineering. Several glycomutants were designed to investigate the influence of N-glycans on BxlB secretion and function. The non-glycosylated mutant (BxlBnon-glyc) showed similar levels of enzyme secretion and activity compared to the wild-type (BxlBwt), while a partially glycosylated mutant (BxlBN1;5;7) exhibited increased activity. Additionally, there was no enzyme secretion in the mutant in which the N-glycosylation context was changed by the introduction of four new N-glycosylation sites (BxlBCC), despite the high transcript levels. BxlBwt, BxlBnon-glyc, and BxlBN1;5;7 formed similar secondary structures, though the mutants had lower melting temperatures compared to the wild type. Six additional glycomutants were designed based on BxlBN1;5;7, to better understand its increased activity. Among them, the two glycomutants which maintained only two N-glycosylation sites each (BxlBN1;5 and BxlBN5;7) showed improved catalytic efficiency, whereas the other four mutants' catalytic efficiencies were reduced. The N-glycosylation site N5 is important for improved BxlB catalytic efficiency, but needs to be complemented by N1 and/or N7. Molecular dynamics simulations of BxlBnon-glyc and BxlBN1;5 reveals that the mobility pattern of structural elements in the vicinity of the catalytic pocket changes upon N1 and N5 N-glycosylation sites, enhancing substrate binding properties which may underlie the observed differences in catalytic efficiency between BxlBnon-glyc and BxlBN1;5. CONCLUSIONS: This study demonstrates the influence of N-glycosylation on A. nidulans BxlB production and function, reinforcing that protein glycoengineering is a promising tool for enhancing thermal stability, secretion, and enzymatic activity. Our report may also support biotechnological applications for N-glycosylation modification of other CAZymes.

Estudo da interação molecular do anticorpo 19CC6CG2 com o antígeno HSBSAG por métodos computacionais / Study of the molecular interaction of the 19CC6CG2 antibody with the HSBSAG antigen by computational methods

A hepatite B é um problema de saúde global: só no ano de 2015 foram quase 900 mil óbitos decorrentes de complicações relacionadas à infecção com o vírus HBV. No Brasil, a situação também é grave: no ano de 2000 até o ano de 2015 o número de óbitos atingiu 13 mil. O diagnóstico da doença e o tratamento dos pacientes crônicos podem ser feitos através do uso de imunoglobulinas que tem afinidade pelo antígeno de superfície do vírus, HBsAg. Portanto, estratégias que busquem diminuir os custos de produção de imunoglobulinas anti-HBsAg ou aumentar sua afinidade frente a este antígeno são desejáveis em âmbitos nacional e global. O estudo de biomoléculas através de técnicas computacionais tem produzido bons resultados, capazes de orientar estudos experimentais, economizando tempo e recursos e, frequentemente, resolvendo problemas biológicos. Dentre estas técnicas computacionais, destaca-se o cálculo de energia livre. A aplicação do cálculo de energia livre a complexos anticorpo-antígeno pode fornecer informações detalhadas sobre a afinidade do anticorpo frente ao antígeno. Neste trabalho, estudamos a interação do anticorpo 19CC6CG2, desenvolvido no Laboratório de Tecnologia de Anticorpos Monoclonais de Bio-Manguinhos, com o HBsAg através de duas técnicas de cálculo de energia livre: MM-PBSA e Adaptive Biasing Force

Embora a primeira técnica tenha fornecido um valor de ΔG de ligação de -12 kcal/mol, a análise mais robusta através do segundo método mostrou um ΔG de dissociação de -7,6 kcal/mol. Adicionalmente, foram propostas mutações na estrutura do anticorpo visando ao aumento da sua afinidade pelo antígeno. O anticorpo mutante foi então modelado in silico e a sua afinidade frente ao HBsAg foi mensurada através da técnica de ABF. Resultados preliminares mostraram um valor de ΔG de ligação de -4,2 kcal/mol. As mutações na estrutura do anticorpo favoreceram a formação de ligações hidrogênio e pontes salinas intermoleculares mais estáveis nas simulações de dinâmica molecular. No entanto, simulações mais longas e/ou o aumento da dimensionalidade do espaço através de variáveis coletivas no cálculo de ABF, podem melhorar a convergência do método, tornando evidente se as mutações aqui propostas são favoráveis à afinidade do anticorpo 19CC6CG2 contra o HBsAg. (AU)

Purification and primary structure of a novel mannose-specific lectin from Centrolobium microchaete Mart seeds.

This study aimed to purify and characterize a novel mannose-binding lectin from the seeds of Centrolobium microchaete. Centrolobium microchaete lectin (CML) was purified by affinity chromatography in mannose-Sepharose-4B column. CML agglutinated rabbit erythrocytes and was inhibited by D-mannose, α-methyl-D-mannoside, D-glucose, N-Acetyl-D-glucosamine and sucrose. The lectin was stable at pH 7.0 and 8.0 and temperatures up to 60°C. The monomeric form of CML showed approximately 28kDa, and its native form is probably a homodimer, as determined by gel filtration chromatography. The primary structure of CML was determined by tandem mass spectrometry that showed CML as a protein with two distinct forms (isolectins CML-1 and CML-2) with 246 and 247 residues, respectively. CML-2 possesses one residue of Asn more than CML-1 in C-terminal. The primary structure of CML agrees with the molecular weights found by electrospray ionization mass spectrometry: 27,224 and 27,338Da for CML-1 and CML-2, respectively. CML is a metal-dependent glycoprotein. Moreover, the glycan composition of CML and its structure were predicted.