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A mutant Trypanosoma rangeli sialidase, Tr7, expressed in Pichia pastoris, exhibits significant transsialidase activity, and has been used for analytical-scale production of sialylated human milk oligosaccharides. Mass spectrometry-based site-specific N-glycoprofiling of Tr7 showed that heterogeneous high-mannose type N-glycans were present at all the five potential N-linked glycosites. N-linked glycans in Tr7 were predominantly neutral oligosaccharides with compositions Man8-16GlcNAc2, but also mono- and di-phosphorylated oligosaccharides in the forms of Man9-15P1GlcNAc2 and Man9-14P2GlcNAc2, respectively. Some phosphorylated N-linked glycans further contained an additional HexNAc, which has not previously been reported in P. pastorisexpressed proteins. We compiled a method pipeline that combined hydrophilic interaction liquid chromatography enrichment of glycopeptides, high accuracy mass spectrometry and automated interpretation of the mass spectra with in-house developed "MassAI" software, which proved efficient in glycan site microheterogeneity analysis. Functional analysis showed that the deglycosylated Tr7 retained more than 90% of both the sialidase and trans-sialidase activities relative to the glycosylated Tr7. [ABSTRACT FROM AUTHOR]

AbrB is a global gene regulator involved in transition phase phenomena in B acillus subtilis. It participates in a complex regulatory network governing the expression of stationary-phase functions. AbrB was previously found to be phosphorylated on serine 86 located close to its C-terminal oligomerization domain. Here we report that AbrB can be phosphorylated by three B . subtilis serine/threonine kinases expressed during the transition and stationary phase: PrkC, PrkD and YabT. Our in vitro findings suggest that AbrB phosphorylation impedes its DNA binding and abolishes binding cooperativity. In vivo we established that a phospho-mimetic mutation abrB S86 D leads to a significant loss of AbrB control over several key target functions: exoprotease production, competence development and sporulation. A wider transcriptome analysis of abrB S86 D and S86 A mutant strains revealed deregulation of a large number of target genes. We therefore propose that AbrB phosphorylation serves as an additional input for fine-tuning the activity of this ambiactive gene regulator. [ABSTRACT FROM AUTHOR]

This paper reports rational engineering of Trypanosoma rangeli sialidase to develop an effective enzyme for a potentially important type of reactivity: production of sialylated prebiotic glycans. The Trypanosoma cruzi trans-sialidase and the homologous T. rangeli sialidase has previously been used to investigate the structural requirements for trans-sialidase activity. We observed that the T. cruzi trans-sialidase has a seven-amino-acid motif (197–203) at the border of the substrate binding cleft. The motif differs substantially in chemical properties and substitution probability from the homologous sialidase, and we hypothesised that this motif is important for trans-sialidase activity. The 197–203 motif is strongly positively charged with a marked change in hydrogen bond donor capacity as compared to the sialidase. To investigate the role of this motif, we expressed and characterised a T. rangeli sialidase mutant, Tr13. Conditions for efficient trans-sialylation were determined, and Tr13's acceptor specificity demonstrated promiscuity with respect to the acceptor molecule enabling sialylation of glycans containing terminal galactose and glucose and even monomers of glucose and fucose. Sialic acid is important in association with human milk oligosaccharides, and Tr13 was shown to sialylate a number of established and potential prebiotics. Initial evaluation of prebiotic potential using pure cultures demonstrated, albeit not selectively, growth of Bifidobacteria. Since the 197–203 motif stands out in the native trans-sialidase, is markedly different from the wild-type sialidase compared to previous mutants, and is shown here to confer efficient and broad trans-sialidase activity, we suggest that this motif can serve as a framework for future optimization of trans-sialylation towards prebiotic production. [ABSTRACT FROM AUTHOR]