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Proteolysis of the type III glutamine synthetase from Bacteroides fragilis causes expedient crystal-packing rearrangements.

ABSTRACT: This work details the intentional modifications that led to the first structure of a type III glutamine synthetase enzyme (GSIII). This approach followed the serendipitous discovery of digestion caused by an extracellular protease from a contaminating bacterium, Pseudomonas fluorescens. The protease only cleaves the GSIII protein at a single site, leaving the oligomer intact but allowing the protein to crystallize in a different space group. This transition from space group P1 to space group C222(1) is accompanied by improved growth characteristics, more reproducible diffraction and enhanced mechanical stability. The crystallographic analyses presented here provide the structural basis of the altered molecular packing in the full-length and digested crystal forms and suggest modifications for future structural studies.

SUBMITTER: van Rooyen J

PROVIDER: S-EPMC3053163 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Proteolysis of the type III glutamine synthetase from Bacteroides fragilis causes expedient crystal-packing rearrangements.

van Rooyen Jason J Belrhali Hassan H Abratt Valarie V Sewell B Trevor BT

Acta crystallographica. Section F, Structural biology and crystallization communications 20110225 Pt 3

This work details the intentional modifications that led to the first structure of a type III glutamine synthetase enzyme (GSIII). This approach followed the serendipitous discovery of digestion caused by an extracellular protease from a contaminating bacterium, Pseudomonas fluorescens. The protease only cleaves the GSIII protein at a single site, leaving the oligomer intact but allowing the protein to crystallize in a different space group. This transition from space group P1 to space group C22 ...[more]

PMID: 21393843

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Project description:In this study, we show that Bacteroides species utilize Fe(III)-xenosiderophores as the only source of exogenous iron to support growth under iron-limiting conditions in vitro anaerobically. Bacteroides fragilis was the only species able to utilize Fe(III)-ferrichrome while Bacteroides vulgatus ATCC 8482 and Bacteroides thetaiotaomicron VPI 5482 were able to utilize both Fe(III)-enterobactin and Fe(III)-salmochelin S4 as the only source of iron in a dose-dependent manner. We have investigated the way B. fragilis assimilates Fe(III)-ferrichrome as initial model to understand the utilization of xenosiderophores in anaerobes. B. fragilis contains two outer membrane TonB-dependent transporters (TBDTs), FchA1 and FchA2, which are homologues to Escherichia coli ferrichrome transporter FhuA. The disruption of fchA1 gene had only partial growth defect on Fe(III)-ferrichrome while the fchA2 mutant had no growth defect compared to the parent strain. The genetic complementation of fchA1 gene restored growth to parent strain levels indicating that it plays a role in Fe(III)-ferrichrome assimilation though we cannot rule out some functional overlap in transport systems as B. fragilis contains abundant TBDTs whose functions are yet not understood. However, the growth of B. fragilis on Fe(III)-ferrichrome was abolished in a feoAB mutant indicating that Fe(III)-ferrichrome transported into the periplasmic space was reduced in the periplasm releasing ferrous iron prior to transport through the FeoAB transport system. Moreover, the release of iron from the ferrichrome may be linked to the thiol redox system as the trxB deletion mutant was also unable to grow in the presence of Fe(III)-ferrichrome. The genetic complementation of feoAB and trxB mutants completely restored growth on Fe(III)-ferrichrome. Taken together, these findings show that Bacteroides species have developed mechanisms to utilize ferric iron bound to xenosiderophores under anaerobic growth conditions though the regulation and role in the biology of Bacteroides in the anaerobic intestinal environment remain to be understood.

Dataset Information

Proteolysis of the type III glutamine synthetase from Bacteroides fragilis causes expedient crystal-packing rearrangements.

Publications

Proteolysis of the type III glutamine synthetase from Bacteroides fragilis causes expedient crystal-packing rearrangements.

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