is an opportunistic pathogen that forms chronic biofilm infections in the lungs of cystic fibrosis (CF) patients. Following initial colonization, P. aeruginosa
converts over time in the oxidative environment of the CF lung to a mucoid phenotype, which is characterized by the overproduction of the exopolysaccharide alginate (Govan & Deretic, 1996
; Breidenstein et al.
). Alginate is a random linear polysaccharide composed of β-(1–4)-linked d
-mannuronate and its C5 epimer α-l
-guluronate (Evans & Linker, 1973
Alginate is synthesized by the 12 proteins encoded on the algD
operon, AlgD/8/44/K/E/G/X/L/I/J/F/A, and AlgC. AlgC is also involved in rhamnolipid and LPS metabolism and is not linked to the algD
operon. The biosynthesis of alginate begins with d
-fructose 6-phosphate, which is converted to GDP-d
-mannuronic acid in the cytoplasm by the enzymes AlgA, AlgC and AlgD (Franklin et al.
). Polymerization occurs on the cytoplasmic face of the inner membrane and both the putative glycosyl transferase Alg8 and the bis-(3′,5′)-cyclic dimeric guanosine monophosphate-binding Alg44 are required (Oglesby et al.
; Merighi et al.
; Remminghorst & Rehm, 2006a
; Remminghorst et al.
). Within the periplasm, residues in the polymannuronate polymer can be modified by addition of acetyl groups to their O2′ and/or O3′ hydroxyls through the concerted action of AlgI, AlgJ and AlgF (Franklin & Ohman, 2002
), epimerized to α-l
-guluronate by AlgG (Franklin et al.
) or remain unaltered. As the polysaccharide passages through the periplasm to AlgE, the outer membrane β-barrel porin, it is believed to be protected by a multi-protein complex consisting of Alg44, AlgG, AlgK, AlgX and AlgL (Franklin et al.
). While the function of AlgX is unknown, the presence of multiple copies of the tetratricopeptide-like protein–protein interaction motif in AlgK has led to the proposal that it is a scaffold protein that assembles the periplasmic biosynthetic complex (Keiski et al.
). AlgL is an alginate lyase which appears to have two roles in alginate biosynthesis: (i) as part of the multi-protein alginate-secretion complex required for export of the polymer (Jain & Ohman, 2005
; Albrecht & Schiller, 2005
) and (ii) depolymerizing alginate that escapes into the periplasm (Bakkevig et al.
; Jain & Ohman, 2005
AlgL is a 40 kDa poly-(β-d
-mannuronate) lyase that preferentially degrades deacetylated polymannuronate via
a β-elimination reaction, resulting in an unsaturated uronic acid at the nonreducing end of the molecule (Linker & Evans, 1984
). AlgL is an endolytic enzyme that cleaves the 1–4 glycosidic linkage, resulting in disaccharides and trisaccharides as its major products (Wong et al.
; Linker & Evans, 1984
). The Phyre
structure-prediction server (Kelley & Sternberg, 2009
) suggests that AlgL has an α6
-barrel fold and is similar to Sphingomonas
spp. alginate lyase A1-III (Yoon et al.
). This structure prediction places AlgL in polysaccharide family 5 (Lombard et al.
). Using site-directed mutagenesis and residue-conservation analysis, several residues, including His202 and Tyr256, have been suggested to play a role in AlgL activity; however, the exact mechanism of catalysis and the molecular determinants of substrate specificity are not yet known (Albrecht & Schiller, 2005
; Preston et al.
In addition to its enzymatic function, AlgL appears to be required for alginate export, as alginate accumulates in the periplasm of algL
deletion strains (Jain & Ohman, 2005
; Bakkevig et al.
). In vivo
, point mutants that disrupt lyase activity result in a non-mucoid phenotype, indicating that AlgL activity is crucial for alginate export and/or biosynthesis (Bakkevig et al.
; Albrecht & Schiller, 2005
). Thus, the scaffolding complex responsible for the export of alginate across the periplasm is hypothesized to include AlgL (Franklin et al.
To understand the catalytic mechanism of AlgL and the role that it plays in the overall process of alginate biosynthesis and export, we have undertaken structural studies of this protein. Here, we describe the overexpression, purification and crystallization of AlgL.