The gellan gum biosynthetic genes gelC and gelE encode two separate polypeptides homologous to the activator and the kinase domains of tyrosine autokinases.
The high-molecular-weight exopolysaccharide gellan is an important commercial gelling agent produced in high yield by the Gram-negative bacterium Sphingomonas elodea ATCC 31461. The cluster of genes required for gellan biosynthesis contains the genes gelC and gelE. These encode for two polypeptides homologous to the activator domain and the kinase domain, respectively, of bacterial autophosphorylating tyrosine kinases involved in polysaccharide chain length determination. The GelC/GelE pair is an exception to the biochemically characterized Gram-negative tyrosine autokinases since it consists of two polypeptides instead of a single one. The deletion of gelC or gelE resulted in the abolishment of gellan in the culture medium confirming their role in gellan biosynthesis. In addition, ATP-binding assays confirmed the predicted ATP-binding ability of GelE. Interestingly, GelE contains an unusual Walker A sequence (ASTGVGCS), where the invariant lysine is replaced by a cysteine. This residue was replaced by alanine or lysine and although both mutant proteins were able to restore gellan production by complementation of the gelE deletion mutant to the production level observed with native GelE, only the mutated GelE where the cysteine was replaced by alanine was demonstrated to bind ATP in vitro. The importance of specific tyrosine residues present in the C-terminal domain of GelE in gellan assembly was also determined. The tyrosine residue at position 198 appears to be essential for the synthesis of high-molecular-weight gellan, although other tyrosine residues may additionally contribute to GelE biological function.
Moreira
Leonilde M
LM
Centro de Engenharia Biológica e Química, Instituto Superior Técnico, PT-1049-001 Lisboa, Portugal. lmoreira@ist.utl.pt
Hoffmann
Karen
K
Albano
Helena
H
Becker
Anke
A
Niehaus
Karsten
K
Sá-Correia
Isabel
I
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D011135:Polysaccharides, Bacterial / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D020578:Sphingomonas / Q000201:enzymology* / Q000235:genetics; D014443:Tyrosine / Q000378:metabolism
D000596:Amino Acids; D001426:Bacterial Proteins; D011135:Polysaccharides, Bacterial; D014443:Tyrosine; C048288:gellan gum; D011505:Protein-Tyrosine Kinases