Novel citramalate lyase with ATP cofactor in pathogenic enterobacteria

Successful expansion of pathogenic cells requires metabolic pathways that allow efficient utilization of nutrients provided in the intestines of the hosts. The genomes of 15 % of sequenced enterobacteria, all pathogens including E. coli O157:H7 (EHEC) [1,2], contain a gene cluster for the fermentation of glutamate, an amino acid abundant in the gut. This cluster encodes a novel citramalate lyase that requires either ATP or ADP as a cofactor. Structural analysis of the enzyme revealed that although ATP is neither hydrolyzed nor does it directly involved in the citramalate lyase reaction, it is involved in the formation of the reaction site and in the coordination of substrate and products by coordinating Mg2+ ions that allow the formation of the substrate binding site in addition to some amino acid residues, all of which involved in the catalytic mechanism are conserved. We identified the corresponding amino acid residues involved in catalysis and in the binding of citramalate and ATP and confirmed their function via kinetic analysis of the respective exchange variants. Some residue substitutions resulted in loss of or reduced interaction with ATP or (S)-citramalate, as demonstrated by thermal shift assays.

This enzyme is the prototype of a novel and diverse family of C-C lyases found in 19 % of bacterial genomes and spanning over 82 bacterial phyla. The enzyme structure is simpler than that of conventional citrate lyases. In addition, the unusual cofactor role of ATP gives the enzyme its plasticity, allowing easy horizontal transfer and adaptation to novel functions. In conclusion, the novel family of enterobacterial C-C-lyases widely distributed in different bacterial groups.

[1] Kato &. Asano, Arch Microbiol 168:457 (1997).

[2] Kronen & Berg, PLOS One 12:e0145098 (2015).