Enterobacterial citramalate lyase is a prototype enzyme for a novel C-C-lyase family.

Many enterobacteria are capable of fermenting glutamate, most of which are pathogenic, such as E. coli O157:H7 (EHEC) strains [1,2]. Glutamate fermentation in these organisms proceeds via the methylaspartate pathway, in which glutamate is isomerized to methylaspartate, which is then converted to (S)-citramalate and cleaved to pyruvate and acetate. Here we show that these enterobacteria possess a novel (S)-citramalate lyase. This enzyme is highly specific to citramalate and requires ATP for its activity, whereas ATP is not hydrolyzed during the reaction. The structural analysis of the enzyme revealed that although ATP does not directly take part in the citramalate lyase reaction, it is involved in the formation of the reaction site and in the coordination of substrate and products. 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. The interaction of the wildtype enzyme and its exchange variants with ATP was confirmed via the thermal shift assay. Interestingly, the enzyme was also active with ADP instead of ATP, although its activity was an order of magnitude lower than with ATP. The proposed enzyme mechanism highlighted participation of ATP in the coordination of Mg2+ that is involved in the binding of citramalate. The hidden Markov model analysis revealed the existence of multiple citramalate lyase homologs that probably catalyze various reactions. Indeed, the homologous protein from Bacillus oleivorans was shown to catalyze ATP-dependent 2-methylisocitate cleavage. To sum up, the enterobacterial ATP-dependent (S)-citramalate lyase is a prototype enzyme for a novel family of C-C-lyases widespread in different bacterial groups.

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

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