Exploring the structure and function of divergent Malyl CoA lyases from bacteria and archaea

LÓPEZ, MARÍA BELÉN; ZERDA MOREIRA, ANDREA; GONZALEZ, JAVIER MARCELO

Rosario

Congreso; L Reunión Anual de la Sociedad Argentina de Biofísica; 2022

Climate change resulting from the accumulation of greenhouse gases is a matter of global concern. Synthetic Biology constitutes a possible solution to this problem by generation of artificial metabolic pathways using enzymes for assimilation of one-carbon compounds such as Malyl CoA lyase (MCL). MCL is a promiscuous C-C bond lyase that functions in different pathways of central carbon metabolism in bacteria and haloarchaea. In the Serine pathway of Methylobacterium extorquens (Mex) this hexameric enzyme catalyzes an essential step in this RuBisCO-independent CO2 fixation pathway, producing glyoxylate and acetyl-CoA by reversible aldol cleavage of CoA thioester, malyl-CoA. The methylaspartate cycle, an anaplerotic acetate assimilation pathway was recently described in Haloarcula marismortui (Hma), where there is also an MCL, but its sequence has only 25.5% sequence ID as compared with that of Mex, despite the fact that both catalyze the same reactions. Herein, we applied a series of bioinformatic tools to elucidate the structure-function relationships of both surrogates. Sequence similarity network (SSN) was calculated for the family PF03328 and their sequences were retrieved from UniProt. Looking at the distribution of the superfamily along the SSN it is observed that the vast majority of enzymes are located in well-defined clusters. The clusters of interest where HmaMCL and MexMCL are found were selected and a phylogenetic tree was generated with the ML algorithm. The structure HmaMCL was modeled with ColabFold and compared with available experimental MCL structures. A high fold conservation can be witnessed, with low r.m.s.d values for 3D alignment, even though the percentage of sequence ID in all cases is less than 30 %. The comparative analysis between the active sites showed a high conservation of the catalytic amino acids E148, D177 and L253, and variation in V176 and H254, which could be associated with substrate specificity. Molecular docking with substrates and products was conducted with DockThor, showing a good resemblance with the R. sphaeroides CoA + oxalate complex. Elucidating the molecular traits behind apparently different enzymes catalyzing the same reaction will enable us to understand their reaction mechanisms and their roles in the corresponding metabolic pathways.