Unusual structural and spectroscopic properties of an alanine transaminase from Escherichia coli

Determining the functions of proteins encoded by genome sequences represents an important challenge of contemporary biology. Recently we became interested in the characterization of the protein YfdZ from Escherichia coli, an ‘orphan enzyme’, for which the catalyzed reaction has not been assigned with certainty. Two recent reports suggest that this enzyme is an alanine aminotransferase (1, 2). Biochemical characterization of YfdZ was performed in our laboratory by overexpressing and purifying it in E. coli as recombinant protein with a C-terminal hexa-histidine tag. Preliminary information on YfdZ catalytic properties, obtained by thin layer chromatography (TLC), confirmed its alanine transaminase activity. Hence, we developed a quantitative enzymatic assay for the alanine-forming reaction and collected kinetic data with the aim of determining the kinetic constants. Furthermore, the kinetic analysis allowed us to detect pyruvate formation (unfavourable direction), not detected by preliminary TLC tests. Interestingly, the spectroscopic analysis showed anomalies in the binding of pyridoxal-5’-phosphate (PLP). Although YfdZ performs a reaction dependent on PLP and meets the necessary requirements for the binding of the cofactor, the spectrum of the purified enzyme is devoid of PLP. Reconstitution of YfdZ with PLP affects its spectroscopic and catalytic properties. Indeed the cofactor needs to be added to the reaction mixture, otherwise no catalytic activity is detectable. By means of SDS-PAGE, we noticed a strong tendency of the enzyme to dimerize in the absence of an excess of reducing agents. This is probably due to the formation of intermolecular-disulfide bond(s) involving one or more of the 4 cysteine residues in the protein. Notably, in the presence of dithiothreitol (DTT), a reducing agent, YfdZ activity is positively affected. Therefore DTT acts sinergically with PLP. By gel filtration chromatography we found that YfdZ exists as a dimer in solution and its quaternary structure does not change in the presence of DTT. Hence, the disulfide bridges among monomers do not alter the dimeric assembly of the enzyme, rather they seem to restrict the conformational changes associated to the enzymatic activity.