Pyridoxal-5′-phosphate (PLP) is a ubiquitous cofactor of over 238 distinct enzyme reactions including decarboxylation, transamination, racemization, elimination and substitution in amino acids, amines or oxoacid substrates. While the basic chemistry of the multiple mechanistic steps involving the cofactor and the enzyme are universal and well understood, the structural basis of the mechanism of action and the roles of active site residues in diverse PLP-dependent enzymes are poorly understood due to the absence of relevant structural information. In this study, structure-function relationships in the Group II PLP-dependent decarboxylases was studied on a model amino acid decarboxylase from Methanocaldococcus jannaschii, MjDC, using a combination of activity assays, UV-vis spectroscopy and crystal structures of the wild-type and several variants.

First, the crystal structure of the internal aldimine form was determined and using a comparative analysis of the structure of the free PLP aldehyde bound form of the enzyme, the structural basis of the mechanism of internal aldimine formation in MjDC was elucidated. The results permit in part to fill the gap of knowledge regarding Schiff base formation in enzymatic environments, which hitherto mostly have been derived from computational studies. Additionally, using mutational and comparative analysis of homologous structures of Group II decarboxylases, the importance of the catalytic loop dynamics involved in a close-to open conformational change during the apo-to-holo transition was revealed.

Next, key reaction intermediates, in productive modes, were obtained in-crystallo for the first time in Group II PLP-dependent decarboxylases. The Schiff base variant K245A, employed to trap the PLP bound form, and the Dunathan and quinonoid intermediates, allowed direct observations of the active site interactions and changes therein. The structures reveal rearrangement of a conserved Arg371 leading to the formation of a binding pocket for a water. The water-mediated interaction between the Arg371 and the carboxylate of the Dunathan intermediate appears to directly stabilize the alignment and facilitate the release of CO2 to yield the quinonoid intermediate. Modeling reveals that a conformational change of the catalytic loop to a closed-form controls a network of hydrogen bond interactions between conserved residues Tyr273ʹ, His132 and a water molecule that acts to protonate the quinonoid Cα. The results, along with previous findings, exhaustive structural comparisons, modelling and mutational analysis provide a significant foundation to elucidate mechanistic roles of residues that govern reaction specificity and catalytic properties in PLP-dependent decarboxylation. Further, a novel extended H-bond network around the active site that may modulate the stereoelectronic profile of PLP during catalysis was identified.

Finally, the evolution of substrate specificity in the Group II family with specific focus on the structural basis of discrimination in a highly conserved active site pocket is described.



Publication:

[1] Subash Chellam Gayathri and Narayanan Manoj. (2019). ‘Structural insights into the mechanism of internal aldimine formation and catalytic loop dynamics in an archaeal Group II decarboxylase’. J Struct Biol, 208(2), 137-151.