Alterations within fumarase C reveal newly observed SS Loop conformations

Abstract

Fumarase belongs to a larger superfamily of enzymes including aspartase, adenylosuccinate lyase, arginosuccinate lyase, and 3-carboxy-cis, cis-muconate lactonizing enzyme. Each enzyme member shares a common quaternary structure and harbors a flexible active site loop, termed the SS Loop. The SS Loop lies within the fumarase superfamily signature sequence 317GS318SxMxK324xN326xxPxE331 and plays a fundamental role during catalysis. Recently, human fumarase deficiencies have been linked to renal cell carcinoma and benign uterine tumor formation. In this study we site-selectively altered SS Loop residues serine 318, lysine 324, asparagine 326 and glutamic acid 331 to alanine. The fumarase C SS Loop variants were characterized functionally and structurally. Functionally, steady-state kinetic experiments were conducted in the classic Krebs Cycle sense (fumarate to S-malate) and in the reductive biosynthetic direction (S-malate to fumarate). Structurally, the single-site SS Loop variants were analyzed via circular dichroism and x-ray crystallography. The steady-state kinetic results report minimal effect on the S-malate and fumarate Km values, while reducing kcat significantly. We have placed the kcat reduction into two categories: (1) diminished charge-relay influence and (2) SS Loop immobility. Structurally, the SS Loop single-site alanine variants have similar thermal and chemical denaturation profiles. Thus, we suggest the overall homotetrameric quaternary structure, a requisite for a functional multi-subunit active site, is intact. X-ray crystallographic results for both the serine 318 to alanine (S318A) and lysine 324 to alanine (K324A) variants report a newly discovered fumarase C active site architecture. The SS Loop in both the S318A and K324A active sites undertakes two disparate conformations leading to two unique active sites. These two active sites are non-crystallographically related and undertake ordered-open and ordered-closed conformations. Thus, alteration of serine 318 and lysine 324 has facilitated crystallographic order within the SS Loop region. Ordering and closure of the S318A SS Loop has created an electrostatic gate between the multi-subunit active site and an auxiliary single-subunit citrate binding site. Adaptation within the K324A crystal structure includes additionally ordered active site water molecules. Effectively, the newly observed water molecules re-establish electrostatic interactions between serine 98, threonine 100, asparagine 141, histidine 188 and the previously observed active site water. Our structural and functional results further establish the importance of SS Loop conformational mobility within the fumarase superfamily. S318A SS Loop and domain 3 movement lead to closure of the fumarase C active site. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.