Crystal structures of Cg1458 reveal a catalytic lid domain and a common catalytic mechanism for the FAH family

Abstract

Cg1458 was recently characterized as a novel soluble oxaloacetate decarboxylase. However, sequence alignment identified that Cg1458 has no similarity with other oxaloacetate decarboxylases and instead belongs to the FAH (fumarylacetoacetate hydrolase) family. Differences in the function of Cg1458 and other FAH proteins may suggest a different catalytic mechanism. To help elucidate the catalytic mechanism of Cg1458, crystal structures of Cg1458 in both the open and closed conformations have been determined for the first time up to a resolution of 1.9 Å (1 Å=0.1 nm) and 2.0 Å respectively. Comparison of both structures and detailed biochemical studies confirmed the presence of a catalytic lid domain which is missing in the native enzyme structure. In this lid domain, a glutamic acid-histidine dyad was found to be critical in mediating enzymatic catalysis. On the basis of structural modelling and comparison, as well as large-scale sequence alignment studies, we further determined that the catalytic mechanism of Cg1458 is actually through a glutamic acid-histidine-water triad, and this catalytic triad is common among FAH family proteins that catalyse the cleavage of the C-C bond of the substrate. Two sequence motifs, HxxE and Hxx…xxE have been identified as the basis for this mechanism.

Identification of FAH Domain-containing Protein 1 (FAHD1) as Oxaloacetate Decarboxylase

“…3E). To further characterize the ODx activity inherent to FAHD1, we used oxalate, an established inhibitor of Cg1485 (8). Indeed, oxalate caused potent competitive inhibition of oxaloacetate decarboxylation by FAHD1, with an inhibitor constant (K I ) of about 1 M (Fig.…”

“…1A). The crystal structure of Cg1458 was described recently (8), and the importance of eight charged amino acids deemed vital for the enzymatic function was analyzed via mutants, resulting in mostly inactive variants.…”

“…Direct Oxaloacetate Decarboxylase Assay-ODx activity was measured with an Infinite M200 spectrophotometer (Tecan), similar to Ran et al (8). The assay was performed in a disposable UV cuvette (1-cm path length, Brand) at 25°C in 1 ml of assay buffer (50 mM Tris-HCl, 100 mM KCl, 1 mM MgCl 2 , pH 7.4) containing varying amounts of recombinant FAHD1 (3-60 g).…”

“…1C, the known catalytic activities of NagK, Cg1458, and FAHD1 are schematically depicted, as well as the hypothetical ODx activity of FAHD1. The catalytic mechanism of Cg1458 was derived from an x-ray structure with the inhibitor oxalate bound to the active site (8). Binding of the inhibitor induces the closure of a lid region over the binding site ( Fig.…”

“…This can be explained by the fact that these enzymes are part of highly specialized metabolic pathways, involving chemical compounds that higher organisms are unable to convert and utilize for their metabolism (5). One recently identified prokaryotic member of the FAH superfamily found in Corynebacterium glutamicum, referred to as Cg1458, was characterized as a novel soluble oxaloacetate decarboxylase (ODx) (7,8); however, eukaryotic ODx enzymes were not identified so far.…”