Active Monomeric and Dimeric Forms of Pseudomonas putida Glyoxalase I:  Evidence for 3D Domain Swapping

Abstract

3D domain swapping of proteins involves the interconversion of a monomer containing a single domain-domain interface and a 2-fold symmetrical dimer containing two equivalent intermolecular interfaces. Human glyoxalase I has the structure of a domain-swapped dimer [Cameron, A. D., Olin, B., Ridderström, M., Mannervik, B., and Jones, T. A. (1997) EMBO J. 16, 3386-3395] but Pseudomonas putida glyoxalase I has been reported to be monomeric [Rhee, H.-I., Murata, K., and Kimura, A. (1986) Biochem. Biophys. Res. Commun. 141, 993-999]. We show here that recombinant P. putida glyoxalase I is an active dimer (kcat approximately 500 +/- 100 s-1; KM approximately 0.4 +/- 0.2 mM) with two zinc ions per dimer. The zinc is required for structure and function. However, treatment of the dimer with glutathione yields an active monomer (kcat approximately 115 +/- 40 s-1; KM approximately 1.4 +/- 0.4 mM) containing a single zinc ion. The monomer is metastable and slowly reverts to the active dimer in the absence of glutathione. Thus, glyoxalase I appears to be a novel example of a single protein able to exist in two alternative domain-swapped forms. It is unique among domain-swapped proteins in that the active site and an essential metal binding site are apparently disassembled and reassembled by the process of domain swapping. Furthermore, it is the only example to date in which 3D domain swapping can be regulated by a small organic ligand.

Crystal Structure of the C-terminal Peptidoglycan-binding Domain of Human Peptidoglycan Recognition Protein Iα

“…5), despite the inclusion of 1 mM Zn 2ϩ in one of the crystallization buffers (see "Experimental Procedures"). This difference is explained by the substitution of zinc-coordinating residues His 42 and Cys 160 in Drosophila PGRP-LB by Ile 207 and Ser 324 in human PGRP-I␣C. Since zinc is required for the PGN-hydrolyzing amidase activity of Drosophila PGRP-LB and other catalytic PGRPs, the absence of this metal ion in human PGRP-I␣C is consistent with its ability to bind, but not hydrolyze, PGNs.…”

A trypanothione-dependent glyoxalase I with a prokaryotic ancestry in Leishmania major

“…The high degree of sequence similarity and the conservation of the predicted metal-binding residues between the trypanosomatid and the E. coli GLO1 enzymes suggest that the parasite enzymes all might be nickel-dependent. However, the cofactor requirement of a GLO1 enzyme cannot be predicted from sequence alone, because the zinc-dependent Pseudomonas putida and S. cerevisiae enzymes have identical metal-binding residues as those of the E. coli enzyme (31,32). Thus, the unambiguous demonstration of the requirement of the L. major GLO1 for nickel, but not zinc, by metal analysis and reconstitution is important in confirming the unique relationship of these eukaryotic glyoxalases to the E. coli enzyme.…”

Glyoxalase biochemistry

“…The predominant use of the glyoxalase enzymes to detoxify MG and other reactive dicarbonyls has resulted in intense interest in ameliorating our understanding of the structure-function relationships of these enzymes. Early research had shown that Glo1 is a Zn 2+ -activated metalloenzyme when isolated from biological sources such as yeast, mammals, and Pseudomonas putida (7,(50)(51)(52) , and even Mg 2+ were also found to activate the Glo1 isolated from these sources (53). This foundational research indicated a broad metal promiscuity for Glo1, although metal reconstitution experiments were technically challenging at times.…”

“…It has been proposed that the evolution of new structures and functions within this protein family likely arose from a combination of horizontal gene transfer and gene fusion events and possibly gene duplication events (79,81). The possibility of three-dimensional domain swapping has also been proposed (52,54). It is interesting to note that the Glo1 protein fold is also found in several βαβββ structural superfamily members that are involved in antibiotic resistance (82).…”