Modulating glyoxalase I metal selectivity by deletional mutagenesis: underlying structural factors contributing to nickel activation profiles.

Metabolically produced methylglyoxal is a cytotoxic compound that can lead to covalent modification of cellular DNA, RNA and protein. One pathway to detoxify this compound is via the glyoxalase enzyme system. The first enzyme of this detoxification system, glyoxalase I (GlxI), can be divided into two classes according to its metal activation profile, a Zn(2+)-activated class and a Ni(2+)-activated class. In order to elucidate some of the key structural features required for selective metal activation by these two classes of GlxI, deletional mutagenesis was utilized to remove, in a step-wise fashion, a key α-helix (residues 73-87) and two small loop regions (residues 99-103 and 111-114) from the Zn(2+)-activated Pseudomonas aeruginosa GlxI (GloA3) in order to mimic the smaller Ni(2+)-activated GlxI (GloA2) from the same organism. This approach was observed to clearly shift the metal activation profile of a Zn(2+)-activated class GlxI into a Ni(2+)-activated class GlxI enzyme. The α-helix structural component was found to contribute significantly toward GlxI metal specificity, while the two small loop regions were observed to play a more crucial role in the magnitude of the enzymatic activity. The current study should provide additional information on the fundamental relationship of protein structure to metal selectivity in these metalloenzymes.

Ni2+-activated glyoxalase I from Escherichia coli: substrate specificity, kinetic isotope effects and evolution within the ßαßßß superfamily.

The Escherichia coli glyoxalase system consists of the metalloenzymes glyoxalase I and glyoxalase II. Little is known regarding Ni(2+)-activated E. coli glyoxalase I substrate specificity, its thiol cofactor preference, the presence or absence of any substrate kinetic isotope effects on the enzyme mechanism, or whether glyoxalase I might catalyze additional reactions similar to those exhibited by related ßαßßß structural superfamily members. The current investigation has shown that this two-enzyme system is capable of utilizing the thiol cofactors glutathionylspermidine and trypanothione, in addition to the known tripeptide glutathione, to convert substrate methylglyoxal to non-toxic D-lactate in the presence of Ni(2+) ion. E. coli glyoxalase I, reconstituted with either Ni(2+) or Cd(2+), was observed to efficiently process deuterated and non-deuterated phenylglyoxal utilizing glutathione as cofactor. Interestingly, a substrate kinetic isotope effect for the Ni(2+)-substituted enzyme was not detected; however, the proton transfer step was observed to be partially rate limiting for the Cd(2+)-substituted enzyme. This is the first non-Zn(2+)-activated GlxI where a metal ion-dependent kinetic isotope effect using deuterium-labelled substrate has been observed. Attempts to detect a glutathione conjugation reaction with the antibiotic fosfomycin, similar to the reaction catalyzed by the related superfamily member FosA, were unsuccessful when utilizing the E. coli glyoxalase I E56A mutein.

15N-1H HSQC NMR evidence for distinct specificity of two active sites in Escherichia coli glyoxalase I.

Much remains to be elucidated concerning the selectivity mechanism of supposedly identical active sites in oligomeric proteins. Glyoxalase I (GlxI) catalyzes the glutathione-dependent conversion of 2-oxoaldehydes to S-2-hydroxyacylglutathione derivatives. The E. coli GlxI is a Ni(2+)/Co(2+)-activated homodimeric protein containing two symmetric, and dually metallated active sites as characterized by X-ray structure determination. Nevertheless, kinetics and isothermal titration calorimetric (ITC) studies indicate that dimeric GlxI binds to metal ions in a ratio of 1:1 (one metal ion/one dimer) [ Clugston , S. L. , Yajima , R. , and Honek , J. F. ( 2004 ) Biochem. J. 377 , 309 - 316 ]. In the current study, we provide spectroscopic evidence for the nonequivalent metallation of GlxI by use of (15)N-(1)H HSQC NMR titration experiments. (15)N-(1)H HSQC NMR spectra reveal that the local conformations of the two active sites in homodimeric GlxI are initially asymmetric in the apo-form, resulting in functional differentiation, wherein only one active site binds to the Ni(2+) ion, and another active site is observed to be more selective for a potent inhibitor. The current results enhance our understanding of GlxI structure-function relationships and provide a potential new strategy for the development of small molecule inhibitors for this enzyme system.

Microbial glyoxalase enzymes: metalloenzymes controlling cellular levels of methylglyoxal.

The glyoxalase system consists of two enzymes, glyoxalase I and glyoxalase II. This system is important in the detoxification of methylglyoxal. Detailed studies have determined that the glyoxalase I from Escherichia coli, Neisseria meningitidis and Yersinia pestis are maximally activated by Ni2+ and Co2+, and are inactive with Zn2+, a situation quite different from the human glyoxalase I enzyme, which is activated by Zn2+. Recent studies on the Pseudomonas aeruginosa genome have led to the characterization of three different glyoxalase I enzymes, two of which follow a Ni2+/Co2+ activation profile and the third exhibits a human-like preference for Zn2+.

Pseudomonas aeruginosa contains multiple glyoxalase I-encoding genes from both metal activation classes.

The glyoxalase (Glx) system is a critical detoxification enzyme system that is widely distributed in prokaryotic and eukaryotic organisms. Glyoxalase I (GlxI), the first enzyme in the system, is a divalent metal-ion dependent lyase (isomerizing), and its homologs have recently been categorized into two metal activation classes which are either Zn2+-dependent or non-Zn2+ dependent (Ni2+-/Co2+-activated). The latter class encompasses enzymes of predominantly bacterial origin. We have identified two genes in Pseudomonas aeruginosa PAO1 encoding glyoxalase I enzymes in addition to the gloA1 sequence recently reported and characterized. The gloA1 and gloA2 genes encode non-Zn2+ dependent glyoxalase I enzymes and the gloA3 gene remarkably encodes a Zn2+-dependent homolog. To our knowledge this is the first report of a eubacterial species with several GlxI encoding genes, and also of an organism possessing GlxI enzymes from both metal activation classes.

Escherichia coli glyoxalase II is a binuclear zinc-dependent metalloenzyme.

Cytotoxic methylglyoxal is detoxified by the two-enzyme glyoxalase system. Glyoxalase I (GlxI) catalyzes conversion of non-enzymatically produced methylglyoxal-glutathione hemithioacetal into its corresponding thioester. Glyoxalase II (Glx II) hydrolyzes the thioester into d-lactate and free glutathione. Glyoxalase I and II are metalloenzymes, which possess mononuclear and binuclear active sites, respectively. There are two distinct classes of GlxI; the first class is Zn2+-dependent and is composed of GlxI from mainly eukaryotic organisms and the second class is composed of non-Zn2+-dependent (but Ni2+ or Co2+-dependent) GlxI enzymes (mainly prokaryotic and leishmanial species). GlxII is typically Zn2+-activated, containing Zn2+ and either Fe3+/Fe2+ or Mn2+ at the active site depending upon the biological source. To address whether two classes of GlxII might exist, glyoxalase II from Escherichia coli was cloned and overexpressed and characterized. Unlike E. coli GlxI, which is non-Zn2+-dependent, Zn2+ activates the E. coli GlxII enzyme, with no evidence for Ni2+ metal utilization.

Distinct classes of glyoxalase I: metal specificity of the Yersinia pestis, Pseudomonas aeruginosa and Neisseria meningitidis enzymes.

The metalloisomerase glyoxalase I (GlxI) catalyses the conversion of methylglyoxal-glutathione hemithioacetal and related derivatives into the corresponding thioesters. In contrast with the previously characterized GlxI enzymes of Homo sapiens, Pseudomonas putida and Saccharomyces cerevisiae, we recently determined that Escherichia coli GlxI surprisingly did not display Zn2+-activation, but instead exhibited maximal activity with Ni2+. To investigate whether non-Zn2+ activation defines a distinct, previously undocumented class of GlxI enzymes, or whether the E. coli GlxI is an exception to the previously established Zn2+-activated GlxI, we have cloned and characterized the bacterial GlxI from Yersinia pestis, Pseudomonas aeruginosa and Neisseria meningitidis. The metal-activation profiles for these additional GlxIs firmly establish the existence of a non-Zn2+-dependent grouping within the general category of GlxI enzymes. This second, established class of metal activation was formerly unidentified for this metalloenzyme. Amino acid sequence comparisons indicate a more extended peptide chain in the Zn2+-dependent forms of GlxI (H. sapiens, P. putida and S. cerevisiae), compared with the GlxI enzymes of E. coli, Y. pestis, P. aeruginosa and N. meningitidis. The longer sequence is due in part to the presence of additional regions situated fairly close to the metal ligands in the Zn2+-dependent forms of the lyase. With respect to sequence alignments, these inserts may potentially contribute to defining the metal specificity of GlxI at a structural level.

Application of crossover-PCR-mediated deletion-insertion mutagenesis to analysis of the bdhA-xdhA2-xdhB2 mixed-function operon of Sinorhizobium meliloti.

The bdhA-xdhA2-xdhB2 mixed-function operon was used to demonstrate the application of crossover PCR to the construction of in-frame, non-polar deletion-insertion mutations in Sinorhizobium meliloti. Replacement of a 474-bp internal portion of the 774-bp coding sequence of bdhA with a 21-bp in-frame synthetic sequence resulted in loss of the bdhA-encoded d-3-hydroxybutyrate dehydrogenase activity. Such mutants retained the xanthine oxidase activity encoded by xdhA2-xdhB2, thus illustrating the non-polar nature of the mutation. This method of constructing unmarked, in-frame deletions should be generally applicable to functional genomics studies in S. meliloti and other alpha-Proteobacteria.