Methylglyoxal, a typical 2-oxaldehyde, is a ubiquitous metabolite produced in vivo during glycolysis. It inhibits the growth of cells in all types of organism. Primary mechanism protecting cells against its toxicity is the glyoxalase system consisting of glyoxalase I and II. Previous investigation on Escherichia coli showed that it contains glyoxalase III, which is similar to glyoxalase I/II system to convert methylglyoxal into D-lactate but unique in its activity in one step reaction in the absence of any known cofactor(s). However, no gene encoding this enzyme has been identified, no mutants are known in this system and its physiological role is poorly understood. We identified a gene that encodes glyoxalase III in Escherichia coli by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) fingerprinting analysis of the purified endogenous protein converting methylglyoxal into D-lactate. The identified gene, yedU/hchA (heat shock chaperone A), is known to encode the heat shock protein (Hsp31), which has both molecular chaperone and a weak aminopeptidase activities. Its crystal structure, however, has a novel catalytic triad (Cys-His-Asp/Glu) suggesting still unproven function for this protein, in agreement with our observation that it has glyoxalase III activity. The glyoxalase III activity was completely abolished in the strain lacking yedU gene. To give further insight, we have mutated, cloned, purified to homogeneity, and characterized recombinant Hsp31. The purified recombinant Hsp31 showed Km and kcat values of 1.08 mM and 116.6 m-1, respectively, for methylglyoxal. It was generally active over wide range of temperature and pH, ranging from 30-500 C and 6.0-8.0, respectively. Its activity was significantly inhibited by Cu2+, Fe3+ and Zn2+. In consistent with earlier report that glyoxalase III was sensitive to thiol-blocking reagents and Cys185 of Hsp31 is an active nucleophile, substitution of putative catalytic site cysteine by alanine (C185A) completely abrogated the glyoxalase activity. Mutation of another highly conserved residue, Glu77, which resides close to Cys185 in the crystal structure of Hsp31 by about 3.30A distance, had the identical effect, indicating the critical role of these residues in enzymatic catalysis. Furthermore, the protein was separated as two major peaks on anion exchange chromatography and isoelectric focusing has shown several isoforms, indicating the possible posttranslational modification of Hsp31, most likely at or near nucleophilic cysteine residue. Our in-vivo data showed that the expression and thus the activity of yedU is growth phase dependent and reaches at its maximum at stationary phase. Its expression was positively regulated by rpoS (sigmaS) and negatively regulated by the histone like nucleoid-binding protein (H-NS). The physiological analysis of the mutant and overexpressed strains had revealed that the glutathione-independent glyoxalase encoded by yedU has a protective function against methylglyoxal toxicity. The stationary phase E. coli cells lacking yedU gene sensitized to exogenous methylglyoxal whereas its overexpression protected exponentially growing cells against increased concentrations of methylglyoxal. The high degree of sequence and structural similarities, and the ablsolute conservation of critical residues between Escherichia coli Hsp31 and large number of organisms from diverse groups suggested that the glyoxalase III is a ubiquitous enzyme.
Methylglyoxal (2-oxoaldehyde)은 해당과정에서 생성되는 대사산물로서 대부분의 생명체에서 세포의 성장을 방해하는 유독한 산물이다. Methylglyoxal의 독성은 세포의 보호기작중의 하나인 glyoxlase system에 의해서 무독화된다. Glyoxalase system 은 1995년 glyoxalase III 가 발표된 후 새롭게 보고된 내용이 거의 없고 이와 관련된 돌연변이나 이를 암호화하고 있는 유전자도 알려지지 않았기 때문에 생리학적 역할 및 특성이 아직 밝혀지지 않았다. 본 연구에서는 대장균의 crude extract를 이용해 methylglyoxal에서 D-lactate로 전환하는데 관여하는 효소를 정제하여 matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)로 분석한 결과 대장균에 존재하는 yedU 나 hchA (heat shock chaperone A) 를 암호화 하고 있는 대장균의 heat shock protein Hsp31이 glyoxalase III 임을 밝혀내었다. 또한 yedU유전자가 제거된 균주를 이용해 glyoxalase III activity를 다시 확인할 수 있었다. glyoxalase III 인 Hsp31은 molecular chaperone이면서 weak aminopeptidase activity를 갖는 단백질로서 크리스탈 구조에서 Cys, His, Asp/Glu로 구성된 catalytic triad를 이루고 있다. Hsp31의 특성을 알아보기 위하여 제조합 단백질 Hsp31 을 분리 정제한 뒤 효소적 특성을 살펴보았더니 KM, Kcat 값이 각각 1.08mM, 11.6m-1 이었고 30~50°C, pH 6.0~8.0에서 가장 activity가 높았으며 2가 메탈 이온인 Cu2+와 Zn2+에 의해 activity가 감소하는 양상을 보였다. 본 연구에서는 또한 catalytic triad에 해당하는 185번 cystein 잔기를 alanine (C185A)로 돌연변이 시킨 후 glyoxalase activity를 확인함으로써 glyoxalse III 가 thiol blocking reagent에 상당히 민감한 사실을 알 수 있었다. 또한 77번 glutamate와 185번 cysteine 잔기 사이에 3.3 oA 거리 차이를 통해 이 두개의 잔기 역시 효소적 특성에 매우 중요한 사실을 확인하였다. 이와 더불어, 이온 교환 크로마토그래피를 이용해 두개 peak으로 분리한 뒤 isoelectric focusing으로 여러 개의 isoform으로 분리하여 Hsp31의 nucleophilic한 cysteine residue에서 post-translational modification을 예측할 수 있었다. yedU의 발현은 성장 곡선에 dependent하여 stationary phase에서 최대로 발현하며 이는 rpoS에 의해 positive regulatioin, histone like nucleoid-binding protein(H-NS)에 의해 negative regulation을 받는다. stationary phase의 yedU deletion 균주는 methylglyoxal에 inhibition을 받는 반면 yedU 가 암호화되어있는 플라스미드에 의해서 그 기능이 회복되며 glutathione-dependent glyoxalase I이 제거된 log phase에서의 균주의 경우 yedU의 효과를 더욱 확실히 확인할 수 있다. 결론적으로 대장균에서는 대사 작용이 현저히 떨어지는 스트레스와 같은 환경이 주어지면 생성되는 reactive carbonyl compound 에 작용하여 glutathione-independent enzyme 이 생성된다. 염기서열과 구조에 있어서 유사성이 높고 대장균 Hsp31의 중요한 잔기들이 prokaryote과 eukaryote의 잔기들과 상당부분 보존되고 있기 때문에 glyoxalase III 는 ubiquitous enzyme 이라고 말할 수 있다.
