Studies on reduction of S-nitrosoglutathione by human carbonyl reductases 1 and 3

Abstract

Human carbonyl reductases 1 and 3 (CBR1 and CBR3) are monomeric NADPH-dependent enzymes of the short-chain dehydrogenase/reductase superfamily. Despite 72% identity in primary structure they exhibit substantial differences in substrate specificity. Recently, the endogenous low molecular weight S-nitrosothiol S-nitrosoglutathione (GSNO) has been added to the broad substrate spectrum of CBR1. The current study initially addressed whether CBR3 could equally reduce GSNO which was not the case. Neither the introduction of residues which contribute to glutathione binding in CBR1, i.e. K106Q and S97V/D98A, nor the exchange C143S, which prevents a theoretical disulfide bond with C227 in CBR3, could engender activity towards GSNO. However, exchanging amino acids 236–244 in CBR3 to correspond to CBR1 was sufficient to engender catalytic activity towards GSNO. Catalytic efficiency was further improved by the exchanges Q142M, C143S, P230W and H270S. Hence, the same residues previously reported as important for reduction of carbonyl compounds appear to be key to CBR1-mediated reduction of GSNO. Furthermore, for CBR1-mediated reduction of GSNO, considerable substrate inhibition at concentrations >5 K_m was observed. Treatment of CBR1 with GSNO followed by removal of low molecular weight compounds decreased the GSNO reducing activity, suggesting a covalent modification. Treatment with dithiothreitol, but not with ascorbic acid, could rescue the activity, indicating S-glutathionylation rather than S-nitrosation as the underlying mechanism. As C227 has previously been identified as the reactive cysteine in CBR1, the variant CBR1 C227S was generated, which, in comparison to the wild-type protein, displayed a similar k_cat, but a 30-fold higher K_m, and did not show substrate inhibition. Collectively, the results clearly argue for a physiological role of CBR1, but not for CBR3, in GSNO reduction and thus ultimately in regulation of NO signaling. Furthermore, at higher concentrations, GSNO appears to work as a suicide inhibitor for CBR1, probably through glutathionylation of C227.

Introduction

Human carbonyl reductases 1 and 3 (CBR1 and CBR3, or, according to the most recent nomenclature [1], SDR21C1 and SDR21C2) are monomeric NADPH-dependent enzymes belonging to the superfamily of short chain dehydrogenases/reductases (SDR). Although highly similar in primary and tertiary structure (cf. Fig. 1) the enzymes exhibit considerable differences in substrate specificity. The CBR1 substrate spectrum is well described and includes a variety of carbonyl compounds as, e.g. prostaglandins, steroids, quinones, and reactive aldehydes [2], [3], [4], [5], [6]. CBR1 contributes significantly to the phase I metabolism of xenobiotics by reducing substrates such as o-quinones derived from polycyclic aromatic hydrocarbons, anthracyclines, and the major tobacco carcinogen 4-methylnitrosamino-1-(3-pyridyl)-butanone (NNK) [7], [8], [9], [10], [11], [12], [13]. In contrast, CBR3 shows a distinct and much narrower range of substrates and its physiological function remains obscure [10].

In addition to the above mentioned carbonyl-containing compounds, the major intracellular low molecular weight S-nitrosothiol (SNO), S-nitrosoglutathione (GSNO), has recently been identified as a CBR1 substrate, where not a carbonyl, but a nitrosyl bond is reduced [14]. GSNO plays an important role in the dynamic regulation of protein S-nitrosation, the covalent attachment of an NO group to reactive protein cysteines, a post-translational modification mediating a considerable part of NO signaling [15], [16]. GSNO lies in a trans-nitrosation equilibrium with protein SNOs and hence, removal of GSNO from the intracellular pool leads to a concomitant decrease of protein SNOs and thus counteracts NO signaling [17], [18], [19], [20]. Targets for protein S-nitrosation comprise proteins of a variety of pathways, including apoptosis (e.g. caspases, HDM2), arginine metabolism (argininosuccinate synthase, dimethylarginine dimethylaminohydrolase), regulation of cellular redox state (e.g. thioredoxin, bacterial OxyR) and many more [15], [16]. The elucidation of the mechanisms that determine SNO turnover is therefore of high interest.

It is well-established that reduction of GSNO mediated by class III alcohol dehydrogenase (ADH3) inversely correlates with SNO levels [19], [21]. Until the report by Bateman et al. [14], ADH3 was the only GSNO reductase known that reduced the NO moiety, thus not resulting in NO release, but in termination of NO signaling [22]. It is now clear that CBR1 catalyzes the same reaction, albeit apparently with inferior catalytic efficiency [14], [22], [23]. Considering the high similarity between CBR1 and CBR3 in sequence and structure (cf. Fig. 1), the question arises whether CBR3 equally reduces GSNO.

GSNO can act both as S-nitrosating or S-glutathionylating compound and hereby covalently modify proteins, variably leading to activation or inactivation [15], [24]. Interestingly, another GSNO-metabolizing enzyme, namely thioredoxin, is glutathionylated by GSNO, leading to enzyme inactivation [25], [26]. Furthermore, there is evidence in the literature that the CBR1 protein exists in at least three molecular species differing in molecular weight and isoelectric point, suggesting different types of post-translational modification [2].

In the present study we initially sought to assess whether CBR3 shows GSNO reducing activity. As this was not the case, we used site-directed mutagenesis to determine the residues responsible for the lack of activity of CBR3 in comparison to CBR1. Additionally, we investigated whether GSNO led to S-nitrosation (also referred to as S-nitrosylation) or S-glutathionylation of CBR1.