Cloning and characterization of dihydrofolate reductases from deep-sea bacteria

Abstract

Enzymes from organisms living in deep-sea are thought to have characteristic pressure-adaptation mechanisms in structure and function. To better understand these mechanisms in dihydrofolate reductase (DHFR), an essential enzyme in living cells, we cloned, overexpressed and purified four new DHFRs from the deep-sea bacteria Shewanella violacea (svDHFR), Photobacterium profundum (ppDHFR), Moritella yayanosii (myDHFR) and Moritella japonica (mjDHFR), and compared their structure and function with those of Escherichia coli DHFR (ecDHFR). These deep-sea DHFRs showed 33-56% primary structure identity to ecDHFR while far-ultraviolet circular dichroism and fluorescence spectra suggested that their secondary and tertiary structures were not largely different. The optimal temperature and pH for deep-sea DHFRs activity were lower than those of ecDHFR and different from each other. Deep-sea DHFRs kinetic parameters K(m) and k(cat) were larger than those of ecDHFR, resulting in 1.5-2.8-fold increase of k(cat)/K(m) except for mjDHFR which had a 28-fold decrease. The enzyme activity of ppDHFR and mjDHFR (moderate piezophilic bacteria) as well as ecDHFR decreased as pressure increased, while svDHFR and myDHFR (piezophilic bacteria) showed a significant tolerance to pressure. These results suggest that DHFRs from deep-sea bacteria possess specific enzymatic properties adapted to their life under high pressure.

Piezotolerance of the Respiratory Terminal Oxidase Activity of the PiezophilicShewanella violaceaDSS12 as Compared with Non-PiezophilicShewanellaSpecies

“…Under a pressure of 200 MPa, no activities were observed in this study (data not shown), although the soluble enzymes retained 30-60% of their activities under similar conditions. [15][16][17] The reason for this remains unclear, but it is possible that the presence of membrane lipids affects enzyme properties under high pressure. On the other hand, shifts in growth temperature showed no effect on the response of respiratory terminal oxidase activity to pressure and temperature (Fig.…”

“…In this regard, many of the soluble enzymes of piezophiles have been found to be relatively resistant to high pressure. [15][16][17] Hence it is of interest to identify the most critical factors in maintaining the activity of membrane-associated enzymes under high pressure. Is it the properties of membranes and their interaction with proteins, or characteristics of the proteins themselves, that is of paramount significance in this regard?…”

“…Many of the enzyme activities of nonpiezophiles are known to be inhibited under high pressure even if the enzymes are soluble, although comparable enzymes from piezophiles are pressure tolerant. [15][16][17][18] Thus it remains unknown whether the decrease in the activities of the membrane-bound enzymes under high pressure is due to the protein structure itself or to the properties of the membrane associated with the enzyme.…”

Catalysis by Dihydrofolate Reductase from the Psychropiezophile Moritella profunda

“…Recently, it has been shown that the catalytic activity of DHFR from Moritella yayanosii, which is 95 % identical to MpDHFR, has a pH dependence very similar to that reported here for MpDHFR. [43] The K M for both cofactor and substrate varied slightly with pH, with maximum values around physiological pH (Figure 7 B and C). The K M values for DHF are small compared to those for NADPH, although the K M values for both ligands are relatively small at low temperature and pH, and at high pH.…”

Effects of pressure and temperature on the binding of RecA protein to single-stranded DNA

“…Essential proteins must have adapted to the species habitat (19)(20)(21)(22)(23)(24)(25). Adaptation means the preservation of the functionality of proteins.…”