A cold-active and thermostable alcohol dehydrogenase of a psychrotorelant from Antarctic seawater, Flavobacterium frigidimaris KUC-1

Abstract

An NAD(+)-dependent alcohol dehydrogenase of a psychrotorelant from Antarctic seawater, Flavobacterium frigidimaris KUC-1 was purified to homogeneity with an overall yield of about 20% and characterized enzymologically. The enzyme has an apparent molecular weight of 160k and consists of four identical subunits with a molecular weight of 40k. The pI value of the enzyme and its optimum pH for the oxidation reaction were determined to be 6.7 and 7.0, respectively. The enzyme contains 2 gram-atoms Zn per subunit. The enzyme exclusively requires NAD(+) as a coenzyme and shows the pro-R stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of NAD(+). F. frigidimaris KUC-1 alcohol dehydrogenase shows as high thermal stability as the enzymes from thermophilic microorganisms. The enzyme is active at 0 to over 85 degrees C and the most active at 70 degrees C. The half-life time and k (cat) value at 60 degrees C were calculated to be 50 min and 27,400 min(-1), respectively. The enzyme also shows high catalytic efficiency at low temperatures (0-20 degrees C) (k(cat)/K(m) at 10 degrees C; 12,600 mM(-1)min(-1)) similar to other cold-active enzymes from psychrophiles. The alcohol dehydrogenase gene is composed of 1,035 bp and codes 344 amino acid residues with an estimated molecular weight of 36,823. The sequence identities were found with the amino acid sequences of alcohol dehydrogenases from Moraxella sp. TAE123 (67%), Pseudomonas aeruginosa (65%) and Geobacillus stearothermophilus LLD-R (56%). This is the first example of a cold-active and thermostable alcohol dehydrogenase.

Structural and Functional Properties of Isocitrate Dehydrogenase from the Psychrophilic Bacterium Desulfotalea psychrophila Reveal a Cold-active Enzyme with an Unusual High Thermal Stability

“…A high level of global stability has been reported for enzymes from psychrophiles, [33][34][35][36] indicating that a low level of global stability is not a prerequisite for cold-activity. Optimization of catalytic activity at low temperatures in psychrophilic enzymes is known to be caused either through a global loss of stability or flexibility in the active site regions.…”

Liposomal Encapsulation of Yeast Alcohol Dehydrogenase with Cofactor for Stabilization of the Enzyme Structure and Activity

“…Each subunit of YADH includes one NAD + to form their compact and stable complex, which contributes to maintaining a suitable assembly of subunits to give the tetrameric YADH. Furthermore, Kazuoka et al (40) recently reported a similar stabilization effect of NAD + on the activity of ADH from Flavobacterium frigidimaris KUC‐1. Figure 4 shows the time courses of the remaining activity of free YADH at 50 °C for concentrations of 0.01, 2.3 and 3.3 mg/mL and those of the free YADH/NAD + for the YADH and NAD + concentrations of 2.3 mg/mL and 3.9 mM, respectively.…”

Deciphering the Structural Basis of High Thermostability of Dehalogenase from Psychrophilic Bacterium Marinobacter sp. ELB17

“…Interestingly, some thermostable enzymes were also isolated from psychrophilic and mesophilic organisms, e.g., aldehyde dehydrogenase [21], aspartase [22] and alcohol dehydrogenase [23] from Flavobacterium frigidimaris KUC-1; endoglucanase from Fusarium oxysporum L19 [24]; isocitrate dehydrogenase from Desulfotalea psychrophila LSv54 [25]; or haloacid dehalogenase from Psychromonas ingrahamii DSMZ 17664 [26]. However, a paradigm of thermostable enzymes from psychrophiles has not yet been conclusively explained and it is not known why cold-adapted organisms possess highly stable enzymes.…”