ORIGINAL PAPER
Uldis Kalnenieks Æ Nina Galinina Æ Malda M. Toma
Physiological regulation of the properties of alcohol dehydrogenase II
(ADH II) of
Zymomonas mobilis
: NADH renders ADH II resistant to cyanide
and aeration
Received: 16 March 2005 / Revised: 10 May 2005 / Accepted: 17 June 2005 / Published online: 19 July 2005
Ó Springer-Verlag 2005
Abstract The variable cyanide-sensitivity of the iron-
containing alcohol dehydrogenase isoenzyme (ADH II)
of the ethanol-producing bacterium Zymomonas mobilis
was studied. In aerobically grown permeabilized cells,
cyanide caused gradual inhibition of ADH II, which was
largely prevented by externally added NADH. Cyanide-
sensitivity of ADH II was highest in cells grown under
conditions of vigorous aeration, in which intracellular
NADH concentration was low. Anaerobically grown
bacteria, as well as those cultivated aerobically in the
presence of cyanide, maintained higher intracellular
NADH levels along with a more cyanide-resistant ADH
II. It was demonstrated that cyanide acted as a
competitive inhibitor of ADH II, competing with nico-
tinamide nucleotides. NADH increased both cyanide-
resistance and oxygen-resistance of ADH II.
Keywords Alcohol dehydrogenase Æ Cyanide-
resistance Æ Oxygen-resistance Æ Zymomonas mobilis
Introduction
There are two cytoplasmic alcohol dehydrogenase
isoenzymes in the Gram-negative, aerotolerant, ethanol-
producing bacterium Zymomonas mobilis: a zinc-
containing ADH I and an iron-containing alcohol
dehydrogenase isoenzyme (ADH II) (Kinoshita et al.
1985; Neale et al. 1986; Conway et al. 1987). Both of
them are NAD
+
-dependent, and together they carry out
the rapid and efficient synthesis of ethanol, attracting the
attention of researchers over decades (Rogers et al.
1982; Sprenger 1996; Dien et al. 2003). ADH I is
essential for the early stages of culture growth, while
ADH II plays a key role later in the fermentation at high
ethanol concentrations (O’Mullan et al. 1995). No ADH
I-negative mutants have been reported so far, yet ADH
II-negative mutants have been obtained and character-
ized by several authors (Wills et al. 1981; O’Mullan
et al. 1995; Delgado et al. 2002). The absence of ADH II
results in a prolonged generation time of the mutant
strain, as well as impaired growth and ethanol synthesis
during the late exponential and early stationary phases
of culture (O’Mullan et al. 1995).
However, ethanol synthesis might not be the sole
function of ADH II. It seems likely that iron-containing
bacterial alcohol dehydrogenases are involved also in
respiratory metabolism and oxidative stress response.
There are strong indications from aerobic chemostat
experiments (Kalnenieks et al. 2002) that ADH II may
function as a component of the respiratory pathway of
Z. mobilis. Under vigorous aeration, it oxidizes ethanol
and supplies the respiratory chain with NADH, while
ADH I catalyses ethanol synthesis, thus forming an
‘‘ethanol cycle’’. Notably, iron-containing alcohol de-
hydrogenases themselves are sensitive to oxygen. Under
oxic conditions, ADH II looses much of its activity,
apparently due to free radical reactions of oxygen at the
active site iron (Tamarit et al. 1997). The same is true for
E. coli iron-containing isoenzyme ADH-E, homologous
to Z. mobilis ADH II, which also is highly sensitive to
metal-catalyzed oxidation (Membrillo-Herna
´
ndez et al.
2000). It has been demonstrated that ADH-E needs to
be protected against oxidative damage by the chaperone
DnaK during aerobic growth (Echave et al. 2002).
Besides, ADH-E acts as a H
2
O
2
scavenger and, becom-
ing partially inactivated, protects E. coli cells against
hydrogen peroxide stress (Echave et al. 2003).
Recently we have demonstrated another property of
ADH II, traditionally associated with respiratory
metabolism—its sensitivity to inhibition by cyanide
(Kalnenieks et al. 2003). Inhibition of ADH II at
submillimolar cyanide concentrations proceeds gradu-
ally and presumably, reflects slow binding of cyanide to
the active site iron. Moreover, the cyanide-sensitivity of
U. Kalnenieks (&) Æ N. Galinina Æ M. M. Toma
Institute of Microbiology and Biotechnology,
University of Latvia, Kronvalda boulv. 4, 1586 Riga, Latvia
E-mail: kalnen@lanet.lv
Tel.: +371-7-034887
Fax: +371-7-034885
Arch Microbiol (2005) 183: 450–455
DOI 10.1007/s00203-005-0023-2