Bacterial Enzymes and Multi-enzymatic Systems for Cleaning-up Dyes from the Environment - Microbial Degradation of Synthetic Dyes in Wastewaters - page 36

Fig. 7 Screening for decolorization of reactive red 4 (RR4), acid red 299 (NY1), reactive black 5
(RB5), direct blue 1 (CSB) and direct black 38 (CB) at increasing concentrations using growing
cells of P. putida MET94

azo dyes (Mendes et al. 2011b ; Brissos et al. 2014 ). The steady-state kinetic analysis,
using 1,4-benzoquinone or reactive black 5 and NADPH, resulted in a family of
parallel lines in a double reciprocal plot (Mendes et al. 2011b ; Gon

alves et al. 2013 )
which is indicative of a ping-pong bi-bi kinetics as described for other

ç

avin-

dependent azoreductases. The ef ciency for 1,4-benzoquinone is one and two orders

of magnitude higher than azo dyes (V max =50Umg − 1 , K m app = 0.005 mM, k cat
app = 49 min − 1 , k cat /K m = 98

10 5 ), showing that quinones represent most probably
the physiological substrates of this enzyme in P. putida cells.
PpAzoR (PDB code 4C0 W) is a homodimer and its tertiary structure adopts a

×

avodoxin-like fold characterized by a central twisted ve parallel ʲ -sheet con-

nected by ʱ -helices, which ank the sheet from the front and the back (Correia et al.

2011 ; Gon -stands is

identical to structures of azoreductases from E. coli (PDB code 2Z98), Pseudo-
monas aeruginosa (PDB code 2V9C), Enterococcus feacalis (PDB code 2HPV)
and Salmonella typhimurium (PDB code 1T5B). Moreover, it contains the con-
served motif patterns of avin-dependent azoreductases, i.e. the sequence involved
in the binding of FAD/FMN co-factors, the sequence involved in the dimerisation
of the two monomers of the enzyme and the possible putative NAD(P)H binding
motif. The crystal structures of native PpAzoR (1.6

ç alves et al. 2013 ). The arrangement of the ʱ -helices and ʲ

Å ) and PpAzoR complexed

with anthraquinone-2-sulphonate (1.5 ), were solved

revealing the residues and subtle changes that accompany substrate binding and
release. Such changes highlight the

Å ) or reactive black 5 (1.9 Å

ne control of access to the catalytic site and

tune the speci city offered by the enzyme towards different substrates. In particular,

it helps to explain how PpAzoR allows for the accommodation of bulky substrates
explaining its enlarged substrate utilization with similar catalytic ef

ciencies

(Gon alves et al. 2013 ).

The enzymatic activity of PpAzoR was tested using 18 structurally different
synthetic dyes by measuring the decolorization levels after 24 h of incubation under
anaerobic conditions. The results show that PpAzoR exhibits a broad substrate
speci

ç

city with decolorization levels above 80 % for most of the dyes tested
(Fig. 8 ).