DEHALOCOCCOIDES AND REDUCTIVE DECHLORINATION OF CHLORINATED SOLVENTS - Bioaugmentation for Groundwater Remediation - page 63

The rdhA genes encoding the RDase catalytic subunits share common features including a
twin arginine translocation (Tat) consensus sequence that enables secretion of the mature
protein through the cell membrane (see below) and two iron-sulfur cluster binding consensus
sequences. The average length of Dhc rdhA and rdhB genes is 1,491 and 237 bp, respectively.
In Dhc , the rdhB gene is located downstream of rdhA, and both genes are transcribed together
(M¨ller et al., 2004 ). rdhA and rdhB are separated by an intergenic spacer region that ranges
from 2 to 50 bp in length.
The rdhA / rdhB gene clusters can occur with accessory genes; however, most of the Dhc
rdhA / rdhB gene clusters lack one or all of the accessory genes rdhC , rdhD and rdhG . rdhC
and rdhD are located upstream of rdhA . Computational analysis suggests that rdhC encodes
a PAS/PAC sensor histidine kinase and rdhD encodes a putative DNA-binding regulator;
however, these functions have not been experimentally demonstrated. Located in variable
distance downstream of rdhB is a phage-related gene of unknown function, or rdhG ,
which encodes a protein of unknown function. Understanding the details of RDase operon
organization and how individual parts function and interact is relevant for the design of
biomarkers that not only provide information regarding RDase gene presence but also RDase
gene expression (i.e., activity).

2.8 REDUCTIVE

DEHALOGENASES (RDASES)
Reductive dechlorination is catalyzed by RDase enzyme systems encoded by rdhA . Dhc
RDases are monomeric enzyme systems with molecular weights ranging from 48.5 to 63 kDa
(based on putative RDase genes identified in sequenced Dhc genomes). Together with the
isomerases and the methyltransferases, RDases represent one of the three currently recognized
classes of B 12 enzyme systems (Banerjee and Ragsdale, 2003 ). RDase A proteins are very
oxygen sensitive and are irreversibly inhibited by exposure to air. Since obtaining large amounts
of biomass of a Dhc pure culture is impractical, protein purification from Dhc cultures is
challenging. Hence, heterologous expression of Dhc RDase genes in a host that produces
biologically active RDase is a major objective as this would allow detailed study of these
interesting enzyme systems. Cloning and overexpression of the pceA gene from Sulfurospir-
illum multivorans was successful but the recombinant PceA protein had no dechlorinating
activity, likely because of incorrect protein folding and/or the cloning host was unable to
synthesize and incorporate the cobalt-containing corrinoid (i.e., cobalamin) cofactor (Neumann
et al., 1996 ). Only four Dhc RDases, PceA, TceA, VcrA and CbrA, have been partially
characterized but the limited amount of enzyme recovered prevented detailed mechanistic
studies (Table 2.6 ).
The physiological electron carrier(s) that donates electrons to RDases is unknown but
RDase activity can be measured in vitro conveniently with artificial, low potential electron
donors such as reduced methyl viologen (E 0 0 ¼

DEHALOCOCCOIDES

446 millivolts [mV]) (Corbin and Watt, 1990 ;
Neumann et al., 1998 ). The Dhc PCE and TCE RDases also can use reduced benzyl viologen, a
weaker reductant ( 360 mV) than methyl viologen, as an electron donor (Jayachandran et al.,

2004 ; Nijenhuis and Zinder, 2005 ) whereas the PCE RDase of Sulfurospirillum multivorans
cannot, suggesting a difference in reaction mechanism.
Characteristics of the RDase A proteins are a Tat signal sequence and two Fe-S clusters.
The Tat signal sequence suggests RDase trafficking into the periplasmic space following
RDase maturation in the cytoplasm (Sargent, 2001 ). The Tat leader peptide has a length of
approximately 40 amino acids and the mature RDase A protein is 415-514 amino acids long.
Both Fe-S cluster-binding motifs occur in the C-terminal region of RDase A and are likely