Shalley joined the Goodwin laboratory in June 2008. Her
project had two major components, each dedicated to one of the
extended interhelical loops unique to KatG. The first is to
determine the role of Large Loop 2 (LL2) in giving
catalase-peroxidases their striking bifunctional capabilities.
Building on Yongjiang Li's observation that removal of the entire
loop leaves KatG peroxidase activity comparable to wild type.
However, there is little if any catalase activity. Prior to
Shalley's work, the mechanism by which this structure specifically
supported catalase activity was not known. Shalley produced a
series of KatG variants by site-directed and deletion mutagenesis
that specifically targeted the apex of LL2 and found that
interactions between LL2 and the enzyme's C-terminal domain were
essential for controlling the active site heme coordination
environment and H-bonded interactions. Dr. Kudalkar also
evaluated a series of deletion variants targeting Large Loop
1. Her stopped-flow evaluation of those variants demonstrated
that an appropriate deletion not only abrogated catalase activity,
but also facilitated electron transfer from exogenous electron
donors to the enzyme's heme center. The net result was a KatG
with peroxidase activity ten fold greater than other
catalase-negative variants (e.g., Y226F KatG) and rivaling that of
the peroxidase gold-standard, horseradish peroxidase. In
December 2011, Shalley successfully defended her dissertation.
It is entitled
Roles of large loops in catalytic versatility of
catalase-peroxidases: Significance of peripheral structures in
improvising enzyme functions. Dr. Kudalkar engaged in
postdoctoral studies at the Vanderbilt University School of Medicine
under the direction of Dr. Larry Marnett followed by a second
postdoctoral position with Dr. Karen Anderson at Yale University.
Dr. Kudalkar is now an Associate Principal Scientist with Merck in
Doylestown, PA.
Publications and Abstracts
Kudalkar, S.N., Njuma, O.J., Li, Y., Muldowney, M., Fuanta, N.R.,
and Goodwin, D.C. 2015. "A role for catalase-peroxidase large
loop 2 revealed by deletion mutagenesis: Control of active site
water and ferric enzyme reactivity" Biochemistry54,
1648 - 1662.
Kudalkar, S.N., Campbell, R.A., Li, Y., Varnado, C.L., Prescott, C.,
and Goodwin D.C. 2012. Enhancing the peroxidatic
activity of KatG by deletion mutagenesis" J. Inorg. Biochem.116, 106 - 115.
Kudalkar,
S. N., and Goodwin, D. C. 2011.Tracing the Impact of a
Unique Loop in Catalase-peroxidase Catalysis.Annual Meeting of the
American Society for Biochemistry and Molecular Biology,
Washington, D. C.
Kudalkar, S. N., and Goodwin, D. C. 2011. Dependence of
catalytic ability of catalase-peroxidase on intersubunit
interactions.Annual Meeting of the
American Society for Biochemistry and Molecular Biology, Washington,
D. C.
Kudalkar,
S. N., and Goodwin, D. C., 2011.Effects of progressive deletion of a unique loop on
structure and function of catalase-peroxidases.Second Southeast Enzymes
Conference, Atlanta, GA.
Kudalkar,
S. N., and Goodwin, D. C.2010.Impact of intersubunit
interactions on catalytic versatility of catalase-peroxidases.First Southeast Enzyme
Conference, Atlanta, GA.
Goodwin, D.C., Li, Y, Kudalkar, S., Campbell, R., and
Prescott, C. 2009.Roles
of insertional sequences in commandeering an existing enzyme
framework for new catalytic function: A case study in
catalase-peroxidases.Gordon Conference:Enzymes, Coenzymes,
and Metabolic Pathways, Waterville Valley, NH.