Mechanistic Studies of Nicotinic Acid Degradation Enzymes: A Model Pathway for Understanding
Biodegradation of N-Heteroaromatic Molecules in the Environment
Dr. Mark J. Snider, Associate Professor of Chemistry
Environmental Significance: Synthetic N-heterocyclic aromatic molecules (e.g. nicotine, pyridine, quinolones,
etc.), used commonly as solvents, dyes, explosives, pharmaceuticals, herbicides and pesticides, have been
observed to contaminate soil and groundwater, as well as in groundwater that has been in contact with coal tar
refineries and coal-bed natural gas wells. These compounds are well established to be toxic, mutagenic or
carcinogenic and directly pose a threat to soil-dwelling organisms and plants, and through biomagnification,
may also pose a threat to the organisms that consume them. Given that many N-heterocyclic compounds can be
used as a source of biomass and energy by bacteria, a deeper understanding of the mechanisms used to
biodegrade these compounds could enhance remediation efforts. Biodegradation of such compounds is typically
achieved through redox processes catalyzed by hydroxylases and oxidoreductases, in which the organic
substrate is oxidized to less harmful, and sometimes useful, molecules.
Model System: The biodegradation pathway of nicotinate, a common N-heterocyclic molecule in soil, is wellestablished in several bacteria and serves as a useful model system for exploring the enzymatic mechanisms that
have evolved to oxidize and degrade this important class of environmental contaminant. Nicotinate (niacin,
vitamin B3) serves as the source of the nicotinamide moiety of NAD+ and NADP+, coenzymes central to
metabolism in all living species. Nicotinate [1, Figure 1] can also serve as a source of carbon, nitrogen and
energy for a variety of bacteria. Nicotinate catabolism occurs by multiple routes, dependent on oxygen
availability, in which 6-oxo-nicotinate [2] is a common first intermediate. 6-Oxo-nicotinate is subsequently
reduced to 1,4,5,6-tetrahydro-6-oxo-nicotinate [3] and further degraded to pyruvate and propionate by anaerobic
bacteria, or oxidized to either 3-hydroxy-2-pyridone [4] (in Pseudomonas and Bordetella species) or 2,6dihydroxynicotinate [5] (in Bacillus species) where it is finally degraded to the common metabolite, fumarate
[7]. Understanding the precise mechanisms by which these enzymes transform nicotinate would provide greater
insight concerning the general chemical principles that evolved in microorganisms to biodegrade Nheterocycles, in particular the oxygen-insertion and ring-opening of N-heteroaromatic molecules.
[4] NicX
OH
NicC
OH
N
[1] [2] NicAB
O
O2
NADH
OH
2H2O O
*
CO2
NAD+
H2O
FAD
O
MCD [2Fe-2S]
H2O* + O2
+ 2H+
O
O2
H2 O*
H+
NH
O
Pseudomonas / Bordetella
NicD
O
H
HCOOH
O
O
Bacillus
N
H
OH
*
[5] O2
O
CO2
N
H
OH
O
NH
O
O
O
COO
OOC
NicF
O
NH4+
O
O
O
O
O
O
O
[7] HCO31H2O
O
O
COO
NH4+
H2O
NicE
H
OH
2 H2O
[6] O
OH
OH
1/2 O2
O
NH2
O
H2 O2
Eubacterium barkeri
& proteobacteria
N
H
O2
N
H
1/2 O2
Mo
[2Fe-2S]
no O2
O
O
O
N
H
[3] O
Fe2+
O
COO
H
Figure 1: Nicotinate degradation pathways in aerobic (e.g. Pseudomonas, Bordetella, Bacillus) and anaerobic (e.g.
Eubacterium barkeri) bacteria. Putative [bracketed] intermediates of the Bacillus pathway have not been confirmed.
Project Goal: Current understanding of the degradation of nicotinate was established from early biochemical
investigations (ca 1950) of the native enzymes isolated from bacteria. Only recently have gene clusters that
code for the enzymes been identified in bacteria that inhabit rhizhospheres (e.g. Pseudomonas, Bordetella, and
Eubacterium barkeri) to enable a more detailed understanding of the structural and chemical basis for these
mechanisms. In fact, students in the Snider lab were the first to clone the nicF gene, study the mechanism of the
enzyme it encodes (maleamate amidohydrolase), and to determine this enzyme’s structure at high resolution.1
Students in the Snider lab continue to clone and characterize the genes of these pathways, with current emphasis
on the mechanisms and structural determinants of substrate specificity of NicC and NicF. Moreover, Zachary
Harvey, a former research student in Snider’s lab, recently sequenced the novel genomome of Bacillus niacini, a
common soil dwelling bacteria, and identified a novel gene cluster that we have hypothesized to code for the
enzymes of nicotinate catabolism.2 Current students are cloning these novel genes to experimentally determine
whether our hypothesis is correct and to develop methods (HPLC-UV, NMR and LC-MS/MS) to more precisely
determine the chemical structures of the two hypothetical intermediates in that metabolic pathway (bracketed
species of Figure 1). Our overall goal in these projects is to establish the mechanisms of the novel enzyme
catalysts to better understand the chemistries that evolved to degrade N-heterocyclic molecules in the
environment.
Summer research students in the Snider lab gain the following skills:
I.
II.
III.
IV.
V.
VI.
Molecular biology techniques (e.g. PCR) to clone genes into plasmid DNA vectors that enable their
enzyme products to be over-expressed in E. coli.
Affinity and gel filtration chromatography to purify enzymes from the cultures of E. coli.
Kinetic assays using spectrophotometry or isotherm titration calorimetry to characterize the activity
of the enzymes.
Analytical techniques, such as HPLC-UV, to isolate the products of the enzyme-catalyzed reactions
to establish the chemical structures, by mass spectrometry (MS) and nuclear magnetic resonance
(NMR), of the intermediates of the metabolic pathways.
Fluorescence spectroscopy and isothermal titration calorimetry to characterize the binding of
alternative substrates and inhibitors by the enzymes.
Communication skills (oral and written) necessary to work effectively as part of a team on a
complex scientific problem.
1 Kincaid,
V., Sullivan, E., Klein, R., Noel, J., Rowlett, R., and Snider, M. J. Structure and catalytic mechanism of
nicotinate (vitamin B3) degradative enzyme maleamate amidohydrolase from Bordetella Bronchiseptica RB50.
Biochemistry 2012, 51, 545-554.
2
Harvey, Z. H. and Snider, M. J. Draft Genome of the Nicotinate-Metabolizing Soil Bacterium Bacillus niacini (DSM
2923). Genome Annoucement – manuscript in preparation.