Structural and kinetic isotope effect studies of nicotinamidase (Pnc1) from saccharomyces cerevisiae

Article

pubs.acs.org/biochemistry

Structural and Kinetic Isotope Effect Studies of Nicotinamidase

(Pnc1) from Saccharomyces cerevisiae

Brian C. Smith,^‡,§Mark A. Anderson,^†Kelly A. Hoadley,^‡James L. Keck,^‡W. Wallace Cleland,*^,†

and John M. Denu*^,‡

^†Institute for Enzyme Research, Department of Biochemistry, University of Wisconsin, 1710 University Avenue, Madison, Wisconsin

53726, United States

^‡Department of Biomolecular Chemistry, University of Wisconsin, 553 Medical Sciences Center, 1300 University Avenue, Madison,

Wisconsin 53706, United States

S

* Supporting Information

ABSTRACT: Nicotinamidases catalyze the hydrolysis of

nicotinamide to nicotinic acid and ammonia. Nicotinamidases

are absent in mammals but function in NAD⁺salvage in many

bacteria, yeast, plants, protozoa, and metazoans. We have

performed structural and kinetic investigations of the

nicotinamidase from Saccharomyces cerevisiae (Pnc1). Steady-

state product inhibitor analysis revealed an irreversible reaction

in which ammonia is the first product released, followed by

nicotinic acid. A series of nicotinamide analogues acting as

inhibitors or substrates were examined, revealing that the

nicotinamide carbonyl oxygen and ring nitrogen are critical for binding and reactivity. X-ray structural analysis revealed a covalent

adduct between nicotinaldehyde and Cys167 of Pnc1 and coordination of the nicotinamide ring nitrogen to the active-site zinc

ion. Using this structure as a guide, the function of several residues was probed via mutagenesis and primary ¹⁵N and ¹³C kinetic

isotope effects (KIEs) on V/K for amide bond hydrolysis. The KIE values of almost all variants were increased, indicating that

C−N bond cleavage is at least partially rate limiting; however, a decreased KIE for D51N was indicative of a stronger

commitment to catalysis. In addition, KIE values using slower alternate substrates indicated that C−N bond cleavage is at least

partially rate limiting with nicotinamide to highly rate limiting with thionicotinamide. A detailed mechanism involving

nucleophilic attack of Cys167, followed by elimination of ammonia and then hydrolysis to liberate nicotinic acid, is discussed.

These results will aid in the design of mechanism-based inhibitors to target pathogens that rely on nicotinamidase activity.

infectious phenotype of Borrelia burgdorferi, the bacterium that

causes Lyme disease.⁹In Brucella abortus, which causes abortion

in domestic animals and undulant fever in humans, the B.

abortus nicotinamidase is essential for replication.¹⁰Further-

more, erythrocytes infected with Plasmodium falciparum, a

parasite that causes malaria in humans, displayed increased

nicotinamidase activity and NAD⁺synthesis.³It is likely that

other human pathogens require nicotinamidase activity for

viability, because many, including P. falciparum, do not possess

the genes necessary for de novo NAD⁺synthesis.

Current tuberculosis treatments target the nicotinamidase

(PncA) of Mycobacterium tuberculosis, which hydrolyzes the

prodrug pyrazinamide to the active form, pyrazinoic acid.

Pyrazinamide, when administered in combination with

isoniazid and rifampin, forms the current short course

treatment recommended by the World Health Organization¹¹

and shortens tuberculosis treatment from 9 to 6 months.

Pyrazinoic acid displays its toxicity by inhibiting M. tuberculosis

icotinamidases (EC 3.5.1.19) are amidohydrolases that

N_{catalyze the hydrolysis of nicotinamide to nicotinic acid}

and ammonia (Scheme 1). Nicotinamidases play a central role

Scheme 1. General Reaction Catalyzed by Nicotinamidases

in the NAD⁺salvage pathway¹of multiple species of bacteria,

yeast,²protozoa,³and plants⁴and are present in many

metazoans such as Drosophila melanogaster⁵and Caenorhabditis

elegans.^6,7However, mammals do not encode a nicotinamidase

but instead use nicotinamide phosphoribosyltransferase to

convert nicotinamide directly to nicotinamide mononucleotide,

which is then recycled to NAD⁺.⁸

The importance of nicotinamidase activity in the NAD⁺

salvage pathways of human pathogens, combined with the

absence of a nicotinamidase in human NAD⁺salvage pathways,

suggests that nicotinamidases are potential drug targets. Indeed,

nicotinamidase was shown to be an essential enzyme for the

Received: October 6, 2011

Revised: December 15, 2011

Published: December 19, 2011

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trans-translation through binding ribosomal protein S1.¹²

Mutations in the M. tuberculosis PNCA gene are associated

with clinical resistance to pyrazinamide.¹³⁻¹⁶

7.5. Pooled fractions containing Pnc1 were concentrated, and

protein concentrations were determined using the Bradford

method³⁴using bovine serum albumin as a standard.

Mutagenesis of Pnc1. Pnc1 mutagenesis was performed

using the QuikChange site-directed mutagenesis kit (Strata-

gene) according to the manufacturer's protocol. The primers

used for mutagenesis are listed in Table S1 of the Supporting

Information.

Nicotinamidases have also been implicated in increasing the

life span of Saccharomyces cerevisiae,¹⁷⁻²⁰C. elegans,⁷and D.

melanogaster.⁵The observed life span extension in these

organisms is potentially mediated by increasing the activity of

sirtuin NAD⁺-dependent deacetylases by decreasing cellular

nicotinamide levels and increasing NAD⁺levels. In support of

this hypothesis, sirtuin overexpression was reported to increase

life span of S. cerevisiae,^21,22C. elegans,²³and D. melanogast-

er,^24,25and nicotinamide is a potent sirtuin product

inhibitor.^19,26−28However, a recent report revealed that the

apparent life span extension through sirtuin overexpression in

C. elegans and D. melanogaster was abolished when a more

appropriate genetic background was used.²⁹

Despite the importance of nicotinamidases in diverse

biological processes, their precise mechanism of catalysis has

yet to be fully elucidated.³⁰⁻³³Understanding the chemical

mechanism and nature of the transition state of the reaction

catalyzed by nicotinamidases would aid the design of inhibitors

or prodrugs (such as pyrazinamide) that target nicotinamidases

for antimicrobial applications. Here, we establish the overall

kinetic mechanism of the eukaryotic nicotinamidase from S.

cerevisiae (Pnc1) through product inhibition analysis. We then

show that ketone- and aldehyde-containing nicotinamide

analogues are Pnc1 inhibitors. Using this knowledge, we

obtained the first crystal structure of a eukaryotic nicotinami-

dase with a nicotinamide analogue bound in the active site. This

structure suggested several residues potentially involved in

catalysis, and the steady-state kinetic parameters of several Pnc1

mutants were determined. Steady-state kinetic parameters with

alternate nicotinamide analogue substrates were also measured.

We then further delineated the mechanism by determining the

primary ¹⁵N and ¹³C kinetic isotope effects (KIEs) of the C−N

bond breaking and the pH dependence of the reaction. To

determine if C−N bond cleavage is partially or fully rate

limiting, we compared the ¹⁵N and ¹³C KIEs for nicotinamide

hydrolysis by Pnc1 with those determined using slow substrate

analogues. In addition, the KIE values for nicotinamide

hydrolysis by several Pnc1 mutants were compared, allowing

for the validation of the proposed catalytic function of active-

site residues. These results suggest specific roles for several

Pnc1 residues during catalysis as well as structure−activity

relationships for Pnc1-catalyzed hydrolysis of nicotinamide

analogues.

Determination of Kinetic Parameters. Nicotinamidase

activity was measured continuously using an enzyme-coupled

assay with glutamate dehydrogenase using a Multiskan Ascent

microplate reader (LabSystems, Franklin, MA). This assay is

slightly modified from that described by Su et al.³⁵Typical

assay mixtures contained 1.25 μM to 3.2 mM nicotinamide or

analogue, 0.2 mM NADPH, 3.3 mM α-ketoglutarate, 50 nM to

10 μM wild-type (WT) or mutant Pnc1, and 3 units of

glutamate dehydrogenase from bovine liver in 50 mM sodium

phosphate (pH 7.5). Assays were conducted in a final volume

of 300 μL per well in a clear, flat-bottom, 96-well plate. All

assay components except Pnc1 were preincubated at 25 °C for

5 min or until the absorbance at 340 nm stabilized, and the

reaction was initiated by the addition of Pnc1. The rates were

monitored continuously for NADPH consumption at 340 nm.

Rates were determined from the slopes of the initial linear

portion of each curve using an extinction coefficient for

NADPH of 6.22 mM⁻¹cm⁻¹and a path length of 0.9 cm for

300 μL reactions. The background rates of reactions lacking

Pnc1 resulting from the spontaneous formation of ammonia

were subtracted from the initial velocities of the Pnc1-catalyzed

reactions.

Determination of K_iValues. Assay mixtures contained

6.7−200 μM nicotinamide, 0.2 mM NADPH, 1 or 3.3 mM α-

ketoglutarate, 100 nM to 0.5 μM WT Pnc1, and 3 units of

glutamate dehydrogenase from bovine liver in 50 mM sodium

phosphate (pH 7.5). For nicotinic acid inhibition, 200 μM to

1.2 mM nicotinic acid was used. For nicotinaldehyde inhibition,

1−6 μM nicotinaldehyde was used. For 3-acetylpyridine

inhibition, 300 μM to 1.8 mM 3-acetylpyridine was used.

Benzaldehyde and pyrazinoic acid were initially dissolved in

DMSO, and concentrations of 5−10 mM were used [final

DMSO concentration of 10% (v/v)]. Assays were run and

analyzed as detailed under Determination of Kinetic Parame-

ters. Initial velocity data were fit in Kinetasyst (Intellikinetics,

State College, PA) to competitive inhibition patterns (eq 1 or

2) based on the algorithms defined by Cleland.³⁶All data were

displayed using Kaleidagraph (Synergy Software, Reading, PA).

V

[S]

max

EXPERIMENTAL PROCEDURES

v =

■

⎛

⎜

⎞

[I]

Expression and Purification of Pnc1. Yeast Pnc1 cloned

into pET-16b or pET-17b^20,32was transformed into Escherichia

coli strain BL21(DE3). A single transformant colony was grown

in 1.0 L of 2×YT medium at 37 °C to an OD₆₀₀of ∼0.6−0.8.

IPTG was added to a final concentration of 0.5 mM, and

expression was continued for 6−8 h at 25 °C. Cells were

harvested by centrifugation and stored at −20 °C. Cellular

pellets were resuspended in buffer A [50 mM NaH₂PO₄(pH

7.5), 300 mM NaCl, 1 mM β-mercaptoethanol, and 1 mM

phenylmethanesulfonyl fluoride] containing 30 mM imidazole

and lysed by sonication. Recombinant Pnc1 was purified using

immobilized metal affinity chromatography with a Ni²⁺-

nitrilotriacetic column. The column was washed with buffer A

containing 30 mM imidazole, and bound proteins were eluted

with a gradient from 30 to 500 mM imidazole in buffer A at pH

⎟

⎠

K

1 +

+ [S]

_max[S]

m

K

⎝

(1)

is

V

log(v) = log

⎛

⎜

⎞

[I]

K_is

⎟

K_m1 +

+ [S]

⎝

⎠

(2)

Protein Crystallization and Structure Determination.

For crystallization trials, the protein was purified as described

above, with the addition of a Sephacryl S-300 size-exclusion

step. Protein was dialyzed against buffer containing 15 mM Tris

(pH 7.5), 50 mM NaCl, 4 mM MgCl₂, 10 mM sodium citrate,

and 5% glycerol, and the inhibitor nicotinaldehyde was added at

a 4/1 molar ratio. The protein (5 mg/mL) was mixed with

mother liquor [1.6 M NaOAc, 10% ethylene glycol, and 0.1 M

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HEPES (pH 7.4)] at a 1/1 (volume) ratio. Crystals were

formed by hanging drop vapor diffusion. Crystals were

transferred to a cryoprotectant solution [1.5 M NaOAc, 20%

ethylene glycol, and 0.1 M HEPES (pH 7.4)] and flash-frozen

in liquid nitrogen.

¹³C Kinetic Isotope Effects. This methodology is a

modification of previous work by Scott and others⁴¹⁻⁴³

(Scheme 2). The solution of the nicotinic acid product was

Scheme 2. Decarboxylation of Nicotinic Acid by Copper

Chromite

Diffraction data were indexed and scaled using HKL2000.³⁷

The structure of Pnc1p with the inhibitor nicotinaldehyde was

determined by molecular replacement (Phaser)³⁸using the apo

Pnc1p structure³²as a search model. The structure was

improved by rounds of manual fitting using Coot³⁹and

refinement using REFMAC5.⁴⁰Coordinate and structure factor

files have been deposited in the Protein Data Bank (entry

3V8E).

reduced to dryness and dissolved in 2 mL of HPLC grade

methanol. The methanol/nicotinic acid solution was added to a

quartz glass tube (25 cm, 9 mm outside diameter, 7 mm inside

diameter), and the methanol was removed under vacuum at a

slightly elevated temperature (40 °C) for several hours. To this

was added 0.5 g of dry copper chromite, and the tube was

evacuated and flame-sealed. The sample was placed in a furnace

and heated at 250 °C for nicotinic acid (232 °C for pyrazinoic

acid, 235 °C for thionicotinic acid, and 240 °C for 5-

methylnicotinic acid) for 3 h and then cooled in a room-

temperature water bath. The tube was cracked, and the CO₂

was distilled through two −130 °C liquid N₂/pentane traps and

finally collected in a third trap at −196 °C. Liquid N₂/pentane

traps were necessary because −78 °C traps were inefficient and

allowed pyridine, a product of the decarboxylation of nicotinic

acid, to contaminate the CO₂sample. The collected CO₂

(∼80% of the total sample) was isolated in the trap and was

not removed from the line at this time.

The final 20% of the CO₂was collected by acidification of

the copper chromite powder. The copper chromite and a small

stir bar were placed in a 30 mL flask equipped with a glass joint,

a side arm with a stopcock, and a septum. The flask was fitted

to a glass stopcock, placed on the high-vacuum line, and

evacuated for at least 15 min. After the top stopcock had been

closed, 2.5 mL of 1 M H₂SO₄was added via syringe through

the septum. CO₂was evolved immediately, and the solution

was stirred for 5 min. A −78 °C bath was used to freeze the

sample, and the CO₂was collected in the same manner

described above and trapped with the first portion of gas. The

entire gas sample was isolated, and the −196 °C trap was

removed and replaced with a −78 °C trap. The entire CO₂

sample was collected in a gas sample tube at −196 °C and was

then analyzed by IRMS. IRMS analysis of the two CO₂portions

individually showed a difference of 1−1.5 δ, so collection of

both was deemed necessary for the most accurate measure-

ment.

When this technique was used to decarboxylate thionicotinic

acid, the δ values were not reproducible. It was assumed that

the sulfur had not been completely removed from the

compound, even though it was heavily acidified. Also, these

samples were not easily evacuated from the IRMS, and a small

residual impurity had to be frozen out of the system at −196 °C

onto molecular sieves. Thus, we were unable to reliably

determine the ¹³C KIE for thionicotinamide.

Fraction of Reaction. The fraction of reaction (f value)

was determined by using a known amount of substrate for the

enzymatic reaction (50 μmol) and then comparing that to the

amount of product NH₃recovered after the reaction. To

double check the f value, the amount of product NH₃was also

compared to the amount of NH₃recovered in 0.1 N H₂SO₄

after steam distillation under basic conditions of the residual

substrate. In all cases, the amount of NH₃was determined by

¹⁵N Kinetic Isotope Effects. Reactions for isotope effect

analysis were conducted in 7.5−10 mL of 20 mM potassium

phosphate (pH 7.5) containing 10 mM nicotinamide or

analogue and 0.75−36 μM WT or mutant Pnc1. Reactions

were quenched by addition of 8 M HCl to a final concentration

of 40 mM. The enzyme was removed by Amicon filtration

(10000 molecular weight cutoff), and the solution was diluted

to 100 mL with distilled water. The pH was adjusted to 6, and

the solution was loaded onto an AG1X8 resin column (Cl⁻

form, 2.5 cm × 30 cm) at a rate of 1 mL/min. The collection of

fractions (8.5 mL/fraction) was started at the time of loading.

When the loading was complete, the products were eluted with

water at a rate of 1 mL/min. NH₄Cl eluted between fractions 3

and 18 and was detected using 50 μL samples of each fraction

added to 50 μL of Nessler’s reagent in 96-well plates. A yellow

color indicated the presence of NH₄Cl. The residual

nicotinamide or analogue generally eluted between fractions

25 and 50 and was detected by being spotted on UV active

TLC plates. The product, nicotinic acid, was eluted with 1 M

HCl from the column and found by UV at 262 nm. The volume

of product NH₄Cl was reduced to 50 mL by rotary evaporation

and the sample purified by steam distillation with 12 mL of 13

M KOH. The NH₃was trapped in 10 mL of 100 mN H₂SO₄

and collected until the total volume reached 50 mL.

The residual substrate was pooled and concentrated to 50

mL. It was then steam distilled at high pH, hydrolyzing the

amide, and the resultant NH₃was trapped in acid. The

hydrolysis of nicotinamide (40−50 μmol) required the addition

of 12 mL of 13 N NaOH to a 50 mL solution. The other

analogues required 13 N NaOH in the following amounts: 10

mL for thionicotinamide, 8 mL for 5-methylnicotinamide, and

3 mL for pyrazinamide. Considerable time was spent

determining the correct amount of base to be used because

too much caused the decomposition of the pyridine ring and

production of non-amide NH₃. This was especially true for

pyrazinamide where the pyrazine is not as stable as pyridine.

The concentration of ammonia for both the product and

residual substrate samples was determined by UV−vis

spectrometry using Nessler’s reagent to establish that no

product NH₄Cl had been lost. The volume of (NH₄)₂SO₄was

then reduced to ∼1 mL by rotary evaporation and the sample

transferred to a flask with a side arm. The side arm was filled

with ∼4 mL of NaOBr. The system was sealed with a stopcock,

and the solution was frozen at −78 °C and subjected to three

freeze−pump−thaw cycles to remove all gases. After the final

thaw, the two solutions were mixed slowly and the liquid was

again frozen at −78 °C. The freshly produced N₂was distilled

through two −78 °C traps and one −196 °C trap and then

collected on molecular sieves at −196 °C. The N₂was analyzed

by IRMS to give the ¹⁵N/¹⁴N ratio.

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comparison of the sample to a standardized curve of NH₄Cl by

UV at 425 nm. The fraction of reaction determined for the

RESULTS

■

Product Inhibition and Irreversibility of the Reaction.

NH₃

versus the initial substrate concentration and the

prod

To establish the kinetic mechanism for S. cerevisiae

nicotinamidase (Pnc1), steady-state product inhibition analysis

was performed. Double-reciprocal analysis of nicotinamidase

inhibition by nicotinic acid and pyrazinoic acid displayed

competitive inhibition patterns with K_ivalues of 120 μM and

6.7 mM, respectively (Table 1 and Figure S1 of the Supporting

fraction of reaction for NH_{3 prod}versus NH_{3 res sub}were generally

within 1−3% of one another.

Kinetic Isotope Effects. Isotope effects were determined

from changes in the ¹⁵N/¹⁴N (or ¹³C/¹²C) natural abundance

ratio in the compound during the reaction. The product and

residual substrate were separated, purified, and converted to N₂

(or CO₂) gas. Each gas sample was analyzed individually by

IRMS to determine its isotopic ratio compared to a known

standard to give δ, defined as

Table 1. K_iValues of Pnc1 Competitive Inhibition by

Nicotinamide Analogues

a

15

⎛

⎜

⎞

⎟

N

sample

14

δ = 1000

− 1

15

N

standard

⎜

⎟

14

⎝

⎠

(3)

To determine the isotope effect, the samples are converted to

an R value defined as

δ

1000

sample

R =

+ 1

(4)

R values for the reaction product, R_p, and residual substrate, R_s,

along with the isotopic ratio of the starting material, R_o, and f,

the fraction of reaction, are used in the following equations to

arrive at the KIE.

a

K_ivalues were determined as described in Experimental Procedures.

All reactions were performed at pH 7.5.

ln(1 − f )

KIE =

Information). The competitive inhibition displayed by nicotinic

acid is consistent with that shown by Gadd et al. for a

nicotinamidase from Micrococcus lysodeikticus⁴⁶but in contrast

to French et al. who observed no inhibition with nicotinic acid

for S. cerevisiae Pnc1.³⁰Double-reciprocal analysis of

nicotinamidase inhibition by ammonium ion using a previously

published HPLC assay⁴⁵yielded noncompetitive inhibition

patterns (data not shown). However, >10 mM ammonium

chloride was required for inhibition, consistent with several

previous reports on nicotinamidases from Mi. lysodeikticus and

yeast.^46,47Therefore, it was unclear if the ammonium ion added

was acting as a product inhibitor or as a denaturant as

noncompetitive inhibition patterns would be predicted in either

case. To determine if the weak inhibition by ammonium ion

could be explained by low reversibility of the reaction, 100 mM

ammonium chloride and 5 mM nicotinic acid were incubated

with 5 μM Pnc1 for 150 min at pH 7.5 and assayed for

nicotinamide formation using an HPLC assay.⁴⁵No nicotina-

mide was detected under these conditions, leading to an upper

estimate for the reverse reaction rate of <10⁻⁵s⁻¹, at least 4

orders of magnitude slower than the forward reaction rate with

nicotinamide (k_cat= 0.69 s⁻¹). Therefore, the Pnc1-catalyzed

reaction is essentially irreversible.

ln(1 − fR /R )

p

o

(5)

(6)

ln(1 − f )

ln[(1 − f )(R /R )]

KIE =

s

o

ln(1 − f )

ln{(1 − f )/[1 − f + f(R /R )]}

p

s

(7)

Dependence of Activity on pH. Reaction mixtures

contained 0.1−3 μM Pnc1 and varying concentrations of

nicotinamide (2.5 μM to 1 mM) or pyrazinamide (20 μM to 2

mM) in 100 μL at 25 °C. TBA buffer (50 mM Tris, 50 mM

BisTris, and 100 mM sodium acetate) was used for the pH

range of 4.0−8.5. ATE buffer (100 mM ACES, 52 mM Tris,

and 52 mM ethanolamine) was used for the pH range of 8.0−

10.5. These buffer mixtures are designed to give a constant

ionic strength over a wide pH range.⁴⁴Reactions were

quenched with 20 μL of 6% (v/v) TFA before 10% of the

substrate was converted to products at intervals of 60−120 s.

The percent product conversion was determined spectrophoto-

metrically by coupling to glutamate dehydrogenase or using a

previously published HPLC assay.⁴⁵Plots of k_catversus pH

were fitted to eq 8:

Inhibition by Nicotinamide Analogues. The product

inhibition analysis described above suggested that nicotinic acid

was the second product released and therefore that an enzyme

intermediate might exist between Pnc1 and nicotinic acid. We

hypothesized that nonhydrolyzable nicotinamide analogues

might trap this intermediate and display potent nicotinamidase

inhibition. All analogues tested displayed competitive inhibition

with K_ivalues ranging from high nanomolar to low millimolar

(Table 1). The competitive inhibition observed with 3-

log v = log[C/(1 + H/K_a+ K_b/H)]

(8)

using KinetAsyst (IntelliKinetics), where C is the pH-

independent value, H is the proton concentration, and K_a

and K_bare the ionization constants of the groups involved in

the reaction.

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acetylpyridine [K_i= 316 μM (Table 1)] was consistent with

several previous reports on nicotinamidases from Mycobacte-

rium phlei,⁴⁸Mi. lysodeikticus (K_i= 160 μM),⁴⁹Tortula cremoris

(K_i= 305 μM),⁵⁰Fleischmann’s yeast (K_i= 65 μM),⁴⁷

Flavobacterium peregrinum,⁵¹and S. cerevisiae (K_i= 46 μM).³⁰

Nicotinaldehyde displayed by far the most potent inhibition

among the analogues tested with a K_ivalue of 940 nM (Table 1

and Figure S1 of the Supporting Information), 10-fold below

the K_mvalue of 9.6 μM for nicotinamide. Potent inhibition by

nicotinaldehyde was previously observed for nicotinamidases

from Mi. lysodeikticus (K_i= 18 nM),⁴⁹S. cerevisiae (K_i= 1.4

μM),³⁰and M. tuberculosis (K_i= 290 nM).³³Benzaldehyde was

the weakest inhibitor tested with a K_iof 20.6 mM, similar to the

previously observed benzaldehyde inhibition of Mi. lysodeikticus

nicotinamidase (K_i= 1.8 mM).⁴⁹

Table 2. X-ray Data Collection and Structure Determination

Statistics

Data Collection

wavelength (Å)

resolution range (high-resolution bin) (Å)

space group

0.97872

30−2.70 (2.75−2.70)

R₃

unit cell

a, b, c (Å)

298.72, 298.72, 112.65

90, 90, 120

100.0 (100.0)

578608

α, β, γ (deg)

completeness (%)

total no. of reflections

no. of unique Reflections

redundancy

101147

5.7 (5.5)

⟨I/σI⟩

19.8 (5.3)

a

R_sym(%)

11.3 (34.8)

Structure of Nicotinaldehyde-Inhibited Pnc1. The

potent inhibition displayed by nicotinaldehyde inspired us to

cocrystallize Pnc1 with nicotinaldehyde. Pnc1 crystallized with

the nicotinaldehyde inhibitor at a 4/1 (nicotinaldehyde/Pnc1)

molar ratio in the same space group with the same unit cell

parameters as the apo structure (Table 2).³²The crystals

diffracted to 2.7 Å resolution, and the structure was determined

by molecular replacement using the apo Pnc1 structure³²as a

search model.³⁸F_o− F_celectron density maps revealed the

location of the inhibitor covalently bound to C167, which was

modeled into the electron density as a modified amino acid

(Figure 1A). Overall, the structure is similar to the apo

structure with a root-mean-square deviation (rmsd) of 0.3 Å²,

and no major structural changes were induced by the formation

of a complex with the inhibitor.

Refinement

resolution (Å)

30−2.70

18.9/21.4

b

R_work/R_free(%)

rmsd

bonds (Å)

0.009

1.23

angles (deg)

Ramachandran statistics (%)

most favored

allowed

89.8

9.9

generously allowed

disallowed

0.3

0.0

no. of atoms

protein

12439

296

water

c

ligand

14

Within the crystal structure, nicotinaldehyde is covalently

bound within the Pnc1 active site through a thiohemiacetal

⟨B factor⟩ (Å²)

a

protein

27.5

23.9

82.7

linkage to C167. Residues D8, K122, and C167 are all within

water

hydrogen bonding distance of one another (Figure 1B) and

form a putative catalytic triad that is conserved throughout all

known nicotinamidases (Figure S2 of the Supporting

Information). In addition to the covalent linkage to C167,

nicotinaldehyde is ligated to the active-site zinc through the

pyridine ring nitrogen (Figure 1). This zinc ligation is

consistent with recent structures of nicotinamidases from

Acinetobacter baumanii and Streptococcus pneumoniae.^31,52The

active-site zinc is also ligated by D51, H53, and H94, consistent

with the previous S. cerevisiae Pnc1 apo structure.³²

Furthermore, we observed evidence of two water molecules

that also coordinate the zinc; one of these is within hydrogen

bonding distance of one conformation of E129 (E129 is present

in two conformations in the structure) (Figure 1C). The

carbonyl oxygen of nicotinaldehyde forms hydrogen bonds to

the backbone amide nitrogens of A163 and C167, consistent

with recent structures of St. pneumoniae PncA.⁵²The pyridine

ring of nicotinaldehyde is also bound within an aromatic cage

consisting of F13, W91, Y131, and Y166 (Figure 1B).

Kinetic Parameters of Pnc1 Active-Site Mutants. The

structure with nicotinaldehyde bound within the Pnc1 active

site and a sequence alignment of prokaryotic and eukaryotic

nicotinamidases (Figure S2 of the Supporting Information)

suggested several conserved residues that may be involved in

catalysis. Therefore, we determined the steady-state kinetic

parameters for Pnc1 mutants of D8, D51, H53, H94, K122, and

C167 (Table 3). The putative base D8 was substituted with Ala,

Asn, or Glu, yielding mutants that displayed k_catvalues 10³−

10⁴-fold lower than that of WT Pnc1 (Table 3). The D8E

mutant exhibited slightly faster rates compared to that of either

c

ligand

a

R_sym= ∑∑_j|I_j− ⟨I⟩|∑I_j, where I_jis the intensity measurement for

reflection j and ⟨I⟩ is the mean intensity for multiply recorded

reflections. R_work/R_free= ∑||F_o| − |F_c||/|F_o|, where R_workand R_freeare

calculated by using the working and free reflection sets, respectively.

The R_freereflections (5% of the total) were held aside throughout

refinement. Ligand refers to seven zinc and seven magnesium ions

(one zinc and one magnesium for each of seven monomers in the

b

c

asymmetric unit).

D8A or D8N. The equivalent D8E mutant in M. tuberculosis

nicotinamidase was previously shown to harbor a 100-fold

lower specific activity compared to that of the wild type.⁵³

When individually substituted with Ala, the zinc-binding

residues D51, H53, and H94 yielded enzymes with 10−50-

fold lower k_catvalues. Similar losses of specific activity (10−

3000-fold) were observed when the corresponding zinc-binding

residues were mutated to Ala in the M. tuberculosis

nicotinamidase.^53,54The D51N mutant was slightly more active

(3-fold) than the D51A mutant. The zinc-binding mutants

displayed K_mvalues that were similar to or lower than those for

Pnc1 WT (Table 3). The nicotinamidase active-site Zn²⁺is

tightly bound, as in addition of 10 mM EDTA did not inhibit

Pnc1 (data not shown), consistent with a previous report on

nicotinamidases from T. cremoris,⁵⁰Mi. lysodeikticus,⁴⁶and M.

tuberculosis.³³Addition of 2 mM ZnCl₂failed to increase the

activity of the wild type (consistent with a previous report³⁰) or

rescue the activity of D51A, D51N, H53A, or H94A mutants

(data not shown), further indicating that Zn²⁺is tightly ligated

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Figure 1. Structure of nicotinaldehyde covalently bound in the active site of yeast Pnc1. (A) F_o− F_celectron density map (3.5σ) showing the

inhibitor and Zn²⁺in a cross-eyed stereoview. The inhibitor (nicotinaldehyde) covalently attached to Cys167 was modeled into the density.

Additional residues that coordinate the Zn²⁺(Asp51, His53, and His94) are also shown. (B) Cross-eyed stereoview of the active site of Pnc1 with

nicotinaldehyde covalently bound. (C) Cross-eyed stereoview of ordered water within the nicotinaldehyde-bound Pnc1 active site.

by D51, H53, and H94 and that these residues are critical for

catalysis. When K122 was replaced with Ala, the k_catwas

decreased by 16-fold, whereas the K122R mutant displayed a

dramatic 770-fold decrease in k_catcompared to that of WT

Pnc1. When the proposed nucleophile C167 was replaced with

Ala, the observed rate was below the detection limit of the

coupled assay (0.0005 s⁻¹), the lowest of any mutant. A

complete loss of activity was also observed when the active-site

Cys was mutated to Ala in the nicotinamidase from M.

tuberculosis.^53,54

was within the range of 6−10 μM obtained from previous

reports on yeast nicotinamidases.^30,47Substitution of the

nicotinamide pyridine ring at position 4 with nitrogen

(pyrazinamide) or addition of a methyl group to position 5

resulted in 3.7- or 2.5-fold faster k_catvalues but lower k_cat/K_m

values due to 16- or 6-fold higher K_mvalues, respectively. The

K_mvalue of 157 μM determined for pyrazinamide was similar

to previously determined values for nicotinamidases from S.

cerevisiae (K_m= 200 μM)²and M. tuberculosis (K_m= 300

μM).³³The K_mvalue of 61 μM determined for 5-

methylnicotinamide was 5-fold lower than a previously

determined value of 360 μM for a nicotinamidase from Mi.

lysodeikticus.⁴⁶Perturbation of the pyridine nitrogen of

nicotinamide by methylation (1-methylnicotinamide) or

Kinetic Parameters of Nicotinamide Analogues as

Alternate Substrates. Kinetic values for the reaction using

substrate analogues produced a wide variation in k_cat/K_mvalues

(Table 4). The K_mvalue of 9.6 μM observed for nicotinamide

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a

Table 3. Steady-State Kinetic Parameters and ¹⁵N and ¹³C KIEs for the Reaction Catalyzed by Pnc1 Mutants

b

−1

k_cat(s⁻¹)

K_m(μM)

k_cat/K_m(M⁻¹s )

¹⁵N KIE

¹³C KIE

wild type

D8A

0.69 0.04

0.0009 0.0001

0.0006 0.0001

0.0070 0.0001

0.020 0.005

0.064 0.012

0.014 0.002

0.020 0.002

0.044 0.023

0.0009 0.0002

<0.0005

9.6 2.1

(7.2 1.2) × 10⁴

1.0122 0.0002

1.0125 0.0004

c

d

NA

ND

c

d

D8N

NA

ND

c

D8E

NA

1.0218 0.0015

1.0166 0.0018

1.0047 0.0006

1.0258 0.0012

1.0075 0.0015

1.0058 0.0006

D51A

D51N

H53A

H94A

K122A

K122R

C167A

<2.4

>8.3 × 10³

>3.5 × 10⁴

<1.8

d

6.5 3.6

<2.7

(2.2 0.9) × 10³

>7.4 × 10³

ND

d

ND

d

<6.5

>6.7 × 10³

ND

c

NA

1.0127 0.0003

1.0172 0.0001

c

d

NA

ND

a

b

Assays were performed as described in Experimental Procedures. All reactions were performed at pH 7.5. k_catand K_mvalues were determined from

c

fitting the data to the Michaelis−Menten equation using Kaleidagraph (Synergy Software). Errors represent the error of the fit to the data. Not

available. Measured rates were too slow for accurate K_mvalues to be determined. Not determined.

d

e

Table 4. Steady-State Kinetic Parameters and ¹⁵N and ¹³C KIEs for the Reaction Catalyzed by Pnc1 with Alternate Substrates

a

k_catand K_mvalues were determined from fitting the data to the Michaelis−Menten equation using Kaleidagraph (Synergy Software). Errors

b

c

represent the error of the fit to the data. Saturation was not obtained; therefore, only k_cat/K_mvalues are reported. No activity was observed above

d

e

the detection limit of the assay, 10⁻⁵s⁻¹. Not determined. Assays were performed as described in Experimental Procedures at pH 7.5.

substitution with carbon (benzamide) resulted in a 8400- or

200-fold decreased k_cat/K_mvalue, respectively, compared to that

of nicotinamide. The activity observed for 1-methylnicotina-

mide was in contrast to the observation of French et al., who

found no activity with S. cerevisiae Pnc1.³⁰We were unable to

accurately determine the k_catvalue for 1-methylnicotinamide as

saturation could not be obtained, but benzamide resulted in a

78-fold lower k_catvalue compared to that of nicotinamide.

Substitution of the amide oxygen of nicotinamide with a sulfur

(thionicotinamide) resulted in a 1400-fold lower k_cat/K_mvalue.

Finally, no activity could be detected above the detection limit

of the HPLC assay (10⁻⁵s⁻¹) for nicotinamide mononucleotide

(NMN⁺) or NAD⁺.

¹⁵N and ¹³C Kinetic Isotope Effects with Substrate

Analogues. We next investigated the first portion of the

nicotinamidase mechanism up to and including the first

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irreversible step (C−N bond cleavage) using kinetic isotope

effects (KIEs). Using slow substrates with Pnc1 allowed us to

determine if and when C−N bond cleavage is rate-limiting for

catalysis. The ¹⁵N and ¹³C KIE values were determined for

Pnc1 with nicotinamide and three substrate analogues: 5-

methylnicotinamide, pyrazinamide, and thionicotinamide

(Table 4). With nicotinamide as the substrate, the ¹⁵N and

¹³C isotope effects were ∼1.2%. Because the upper theoretical

limit for ¹⁵N and ¹³C effects is 3−4%, these primary effects

indicate that C−N bond cleavage is at least partially rate-

limiting. When 5-methylnicotinamide, pyrazinamide, or thio-

nicotinamide was utilized as the substrate, all of the KIEs are

more fully expressed than those with nicotinamide (Table 4).

Also, in a comparison of the ¹⁵N effects versus the k_cat/K_mdata,

there is good correlation between the kinetic data and the KIE

showing that the slower substrates exhibit higher primary KIEs.

The trend is not as well-defined for the ¹³C data, but the KIE

for 5-methylnicotinamide is roughly the same as that of

pyrazinamide when the standard error is taken into

consideration.

¹⁵N and ¹³C Kinetic Isotope Effects of Pnc1 Mutants.

The same KIEs determined with the substrate analogues were

also determined for four Pnc1 mutants, D8E, D51N, D51A, and

K122R, with nicotinamide as the substrate (Table 3). These

Pnc1 mutants were chosen because of the differences observed

in the determined kinetic parameters and their ability to achieve

adequate product conversion to determine KIEs. These KIEs

permitted us to investigate whether the mutated residues are

responsible for the actions discussed here and by

others.^{30,31,33,52,55}For the Zn²⁺ligation mutant, D51N, the

KIE values were both lower than that for WT Pnc1, whereas a

higher ¹⁵N KIE and a lower ¹³C KIE were observed for D51A

compared to those of WT Pnc1. For the K122R mutant, the

¹⁵N KIE was exactly the same as that determined for the WT

enzyme while the ¹³C effect is only ∼0.4% higher. Finally, the

D8E mutant displayed increased KIEs that were very close to

the maximum observed with the substrate analogues.

Figure 2. pH−rate profiles. Reaction rates were determined as

described in Experimental Procedures. (A) Effect of pH on the k_catof

Pnc1-catalyzed hydrolysis of nicotinamide. (B) Effect of pH on the k_cat

of Pnc1-catalyzed hydrolysis of pyrazinamide.

pH Dependence of Kinetic Parameters. Several residues

within the Pnc1 active site may require a particular ionization

state for catalysis. Within Pnc1, these include D8, K122, and

C167. To provide evidence of the involvement of these residues

during catalysis, we determined the effect of pH on the k_cat

values of Pnc1. First, the stability of Pnc1 at pH extremes was

determined to ensure any changes in rate were caused by the

protonation states of active-site residues and not global protein

unfolding. Determination of Pnc1 k_catvalues with saturating

nicotinamide over a pH range of 4.5−10.5 showed no critical

ionizations but did show a slight pH-dependent rate change

that varied only 4-fold between the pH extremes (0.45 s⁻¹at

low pH and 1.87 s⁻¹at high pH) (Figure 2A). Similarly, the

k_cat/K_mprofile did not reveal any critical ionizations (data not

shown). The lack of a critical ionization using nicotinamide as

the substrate was consistent with previous reports on

nicotinamidases from T. cremoris and M. tuberculosis.^33,50

The higher KIE values and slower k_cat/K_mvalues for

pyrazinamide compared to those for nicotinamide (Table 4)

indicated that C−N bond cleavage was only partially rate

limiting for nicotinamide but nearly fully rate limiting for

pyrazinamide. Therefore, we hypothesized that the pH profile

for pyrazinamide would display critical ionizations involved in

C−N bond cleavage. Indeed, Pnc1 k_catvalues for pyrazinamide

revealed two critical ionizations over a pH range of 4 −10, one

unprotonated for activity and another with an apparent pK_b

value of 9.0 0.2 that must be protonated for activity (Figure

2B). The residues responsible for these ionizations are

discussed below.

DISCUSSION

■

Kinetic Mechanism of Nicotinamidase. Although several

nicotinamidase chemical mechanisms have been proposed,³⁰⁻³³

the kinetic mechanism had not been investigated in detail.

Here, we determined that Pnc1 follows an ordered uni-bi

kinetic mechanism (Figure 3) in which nicotinamide binding is

Figure 3. Proposed kinetic mechanism of the nicotinamidase reaction.

E represents the unmodified Pnc1 enzyme, F the covalent thioester

intermediate between nicotinic acid and C167, NAM nicotinamide,

and NA nicotinic acid.

followed by formation of a thioester intermediate between

C167 and nicotinic acid. Evidence supporting this thioester

with an apparent pK_avalue of 5.1

0.2 that must be

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Figure 4. Putative route of access of nicotinamide to the Pnc1 active site. (A) A narrow tunnel exists in the Pnc1 structure from the active site to the

protein surface. This tunnel was discovered using Caver.^66,67(B) The C-terminal loop and helix of the nicotinaldehyde−Pnc1 structure make up the

most dynamic portion of the protein. All seven Pnc1 monomers in the asymmetric unit were aligned and represented using the B factor putty preset

of Pymol.⁶⁸The range of B factors in the structure are represented as thin (low B factor) to thick (high B factor) main chains and a color gradient

from blue (low B factor) to red (high B factor). (C) Alignment of all published nicotinamidase structures reveals that the C-terminal loop and helix

containing I192 make up the most dynamic portion of the structures. The nicotinamidases shown are from Pyrococcus horikoshii (PDB entry 1IM5,

yellow), M. tuberculosis (PDB entry 3PL1, orange), St. pneumoniae (PDB entry 3O91, blue), Acinetobacter baumannii (PDB entry 2WT9, pink), and

S. cerevisiae (this work, green).

intermediate is discussed below. Ammonia is released, and then

the thioester intermediate is hydrolyzed to form nicotinic acid,

which is then released. The product inhibition pattern expected

for an ordered uni-bi mechanism is one in which the second

product released acts as a competitive inhibitor versus

nicotinamide and the first product released acts as a

noncompetitive inhibitor versus nicotinamide.⁵⁶Therefore,

nicotinic acid is the second product released on the basis of

competitive inhibition patterns displayed versus nicotinamide

(Figure S1 of the Supporting Information). The non-

competitive inhibition displayed by ammonium chloride is

also consistent with ammonia being the first product released,

but we were unable to confirm that the inhibition was specific

to the Pnc1 active site or that the inhibition was caused by NH₃

2.6 Å. This Zn²⁺ligation is important for binding and/or

activating nicotinamide for catalysis as mutation of D51, H53,

or H94 decreased the k_catvalue 11−49-fold. The importance of

the Zn²⁺ligation is further shown by the alternate substrate

benzamide for which the pyridyl nitrogen is replaced with a

carbon. Using benzamide as an alternate substrate reduced the

k_cat/K_mvalue 200-fold compared to that of nicotinamide (Table

4). This rate difference can partially be explained by the

observed 7-fold slower pseudo-first-order rate of benzamide

hydrolysis in basic solutions compared to nicotinamide

(0.00408 h⁻¹vs 0.0301 h⁻¹at 25 °C in 0.1 N NaOH).⁵⁷In

addition, benzaldehyde was 22000-fold less potent as an

inhibitor than nicotinaldehyde (Table 1), indicating that the

pyridyl nitrogen plays a significant role in substrate affinity

through Zn²⁺ligation. Besides nicotinamide binding, the Zn²⁺

also acts as a Lewis acid to activate nicotinamide toward

hydrolysis. Interestingly, this method of Lewis acid catalysis

appears to be unique among zinc hydrolases, which typically

facilitate hydrolysis through binding to the carbonyl oxygen of

the substrate.^58,59In nicotinamidases, the Zn²⁺instead acts as a

“vinylogous Lewis acid” activating the amide carbon for

nucleophilic attack by C167 through the electron withdrawing

character of Zn²⁺, which acts through the conjugation of the

pyridine ring instead of directly at the amide oxygen.

+

rather than NH₄.

Nicotinamide Binding May Proceed through a Hydro-

phobic Tunnel. The Pnc1 active site is largely sequestered

from solvent. However, a small tunnel exists from the active site

to the protein surface that is constricted by L20, Y70, and I192

(Figure 4A). I192 appears to be especially important in the

opening and closing of this tunnel as I192 resides on a dynamic

C-terminal loop in Pnc1. The dynamic nature of the C-terminal

loop and helix is exemplified by the high B factors of this region

in the crystal structure (Figure 4B). Furthermore, the C-

terminal helix displayed greater variance than any other portion

of the structure when aligned with previous nicotinamidase

structures^31,52,53,55(Figure 4C). However, Pnc1 does not

undergo significant conformational changes outside of this C-

terminal loop and helix upon nicotinamide binding as shown by

the low rmsd values (0.23−0.38 Å) between the apo³²and

nicotinaldehyde-bound Pnc1 structures. Once nicotinamide

proceeds through this tunnel, binding is stabilized within the

active site through edge−face aromatic interactions between the

aromatic cage residues F13, W91, Y131, and Y166 and the

pyridine ring of nicotinamide (Figure 1B).

The Zn²⁺ligand mutants also displayed the most interesting

KIE results. For D51N Pnc1, both the ¹⁵N and ¹³C KIEs are

∼0.5% compared to ∼1.25% for WT Pnc1 (Table 3). We

believe this is due to an increase in one of the back reaction off

rates. A truncated derivation demonstrating how this is possible

is included in the Supporting Information. Another intriguing

KIE result is that the Zn²⁺ligand mutant, D51A, has a ¹⁵N KIE

that is larger than that of the wild type, while the ¹³C KIE is

smaller. One feasible explanation is that the carbon is bonded

more stiffly than the nitrogen at the transition state in the

D51A mutant. This may be due to the inability of the alanine

residue in the mutant enzyme to be liganded to the Zn²⁺, which

affects the position of the Zn²⁺and nicotinamide substrate in

the active site compared to those of the wild type. If true, the

orientation of the substrate has changed before the nucleophilic

attack by C167, possibly affecting the stiffness of the bond from

Zinc Is Involved in Binding and Activation of

Substrate. The Zn²⁺ligated by D51, H53, and H94 also

stabilizes nicotinamide binding within the active site. Our

structure of nicotinaldehyde bound to Pnc1 revealed that the

pyridyl nitrogen is ligated to the active-site Zn²⁺at a distance of

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carbon to nitrogen in the transition state (Scheme 3). One final

possibility for the difference in KIE for the D51N and D51A

nicotinamidase from M. tuberculosis.³³The lowered pK_aof

C167 is likely due to the positively charged K122 ε-amino

group being within 3.1 Å of the C167 sulfur. The group that

must be protonated (pK_a∼ 9) may reflect the requirement for

protonated K122. The precise positioning of the K122 ε-amine

within the Pnc1 active site is critical as both the Pnc1 K122A

and K122R mutants displayed significantly diminished k_cat

values 16- and 767-fold below that of WT Pnc1, respectively.

For the K122R mutant, the ¹⁵N KIE is exactly the same as that

determined for WT Pnc1 while the ¹³C effect is only ∼0.4%

higher. This may reflect a classic case of nonproductive

substrate binding, which affects the k_catbut not the k_cat/K_m

value. Steric interference between arginine and nicotinamide

most likely increases the amount of nonproductive substrate

binding. However, once the substrate achieves the proper near

attack conformation, the reaction proceeds normally, resulting

in a minimal increase in the KIE. Another possibility is that the

positively charged K122 might lower the pK_avalues of D8 and

C167.^31,33If so, the KIEs of K122R suggest that the positive

charge of arginine is sufficient to maintain the lowered pK_a

values of D8 and C167 in the transition state. If not, the KIE

would be expected to increase because an increased pK_avalue

of either residue would slow proton abstraction and thus C−N

bond cleavage.

Within the Pnc1 active site, D8 is likely the base that initiates

catalysis by abstracting the proton from C167, leading to the

attack at the nicotinamide amide carbon.^30,31,33,52The Pnc1

D8A, D8N, and D8E mutants revealed the importance of both

the charge and positioning of D8 within the Pnc1 active site.

Removal of the charge (D8A and D8N) resulted in 767- and

1150-fold reductions in k_cat, respectively, compared to that of

WT Pnc1. Repositioning the negative charge (D8E) increased

both the ¹⁵N KIE and the ¹³C KIE to very close to the

maximum value [∼2.7% for ¹⁵N (Table 3)] observed with the

substrate analogues and resulted in a 100-fold reduction in k_cat.

Therefore, the extra methylene group of glutamate likely pushes

the carboxyl group out of position to substantially slow proton

abstraction, resulting in slower C−N bond cleavage and the

fullest expression of the KIEs. Finally, nucleophilic attack by

C167 is further favored by the presence of a putative oxyanion

hole (Figure 1B) originally proposed by Fyfe et al.³¹consisting

of the amide backbone nitrogens of the conserved A163 and

invariant C167 (Figure S2 of the Supporting Information).

These nitrogens form hydrogen bonds to the oxygen of the

trapped thiohemiacetal adduct in the structure of nicotinalde-

hyde-inhibited Pnc1.

Scheme 3. Possible Transition States Depicting a More

Stiffly Bound Carbon (lower KIE) and a Less Stiffly Bound

Nitrogen (higher KIE) in the D51A Mutant Compared to

WT

mutants compared to the wild type is the change in charge at

the active site upon mutation of a negatively charged aspartate

residue to the neutral asparagine or alanine. There is

precedence for the requirement of certain charges in and

around the active site for proper substrate binding and

catalysis.⁶⁰With Pnc1, it is unclear how loss of a negative

charge may cause changes in the KIE and kinetic data, but it

may be due to the same factors discussed with the pH profiles.

C167 Is Activated by Active-Site Residues of Pnc1.

Our Pnc1 structure suggested D8, K122, and C167 form a

catalytic triad, which resembles the catalytic triad of the nitrilase

superfamily.⁶¹These three residues are universally conserved

among all nicotinamidases (Figure S2 of the Supporting

Information), and an active-site Cys residue (C167 in Pnc1)

was previously proposed to act as a nucleophile during

catalysis,^{30,31,46,47,50−52,55}attacking the carbonyl carbon of

nicotinamide to form a thioester intermediate. Here, we

present several lines of evidence that are highly consistent

with this nucleophilic role of C167. First, nicotinamide

analogue aldehydes and ketones are competitive inhibitors

versus nicotinamide. Although these aldehydes and ketones are

predicted to form covalent adducts with C167, classical

competitive inhibition patterns and no time-dependent loss of

activity are expected as the reaction with C167 is completely

reversible^49,50,62and infinite concentrations of nicotinamide

would preclude binding of the inhibitor to Pnc1. Nicotinalde-

hyde is a particularly potent inhibitor (K_ivalue of 940 nM), and

the weaker inhibition of 3-acetylpyridine (K_i= 316 μM) is

consistent with the greater reactivity of aldehydes versus

ketones.⁶³We cocrystallized nicotinaldehyde with Pnc1, and

continuous electron density was observed between C167 and

nicotinaldehyde (Figure 1A), indicating a covalent thiohemia-

cetal adduct was present in the crystals. The importance of

C167 was confirmed through mutagenesis as the Pnc1 C167A

mutant lost all detectable nicotinamidase activity (Table 3).

The Pnc1 pH profile using pyrazinamide as a substrate

revealed a critical ionization with an apparent pK_avalue of 5.1

that must be unprotonated and a critical ionization with an

apparent pK_avalue of 9.0 that must be protonated for activity

(Figure 3B). The group that must be unprotonated for activity

might reflect C167, consistent with a previously determined

pK_aof 6.6 for alkylation of cysteine by iodoacetamide with the

Structure−Activity Relationships and Rate-Limiting

Steps of Nicotinamide Analogues. The pH dependence of

nicotinamide hydrolysis displayed no critical ionizations over

the range of 4.5−10.5 (Figure 2A), indicating a step other than

C167 nucleophilic attack and C−N bond cleavage is rate-

limiting for nicotinamide, such as tetrahedral intermediate

breakdown or product release. In addition, the KIEs

determined for nicotinamide were significantly lower than the

maximum observed for the substrate analogues, indicating that

C−N bond cleavage is only partially rate limiting with respect

to the k_cat/K_mvalue. The two critical features of proper

nicotinamide binding and orientation are the ring nitrogen,

which is liganded to the zinc atom, and the nicotinamide

carbonyl oxygen, which is held in place by backbone amide

interactions. Replacing the oxygen with sulfur (i.e., thionicoti-

namide) leads to a >10³-fold decrease in k_cat/K_mand a large

1.4% increase in the ¹⁵N KIE value compared to that of

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Scheme 4. Proposed Chemical Mechanism of Pnc1

nicotinamide. Therefore, the overall rate-limiting step likely

shifts from product release for nicotinamide to C−N bond

cleavage for thionicotinamide. Oxygen is more electronegative

(3.44) than sulfur (2.58) (Pauling Scale),⁶⁴which can affect the

reaction in two possible ways. First, the hydrogen bonding is

weaker for a thionyl group than a carbonyl, leading to a more

loosely bound substrate (i.e., the interaction with the backbone

amides of A163 and C167). Second, the decrease in

electronegativity directly impacts the electrophilicity of the

thionyl amide carbon, thus diminishing its reactivity when

attacked by C167. In addition, the larger size of sulfur and the

longer bond length of CS versus CO bonds may also

sterically clash with the enzyme and result in nonoptimal

substrate positioning. Any of these reasons would lead to a

decreased forward commitment factor of the reaction and thus

an increase in the observed ¹⁵N and ¹³C KIEs.

Addition of a methyl group on the ring nitrogen (i.e., 1-

methylnicotinamide) and replacement of N1 with carbon (i.e.,

benzamide) both block the interaction with the bound zinc

atom, resulting in a dramatic decrease in k_catand k_cat/K_mvalues

(see above). Methylation at the C5 position (i.e., 5-

methylnicotinamide) is tolerated, yielding a substrate that

displays modest effects on k_cat/K_mvalues but not on k_catvalues

compared to those of nicotinamide. The higher KIE values for

5-methylnicotinamide (and all the analogues tested) compared

to that of nicotinamide indicated that 5-methylnicotinamide is

not as “sticky” as nicotinamide (i.e., the ratio of substrate

dissociation to forward reaction is greater for the analogues).

Thus, C−N bond cleavage is almost entirely rate limiting in

regard to the k_cat/K_mvalue. Therefore, the extra methyl group

of 5-methylnicotinamide is most likely sterically interacting

with a nearby residue (possibly Y166), hindering achievement

of the proper orientation for optimal catalysis. However, the

steric hindrance of the methyl group of 5-methylnicotinamide

also results in a more loosely bound substrate (higher K_m) and

a higher k_catvalue likely because of faster product release.

Therefore, 5-methylnicotinic acid release, not C−N bond

breaking, is the overall rate-limiting step reflected in the k_cat

value. Consistent with the high specificity of nicotinamidases

toward nicotinamide, NAD⁺salvage pathway metabolites

NMN⁺and NAD⁺are not substrates.

Pyrazinamide displayed a 4.5-fold lower k_cat/K_mvalue than

nicotinamide but a 3.7-fold higher k_catvalue (Table 4). The size

and geometry of the pyrazine ring are nearly identical to those

of the pyridine ring of nicotinamide, so the additional nitrogen

in the pyrazine ring slows the binding and chemical steps

reflected in the k_cat/K_mvalue (substrate binding, nucleophilic

attack of C167, and release of ammonia) through inductive

effects or unfavorable interaction of the unshared pair of

electrons with nearby aromatic residues involved in substrate

binding and positioning, leading to a more loosely bound

substrate. However, the higher k_catvalue indicates that

pyrazinamide reacts faster in the chemical steps after release

of ammonia such as thioester hydrolysis and pyrazinoic acid

release. Indeed, the 56-fold higher K_ivalue of pyrazinoic acid

compared to that of nicotinic acid (Table 1) is consistent with a

higher off rate constant for pyrazinoic acid and an overall rate

enhancement due to an increased rate of rate-limiting product

release. The 4.5-fold lower k_cat/K_mvalue for pyrazinamide is

due to slower and nearly fully rate limiting amide C−N bond

cleavage as revealed by the higher ¹⁵N and ¹³C KIE values of

2.3 and 2.0%, respectively, determined for pyrazinamide,

compared to values of 1.2 and 1.3%, respectively, determined

for nicotinamide.

Chemical Mechanism of Nicotinamidase. Previous

nicotinamidase studies indicated that there are three residues

that directly chelate the Zn²⁺at the active site (D51, H53, and

H94 in Pnc1) and three that are involved as a catalytic triad

(C167, D8, and K122 in Pnc1).^31,32,55Here, our data suggest

specific roles for each of these residues and other active-site

residues during catalysis (Scheme 4). In the first step of the

mechanism, the substrate binds at the active site, replacing one

of two equatorial water molecules ligated to the Zn²⁺, and is

stabilized by edge−face interactions with the aromatic residues

F13, W91, Y131, and Y166. The nitrogen of the pyridine ring is

liganded to the Zn²⁺at the active site, and the proton is

removed from C167 by D8, forming a thiolate that then attacks

the amide carbonyl carbon creating a tetrahedral intermediate.

The recently abstracted proton is transferred to the amino

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Biochemistry

Article

portion of the amide, which is then ejected as NH₃as the

intermediate collapses and the C−N bond is broken. Although

this final step before C−N scission is shown as concerted in

Scheme 4, proton transfer and C−N cleavage may be in fact

stepwise.

AUTHOR INFORMATION

Corresponding Author

*W.W.C.: e-mail, cleland@enzyme.wisc.edu; phone, (608) 262-

1373; fax, (608) 265-2904. J.M.D.: e-mail, jmdenu@wisc.edu;

phone, (608) 265-1859; fax, (608) 262-5253.

■

At the next stage of the mechanism, there is some

disagreement about how the reaction moves forward. It has

been hypothesized that the Zn²⁺-ligated water is in the proper

position to attack the thiol ester,⁵²but our crystal structure

indicates the water is too far (∼5−7 Å) from the carbonyl

carbon to be effective without a significant conformational

rearrangement or dissociation from the Zn²⁺. Another option is

that the Zn²⁺-ligated water is deprotonated and can then

deprotonate a bulk solvent water molecule that attacks the thiol

ester.³⁰The final possibility is that a bulk solvent water

molecule is deprotonated by D8 and then acts as the

nucleophile, re-forming a tetrahedral intermediate.^31,33We

favor the third option as D8 is in the proper position to

accomplish this deprotonation. Finally, the intermediate

collapses to form nicotinic acid; the cysteine thiolate abstracts

the proton from D8, and the catalytic cycle is complete.

It is possible that if the thiol pK_ain Pnc1 is lowered to the

same level as in PncA (pK_a= 6.6),³³presumably by the

interaction with the positive charge of K122, then under the

conditions of our experiments (pH 7.5) ∼90% of the cysteine

would exist in its thiolate form and thus would not require

deprotonation by D8. Accordingly, D8 would be involved in

only the hydrolytic portion of the reaction, removing a proton

from a water molecule and re-forming the tetrahedral

intermediate. The glutamate in the D8E mutant is out of

position relative to the substrate for forming the tetrahedral

intermediate, thus retarding the reaction so that the KIE is fully

expressed in the D8E mutant because the forward commitment

has become very small.

Present Address

^§California Institute for Quantitative Biosciences, University of

California, Berkeley, CA 94720-3220.

Author Contributions

B.C.S. and M.A.A. contributed equally to this work.

Funding

This work was supported by National Institutes of Health

(NIH) Grants GM065386 (to J.M.D.) and GM18938 (to

W.W.C.), by NIH Biotechnology Training Grant NIH 5 T32

GM08349 (to B.C.S.), and by NIH Training Grant in

Molecular Biosciences GM07215 (to K.A.H.).

ACKNOWLEDGMENTS

■

We thank the Advanced Photon Source staff (LS-CAT

beamline) and Katrina Forest for assistance with data collection

and Ken Satyshur and Nick George for help with structure

determination. We also thank Dr. Tonya Zeczycki for her

helpful comments and critique of the manuscript.

ABBREVIATIONS

■

EDTA, ethylenediaminetetraacetic acid; KIE, kinetic isotope

effect; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic

acid; HPLC, high-performance liquid chromatography; IRMS,

isotope ratio mass spectrometry; NAD⁺, β-nicotinamide

adenine dinucleotide; NADPH, β-nicotinamide adenine

dinucleotide 2′-phosphate, reduced form; NMN⁺, nicotinamide

mononucleotide; Pnc1, nicotinamidase from S. cerevisiae; WT,

wild type.

ADDITIONAL NOTE

All amino acid numbering used is based on the Pnc1 S.

cerevisiae nicotinamidase sequence.

■

a

CONCLUSION

■

This detailed characterization of a eukaryotic nicotinamidase

provides critical information about the structure and mecha-

nism of nicotinamidases. In particular, this work provides a

fundamental understanding for the development of mechanism-

based inhibitors and prodrugs that target nicotinamidases to

treat fungal, bacterial, or parasite infections. In addition, results

like those reported here, along with theoretical calculations of

transition-state bond lengths, have been used by others to

design excellent transition-state inhibitors for other enzymes.⁶⁵

Similarly, we anticipate that these findings can lead to the

development of nicotinamidase inhibitors for treating infections

in which nicotinamidase activity is critical for the viability or the

infectious phenotype.

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* Supporting Information

Derivation to explain the KIE observed for D51N Pnc1, table of

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