Mechanism of molybdate insertion into pterin-based molybdenum cofactors.

Mechanism of molybdate insertion into pterin-based

molybdenum cofactors.

Corinna Probst

Jing Yang

Joern Krausze

Thomas W. Hercher

Casseday P.

Richers

Thomas Spatzal

KC Khadanand

Logan J. Giles

Douglas C. Rees

Ralf R.

Mendel

Martin L. Kirk

2,#

Tobias Kruse

1,#

TU Braunschweig, Institute of Plant Biology, 38106 Braunschweig

Department of Chemistry and Chemical Biology, The University of New Mexico, 1 University of

New Mexico, MSC03 2060, Albuquerque, NM 87131

Division of Chemistry and Chemical Engineering 114-96, Howard Hughes Medical Institute,

California Institute of Technology, Pasadena, CA 91125 USA

These authors contributed equally to this work.

Abstract

The molybdenum cofactor (Moco) is found in the active site of numerous important enzymes that

are critical to biological processes. The bidentate ligand that chelates molybdenum (Mo) in Moco

is the pyranopterin dithiolene (molybdopterin, MPT); however, neither the mechanism of

molybdate insertion into MPT nor the structure of Moco prior to its insertion into pyranopterin

molybdenum enzymes is known. Here we report this final maturation step, where adenylated MPT

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To whom correspondence should be addressed: T. Kruse, TU Braunschweig, Institute of Plant Biology, Spielmannstrasse 7, 38106

Braunschweig, Germany. +49-531-3915873, Fax: +49-531-3918128; t.kruse@tu-bs.de; M. L. Kirk, Department of Chemistry and

Chemical Biology, The University of New Mexico, 1 University of New Mexico, MSC03 2060, Albuquerque, NM 87131, USA.

+1-505-277-5992; mkirk@unm.edu.

These authors share senior corresponding authorship

AUTHOR CONTRIBUTIONS

Corinna Probst:

Acquisition, analysis and interpretation of data

Jing Yang:

Acquisition, analysis and interpretation of XAS data; computed reaction coordinate

Joern Krausze:

Analysis and interpretation of data

Thomas W. Hercher:

Acquisition, analysis and interpretation of data

Casseday P. Richers:

Synthesized and characterized the trioxo- and dioxo- molybdenum model compounds (

, and

) and analyzed

spectroscopic data

Thomas Spatzal:

Acquisition, analysis and interpretation of data

Khadanand KC

: Assisted in the collection of XAS data

Logan J. Giles:

Assisted in the collection of XAS data

Douglas C. Rees:

Revision of the article

Ralf R. Mendel:

Revision of the article

Martin L. Kirk:

Conception and design analysis and interpretation of data, drafting the article

Tobias Kruse:

Conception and design, analysis and interpretation of data, drafting the article

C. P.

J. Y

., and

J. K.

contributed equally to this work. All authors discussed the results and commented on the manuscript.

COMPETING INTERESTS STATEMENT

The authors declare no competing interests.

SUPPLEMENTARY

Includes details of experimental data, procedural details, synthesis and characterization data, NMR spectra, the X-ray absorption

spectroscopy (XAS) experiment, XAS data analysis, and electronic structure computations. Supplementary figures and tables are

freely accessible on

nature.com

HHS Public Access

Author manuscript

Nat Chem

. Author manuscript; available in PMC 2021 December 28.

Published in final edited form as:

Nat Chem

. 2021 August ; 13(8): 758–765. doi:10.1038/s41557-021-00714-1.

Author Manuscript

(MPT-AMP) and molybdate are the substrates. X-ray crystallography of the Arabidopsis thaliana

Mo-insertase variant Cnx1E S269D D274S identified adenylated Moco (Moco-AMP) as an

unexpected intermediate in this reaction sequence. X-ray absorption spectroscopy revealed the first

coordination sphere geometry of Moco trapped in the Cnx1E active site. We have used this

structural information to deduce a mechanism for molybdate insertion into MPT-AMP. Given their

high degree of structural and sequence similarity, we suggest that this mechanism is employed by

all eukaryotic Mo-insertases.

Graphical Abstract

Keywords

Molybdenum cofactor; Moco; Moco biosynthesis; Molybdenum insertion; X-ray crystallography;

X-ray absorption spectroscopy; EXAFS

Molybdenum cofactor (Moco) dependent enzymes (Mo-enzymes) are ubiquitous, and are

commonly found in archaea, bacteria, and eukaryota

. These enzymes are involved in

numerous redox reactions that contribute to the global sulfur, carbon and nitrogen cycles

and they are vitally important to a variety of life processes

. This last point is underscored

by the fact that specific Moco dependent enzymes are essential to human life. A lack of

sulfite oxidase (SOX) activity in humans results in the accumulation of highly toxic sulfite,

which subsequently leads to the onset of severe neurological disorders and eventual early

childhood death

. Regarding their global importance, the Moco dependent nitrate reductases

(NR) are essential enzymes in autotrophic organisms such as plants, and defects in the NR

structural gene, or within the Moco biosynthesis pathway, cause severe growth

phenotypes

. Moco biosynthesis is catalyzed by an evolutionarily old and highly

conserved multi-step pathway

. Consequently, this pathway is found in the last universal

common ancestor of all life forms (LUCA) where Moco-dependent enzymes likely

functioned to assimilate carbon, catalyze oxygen independent hydroxylation reactions, and

contribute to energy metabolism

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Moco biosynthesis is subdivided into four major steps (Figure 1)

. In the first step of the

pathway, GTP is converted to 3’,8-cH

GTP

, which is subsequently processed to yield

cyclic pyranopterin monophosphate (cPMP

). Work with the model plant

Arabidopsis

thaliana

(

A. thaliana

) showed that cPMP synthesis takes place in the mitochondria, and an

ABC type exporter is required for cPMP export into the cytosol of the cell

. Given the high

degree of functional conservation, this is assumed to be valid for all eukaryotes

. In the

second step of the pathway, cPMP is converted into molybdopterin (MPT). This reaction is

characterized by the sequential introduction of the MPT dithiolene sulfurs

, where after

each reaction cycle the MPT-synthase (precisely its small subunits) have to be re-

sulfurated

. Work on the first and second steps of the Moco biosynthesis pathway were

primarily performed using

E. coli

enzymes, which serve as model enzymes here

Elucidation of the third and fourth steps in the pathway primarily involved the

A. thaliana

molybdenum insertase (Mo-insertase) Cnx1 serving as a model enzyme

. Early work with

fungal Mo-insertase mutants identified these to be functionally rescued when grown in the

presence of millimolar concentrations of sodium molybdate

, rendering molybdate

complexation by the MPT dithiolene to be independent from an enzyme catalyst under

conditions where excess molybdate is available. In the third step, MPT is adenylated by the

Mo-insertase G-domain to yield adenylated MPT (MPT-AMP). MPT-AMP is a substrate of

the Mo-insertase E-domain, which catalyzes the insertion of molybdate into the MPT

dithiolene

. Early

and more recent

work identified this reaction to depend on a

critical subset of steps that must occur prior to the actual molybdate insertion reaction. In

our more recent work, we identified Cnx1E to possess two mutually exclusive molybdate

binding sites, which we assigned as functioning in molybdate binding and insertion,

respectively

. These molybdate binding sites are in close proximity to what we previously

proposed to be the dithiolene chelate component of bound MPT-AMP

. However, a

critical question remains regarding the nature of the individual steps and the energetics that

define the reaction coordinate for molybdate insertion into MPT-AMP. Recently, a structural

analysis of the GephE ADP complex suggested that the active site residue Asp580 is

required for functionality, while earlier genetic work identified the homologous aspartate

residue in the fungal Mo-insertase CnxE (

A. nidulans

) to be vitally important.

Furthermore, the corresponding Cnx1 exchange variant (D274E) was consistently found to

be catalytically inactive

In this work, we describe the identification of an unexpected Moco precursor

viz.

adenylated

molybdenum cofactor (Moco-AMP), which provides detailed insight into the underlying

mechanism of molybdate insertion into the MPT-AMP dithiolene. Remarkably, ATP does

not appear to be required as an energy transfer molecule for the actual molybdate insertion

process, but appears to be required for the positioning of MPT within the Cnx1E active site.

Although the Cnx1E co-structure has been solved here to high resolution (PDB: 6Q32), the

precise nature of the Moco-AMP molybdenum coordination environment could not be

determined. To address this critical structural issue and provide much-needed insight into the

insertion mechanism, we performed a detailed Mo K-edge X-ray absorption spectroscopic

(XAS) study of the Cnx1E Moco-AMP complex. The Cnx1E Moco-AMP X-ray absorption

near-edge structure (XANES) data have been analyzed in the context of XANES data from

small molecule structural analogs of the Moco-AMP Mo site. This comparative analysis

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yielded the discovery of a dioxo-Mo structure for Cnx1E bound Moco-AMP. This structure

is further revealed to be a Mo(VI) [(MPT)MoO

(OH)]

1−

site by extended X-ray absorption

fine structure (EXAFS) experiments, and represents a unique structure observed for any

Moco-dependent pyranopterin Mo enzyme

. Defining the nature of the Mo coordination

geometry has finally allowed us to deduce the mechanism for molybdate insertion into MPT-

AMP; a process that underlies Moco formation. We have thus characterized the Mo-insertase

model enzyme Cnx1E from the plant

A. thaliana

and suggest our proposed mechanism to be

valid for all eukaryotic Mo-insertases, since they are both structurally and functionally

conserved.

Results and Discussion

X-ray crystallography.

The Cnx1E active site loop

was targeted by structure-guided mutagenesis experiments to

yield the substrate accumulating Cnx1E variant S269D D274S (Supplementary Fig. 1 and

2), the structure of which has been determined by X-ray crystallography to 1.51 Å resolution

(PDB: 6Q32; R

free

=0.176; Supplementary Table 1) and is reported here. Within the enzyme-

substrate complex, MPT-AMP is bound in a shallow cavity that is located at the interface

between Cnx1E sub-domains III (SIII) and II

′

(SII

′

) (Fig. 2A, Supplementary Fig. 3 and 4).

Our earlier work identified Cnx1E MPT-AMP binding to correlate with the capability of

Cnx1E to form a dimeric enzyme

, a finding which is fully consistent with the identified

position of MPT-AMP at the interface of the dimeric enzyme as is shown here (Fig. 2A).

Previously, Cnx1E was characterized to bind Moco/MPT with a rather low affinity (

) in

the μM range

. We recently suggested that the low binding affinity for Moco/MPT may be

due to the lack of AMP, which when covalently linked to MPT (MPT-AMP) could function

to anchor MPT within the Cnx1E active site

. Adjacent to the bound MPT-AMP

molecule, we observe additional well-defined electron density that is located within bond-

length distance to the MPT-AMP dithiolene chelate (Fig. 2B, C). The nature of this electron

density is consistent with either a dioxo [(MPT)MoO

(OH)]

1−

species or a trioxo

[(MPT)MoO

]

2−

species with a distorted square-pyramidal coordination geometry. The

presence of a molybdenum atom at the center of this additional electron density is confirmed

by anomalous diffraction data (Fig. 2B, C). This finding was unexpected and led us to the

conclusion that Cnx1E variant S269D D274S accumulates the novel Moco precursor

adenylated Moco (Moco-AMP, Fig. 2D). Analysis of its coordination environment revealed

that the Moco-AMP pterin moiety almost exclusively interacts with subdomain II

′

(Supplementary Fig. 3 and 4), thus complementing the subdomain III AMP binding function

that was suggested to be essential for positioning of the otherwise loosely bound pterin

moiety within the E-domain active site

. Here, molybdate is initially bound in the

vicinity of a strictly conserved set of amino acids

, awaiting its relocation to the

catalytically productive site. In addition to molybdate, various other oxyanions can induce

cooperative MPT-AMP binding to Cnx1E. However, only molybdate is accepted as a

substrate for the insertion reaction

, thus rendering molybdate binding and insertion to be

distinctly separate processes

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Analogous to Moco-AMP, other molybdenum dinucleotide cofactors, including

molydopterin cytosine dinucleotide (MCD) and bis-molybdopterin guanosine dinucleotide

(bis-MGD), are commonly found in the active sites of numerous prokaryotic Mo enzymes

Both MCD and bis-MGD are synthesized in a reaction step that is subsequent to the release

of mature Moco from the bacterial Cnx1E homolog by dedicated enzymes that only exist in

prokaryotes

. Consequently, Moco-AMP constitutes the first example of a Moco

dinucleotide found in eukaryotic organisms. The phosphoric acid anhydride

(phosphoanhydride) that connects the two nucleotides can be characterized by its two

dihedral angles,

and

, which differ among Moco-AMP, MCD, and bis-MGD but are

similar between Moco-AMP and MPT-AMP bound to Cnx1G

(Fig. 3A, Supplementary

Table 2). As a consequence of these phosphoric acid anhydride

and

values, the

nucleotide moieties in MPT-AMP and Moco-AMP are arranged in a “pseudo-

cis

”

conformation relative to an imaginary axis that connects the two phosphorous atoms of the

anhydride bond, while the MCD and bis-MGD are in a “pseudo-

trans

” conformation.

However, the orientation of the AMP moiety with respect to Moco in Cnx1E is different

from that between AMP and MPT in Cnx1G, which requires a rotation by approximately

=120° around the bond formed between the molybdopterin atom C-2 and the C-P carbon

atom connected to the phosphate group (Fig. 3B, Supplementary Fig. 12. This rotation must

occur during the transition of MPT-AMP from Cnx1G to Cnx1E because the conformation

present in Cnx1G cannot be accommodated by Cnx1E

. The nature of the Mo-dithiolene

bonding in Moco-AMP closely resembles that found in Moco-containing enzymes that

belong to the sulfite oxidase family

, and this is apparent by the fact that their chiral

volumes are of the same sign. However, the conformation of the pyranopterin backbone, as

characterized through the dihedral angles

(−26.0°) and

(98.6°), cannot be assigned

clearly to one of the two conformations that are typical for the Mo-enzyme families

(Fig.

3C). In contrast to MCD and bis-MGD, Moco-AMP is relevant only as a cofactor

biosynthesis intermediate. Therefore, the identification of Moco-AMP is in complete

agreement with the latest model of E-domain functionality

. Moco-AMP was found to

be bound by both monomers of the Cnx1E dimer, and this is consistent with the current

model of E-domain ancestry, which suggests that throughout evolution the Mo-insertase G-

domain encoding gene was duplicated

to form subdomain III, which is opposing

subdomain II

′

in the dimer (Fig. 2A). Subdomain III retained the ability to bind AMP but

lost a helical active site element that is part of the G-domain pterin binding site

(Supplementary Fig. 3). This Cnx1G element confines the range of orientations that the

MPT-AMP pterin moiety can adopt. The evolutionary loss of this element within the Cnx1E

subdomain III is expected to enable the pterin in Cnx1E bound MPT-AMP to adopt the

characteristic E-domain conformation, which we suggest is essential for facilitating the

catalyzed insertion reaction

X-ray absorption spectroscopy.

Mo K-edge XANES data have been collected in order to probe the nature of the Mo center

of Moco-AMP in Cnx1E S269D D274S at pH 6 and pH 8 (Fig. 4A). These data display an

intense pre-edge feature at ~20,006 eV built on the rising Mo K-edge at ~20,016 eV. The

intensity of these “oxo edge” pre-edge features has previously been correlated with the

number of oxo donor ligands that are coordinated to the Mo ion

, with a greater number

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of oxo ligands leading to a correspondingly greater pre-edge intensity. Thus, the XAS

intensity in the pre-edge region is expected to be a sensitive probe of the number of Mo-oxo

bonds in Cnx1E bound Moco-AMP, providing a competitive advantage over X-ray

crystallography in discerning terminal oxo ligands from hydroxide and aqua ligands. In

order to more accurately assess the oxidation state and the number of oxo ligands bound to

Mo in Cnx1E bound Moco-AMP, we have compared the enzyme XANES data with sodium

molybdate, Na

MoO

(

), and three synthetic Mo(VI)-dithiolene analog complexes:

[(bdt)MoO

](NEt

)

(

)

, [(bdt)MoO

(OSiPh

Bu)](NEt

) (

)

, and [(bdt)MoO

(OSiPh

)]

(NEt

) (

)

(bdt = benzene-1,2-dithiolate) (see Supplementary Fig. 6-9). The nearly

identical rising edge inflection point energies observed for Cnx1E bound Moco-AMP

proteins and molecules

clearly show that the Mo ion in Cnx1E Moco-AMP is in the

Mo(VI) oxidation state. The strong relationship between the number of oxo ligands bound to

Mo and the Mo K-edge “oxo edge” intensity is quantitatively represented in Fig. 4B, where

the integrated area for each individual pre-edge peak is plotted as a function of the number

of terminal oxo donors. The remarkable similarity between the pre-edge intensities and the

overall XANES structure of Cnx1E bound Moco-AMP,

, and

strongly support a Mo(VI)

cis

dioxo coordination geometry for the Moco-AMP Mo site, which we can evaluate at

higher resolution using Mo K-edge EXAFS (Figure 4C-4E, and Supplementary Fig. 5). The

EXAFS analysis required the inclusion of a single non-oxo light atom scatter in order to

obtain a high-quality fit to the data. Inspection of the real part of the FT EXAFS in Figure

4E shows that this light atom scatterer contributes to the overall shape of this function in the

R + Δ = 2.00 Å – 2.25 Å range, providing opposite phase components with respect to the

Mo-oxo and Mo-S contributions. The 2.03 Å – 2.12 Å bond distance determined for the light

atom scatterer is most consistent with a coordinated hydroxyl ligand (Mo-OH)

since Mo-

aqua bonds are anticipated to be markedly longer (

ca.

2.35 Å)

. Although a weakly

coordinated aqua ligand cannot be completely ruled out, our DFT calculations on

[(MPT)MoO

(OH)]

1−

and (MPT)MoO

(OH

) computational models for Moco yield Mo-

OH and Mo-OH

bond lengths of 1.96 Å and 2.42 Å, respectively, supporting our argument

for a coordinated hydroxide (Supplementary Fig. 10). The bond lengths (R) and the number

of scatterers of a given atom type (N) determined from the Moco-AMP EXAFS analysis are

compared with crystal structure values for trioxo model compound

, dioxo model

compound

, and a dioxo computational model (listed in Supplementary Table 3). EXAFS

parameters for various values of N at pH 6 and pH 8 are presented in Supplementary Tables

4-5. Although there is precedence for a [(MPT)MoO

]

2−

site in the C207S variant of human

sulfite oxidase

we could not obtain satisfactory fits to the EXAFS data for a trioxo

structure. Additionally, the trioxo structure is inconsistent with our analysis of the “oxo-

edge” intensity for Moco in Cnx1E bound Moco-AMP (Figure 4A-4B). In summary, the

combined computational, XANES, and EXAFS data strongly support a

[(MPT)MoO

(OH)]

1−

structure for Moco-AMP, which bears close resemblance to the 5-

coordinate structures determined for model compounds

and

that were previously

characterized by X-ray crystallography

Molybdate insertion into Moco.

Identification of a [(MPT)MoO

(OH)]

1−

structure in Cnx1E S269D D274S allows for a

mechanism to be posited regarding how molybdate is incorporated into Moco (Figure 5A).

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A key component of the mechanism is that molybdate (molybdic acid: pK

= 3.9; pK

=4.4 @ 30C)

must be protonated prior to, or concomitantly with its insertion into the

MPT dithiolene. The side chains of Arg (pK

= 12.5), Ser (pK

= 15.0), and Lys (pK

10.8), which are most proximal to a molybdate oxo in the binding site (3.0 – 4.7Å;

Supplementary Fig. 3 and Fig. 4), could function as proton donors. However, the relatively

high pK

values of these side chains would require that they are significantly lowered within

the Cnx1E active site in order to protonate molybdate. The diacid form of the MPT

dithiolene could also supply the necessary protons, with molybdate properly positioned in

the binding pocket through a combination of hydrogen bonding and electrostatic interactions

involving these amino acids. Although the pK

s for the MPT dithiolene protons are not

known, the corresponding dithiolene pK

values for 1,2-benzenedithiol (bdt) are known

(pK

= 6.5; pK

=10.9), with the dithiolene pK

being markedly lower than the amino

acids in the vicinity of the molybdate binding site. It is known from model chemistry that

dithiolene protons (

e.g.

bdt) can labilize strong Mo-oxo bonds, resulting in the

elimination of water and insertion of Mo into the dithiolene.

As a result, we suggest that in

Cnx1E the MPT dithiolene supplies the two protons that are required to labilize one oxo

ligand from MoO

2−

and eliminate a water molecule. This proposal is based on (1) the need

to deprotonate the dithiolene in order to coordinate Mo, (2) the immediate proximity of the

two dithiolene S-H protons to a single terminal oxo ligand of molybdate, and (3) the

relatively high acidity of the dithiolene protons compared to nearby amino acids. Thus, the

double-protonation of a single molybdate oxygen would function to trigger the release of a

water molecule from molybdate, resulting in Mo insertion and the formation of a trioxo

[(MPT)MoO

]

2−

intermediate which, upon an additional protonation step, leads directly to

the formation of [(MPT)MoO

(OH)]

1−

; a structure that is fully consistent with the results of

our X-ray crystallographic and XAS data. Remarkably, this biosynthetic structure-based

mechanistic proposal for molybdenum incorporation into MPT-AMP bound to Cnx1E is

related to the route by which trioxo-M and dioxo-M inorganic model compounds (M = Mo,

W; Supplementary Fig. 6-8) can be chemically synthesized (Figure 5B)

. For the small

molecule analog synthesis, silyl groups (-SiR

) function as proton analogs and the silylation

reactions used in the synthesis of

effectively mimic the double-protonation of molybdate

by the diprotic acid form of the MPT dithiolene to yield [(MPT)MoO

]

2−

. However, an

important difference between the small molecule synthetic route and the Cnx1E catalyzed

insertion of molybdate is the markedly greater Si-O bond enthalpy in the model system

compared to the H-O bond enthalpy (

e.g

. the formation of (R

Si)

O (model) vs. H

(enzyme)). A lower thermodynamic driving force for the formation of a trioxo

[(MPT)MoO

]

2−

species in Cnx1E likely contributes to the requirement for further

protonation, leading to the more thermodynamically stable [(MPT)MoO

(OH)]

1−

structure

that we observe experimentally.

The elegant chemical synthesis of trioxo and dioxo small molecule analogs of the Cnx1E

Mo site provides a direct chemical precedent for our proposed insertion mechanism

, and

we have evaluated this mechanism in Cnx1E by performing linear transit reaction coordinate

computations at the DFT level of theory. Specifically, these computations have used our new

XAS determined structural details to assess the energetics (total energy and Gibbs free

energy) of the individual steps involved in the insertion mechanism (Figure 5C). The initial

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protonation of molybdate by an MPT dithiolene proton to yield [MoO

(OH)]

1−

is calculated

to be barrierless. This leads to the formation of IM1, which is stabilized by 57.0 kcal/mol

relative to E-S. The second protonation requires an activation barrier of 11.2 kcal/mol and

leads to the formation of the initial [(MPT)MoO

]

2−

product (Mo-P) complex with

elimination of water, which is only stabilized relative to IM1 by 5.8 kcal/mol. The Gibbs

free energies computed along this coordinate display similar behavior with an activation

barrier of 20.7 kcal/mol and Mo-P being slightly destabilized relative to IM1 by 5.0 kcal/

mol. The fact that both IM1 and the trioxo Mo-product are nearly isoenergetic poses a

potential problem with respect to the thermodynamic stability of the trioxo form of Moco.

Therefore, we postulate that a final protonation step of the trioxo Mo-product, which results

in the formation of [(MPT)MoO

(OH)]

1−

, must be critical for the overall catalytic efficiency

of the insertion reaction. This final protonation event will drive the equilibrium to the

[(MPT)MoO

(OH)]

1−

product side, and effectively prevent a back reaction that leads to Mo-

P and the potential for dissociation of Mo from MPT (i.e. IM1). The Cnx1E amino acids

Ser328, Arg369, and Ser400 are located at distances of 2.7, 2.9, and 2.8 Å, respectively, with

respect to Mo-P (measured without regard for the hydrogen atoms). These distances are

within a range compatible with their potential role of protonating [(MPT)MoO

]

2−

However, the lower pK

value for Arg renders Arg369 the most likely candidate to protonate

[(MPT)MoO

]

2−

and form [(MPT)MoO

(OH)]

1−

(Supplementary Fig. 11). We cannot

eliminate the possibility that protonation occurs through Ser328 or Ser400 since their actual

values could be lower than expected in the protein. However, our assessment of the

local protein environment does not provide any evidence that their pK

values are deviate

from literature reference values. We note that the Cnx1E amino acids Arg369, Ser328, and

Ser400 are all strictly conserved among various eukaryotic Mo-insertases

, providing

strong support for their proposed function in the Cnx1E active site.

Moco-AMP as an intermediate of the Moco biosynthesis pathway.

As earlier studies

suggested that MPT-AMP hydrolysis is a prerequisite for molybdate

insertion into the MPT dithiolene, we expected a hydrolysis-inactive Cnx1E variant (

i.e.

Cnx1E S269D D274S) to accumulate both of its substrates: MPT-AMP and molybdate. To

our surprise these substrates were not observed, but rather Moco-AMP was found to be

bound in the active site. This observation proves that molybdate insertion into the MPT

dithiolene is independent of MPT-AMP hydrolysis, and therefore does not require the energy

stored within MPT-AMPs phosphor-anhydride bond. This finding is in line with fact that

premature MPT-AMP hydrolysis would negate AMP’s anchoring function and adversely

affect the efficiency of Cnx1E. However, although Moco-AMP is stable in Cnx1E S269D

D274S, we cannot rule out that MPT-AMP hydrolysis in wildtype Cnx1E occurs

immediately upon molybdate insertion, rendering Moco-AMP as transient intermediate that

quickly proceeds to form Moco. In this respect, Moco-AMP would resemble 3’,8-cH

GTP,

the short-lived precursor of cPMP that was structurally characterized through co-

crystallization with accumulating active site variants

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Conclusions

The

A. thaliana

Mo-insertase Cnx1E functions as molecular scaffold, and is required to align

MPT-AMP and molybdate in a catalytically productive geometry. Within MPT-AMP, the

AMP moiety functions as an anchor to enable the enzyme to properly position MPT in the

active site. While simultaneously maintaining a low Moco/MPT binding affinity

, this

strategy would also allow for efficient release of the Moco product. Upon AMP directed

positioning of MPT within the Cnx1E active site, Mo insertion is initiated by protonation of

the molybdate ion and results in Mo binding to the MPT dithiolene leading to Moco

formation. This mechanistic sequence displays a remarkable similarity to the methods by

which di-oxo and tri-oxo small molecule analogs of Moco are chemically synthesized

indicating that the underlying principles of molybdate insertion into dithiolene ligands are

universal. Subsequent to Mo insertion into MPT-AMP, hydrolysis is required to liberate

Moco from the anchoring AMP molecule (Extended Data Fig. 1). Interestingly, when

molybdate is replaced by its tungstate congener neither the insertion into the MPT dithiolene

chelate nor the hydrolysis reaction occur

. Hence we conclude that Moco formation

affects a minor re-arrangement of the phosphoric acid anhydride bond, and this renders

Moco-AMP compatible with Mg

driven hydrolysis

. Upon hydrolysis of the

phosphoric acid anhydride bond, both AMP and Moco are released from Cnx1E

. When

Moco is biosynthesized under fully-defined

in vitro

conditions, it is physiologically active

and is accepted by various user enzymes. This leads to the conclusion that the Mo first

coordination sphere geometry present in Moco-AMP is also fully compatible with all

cellular downstream processes that Moco is subjected to, including Moco transfer, storage

and insertion into apo-enzymes

. Given the high degree of structural and functional

conservation amongst eukaryotic Mo-insertases

, we expect that the reaction mechanism

described in this work is valid for all eukaryotic Mo-insertases.

METHODS

Cloning of cnx1e Variant s269d d274s –

cnx1e

variant

s269d d274s

was generated by overlap extension PCR and following a

protocol published earlier

. Mutagenesis primers used were

Cnx1E_S269D_D274S_FusB_for (5

′

-ggtggtgttgatatgggagacaggtcattcgtcaagc-3

′

) and

Cnx1E_S269D_D274S_FusA_rev (5

′

-gcttgacgaatgacctgtctcccatatcaacaccacc-3

′

Expression and Purification of Cnx1E –

Expression of Cnx1E wildtype and variant S269D D274S was carried out as described

. For

biochemical characterization Cnx1E was purified under aerobic conditions, following

established protocols

. For crystallization experiments cell lysis and subsequent purification

steps were performed under anaerobic conditions. To enhance the Moco-AMP occupancy,

preceding crystallization, for variant S269D D274S

in vitro

MPT-AMP loading was carried

out essentially as described earlier

Moco metabolite detection and quantification –

Moco metabolite detection and quantification were carried out as described previously

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Inductively coupled Plasma Mass Spectrometry (ICP-MS) –

The molybdenum content of wild type Cnx1E and Cnx1E variants was quantified using an

Agilent 7700 Series ICP-MS machine (Agilent Technologies) as described earlier

Crystallization, data collection and structure determination of Cnx1E variant S269D D274S

–

Prior to crystallization, protein preparations were anaerobically buffer exchanged to 50 mM

Tris-HCl pH 8.0, 150 mM NaCl, 2% (v/v) Glycerol. Cnx1E S269D D274S was crystallized

by sitting-drop vapor diffusion method at 294 K in an anaerobic chamber containing a 95%

Ar / 5% H

atmosphere. Crystals were obtained with a reservoir solution containing 0.1 M

MES pH 6.5, 24 %(v/v) PEG400. To all crystallization solutions 500 μM sodium dithionite

was added. Crystals were harvested and cryo-protection was accomplished by transferring

crystals into reservoir solution containing 10 % (v/v) MPD. Diffraction data were collected

at 12,400 eV (0.99987 Å) and 7,100 eV (1.74626 Å) at the Stanford Synchrotron Radiation

Lightsource (SSRL) and at the Advanced Light Source (ALS, Berkley). Obtained data sets

were indexed and integrated using iMosflm

and XDS

and scaled with StarAniso

taking

into account the anisotropic diffraction. Phase information was derived and transplanted

from the structure of MPT-AMP-free Cnx1E wt (PDB 5G2R

). Structural refinement and

rebuilding were carried out using Buster

and COOT

. Electron density maps were

calculated using FFT (CCP4

). Images of protein structures were generated with PyMol

and UCSF Chimera

. The files containing the structure factors and the structural models

were deposited within the Protein Data Bank with accession number 6Q32 (Cnx1E S269D

D274S). The complete data collection and refinement statistics are summarized in Table S1.

X-ray Absorption Spectroscopy –

X-ray absorption spectroscopy of Mo K-edge (20,000 eV) for Cnx1E S269D D274S

samples were collected at beamline 7-3 of the Stanford Synchrotron Radiation Lightsource

(SSRL). Beamline 7-3 is equipped with a Si(220) double-crystal monochromator and a

rhodium-coated vertical collimating mirror upstream of the monochromator. X-ray

absorption data for the protein samples were collected under the Mo K

fluorescence mode,

while the reference absorption spectra of a Mo metal foil were collected simultaneously

under the transmittance mode. The intensity of the incident X-ray was monitored with an

argon-filled ion chamber and detuned ~20% away from its maximum to remove the adverse

order harmonic. The incident light passed a Sollar slit and a Zr-3 metal filter onto the

sample, and thereafter the florescence emitted from the sample was collected with a 30-

element germanium array detector. The protein samples were freshly prepared and filled into

the Delrin cells (~150 μL), which were subsequently sealed with thin sulfur-free Kapton

tape, and then kept frozen. The temperature for data acquisition was maintained around 10K

with an Oxford Instrument liquid helium flow cryostat. The energy of incident light was

calibrated to the first reflection point (20,000 eV) of the Mo foil reference spectrum. The

energy range for data collection starts from 19,692 eV to k = 18 Å above the Mo K-edge.

Each spectrum in the manuscript is an average of six data sweeps. The X-ray absorption data

were processed with the XAS analysis software DEMETER (version 0.9.25).

Energy

calibrations, averaging, and normalizations were performed using Athena to produce the X-

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ray absorption near edge spectra (XANES). The sample energy was calibrated with respect

to the second inflection point (20,010 eV) of the molybdenum foil spectrum, and the energy

threshold (E0) was set to the second inflection point of the sample (20,016 eV). The

extended X-ray absorption fine structure (EXAFS) analysis was carried out in Artemis, in

which IFEFF6 was imbedded to calculate the theoretical phase and amplitude functions, and

to model the scattering paths. All data sets were fit in k space using k

weighting, and the k-

and R-ranges for fitting models to the data were k = 2.5 - 12 and R = 1 - 3, respectively. S0

was defined as 0.97 and the phase correction was the Mo-oxo scattering path. All other

parameters were allowed to float. The resulting fit quality was judged by the R factor. No

data smoothing or further data manipulation was performed.

Computational Studies Addressing Mechanism –

All model structures were geometry optimized using Gaussian 09, (revision C.01)

at the

density functional theory (DFT) level utilizing the B3LYP hybrid exchange-correlation

functional. A 6-31g* basis set was used for all light atoms, and the LANL2DZ basis set with

a LANL2 effective core potential was used for Mo. Solvation effects were taken into account

using the polarizable continuum model (PCM) in the SCRF method with a modified

dielectric value of 4 to mimic the protein environment. Transition state searches using the

QST3 method and a single negative frequency was observed for the TS structure.

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Extended Data

Extended Data Fig. 1. The proposed mechanism of molybdate insertion into the MPT dithiolene

moiety.

(A) Ground state of Cnx1E with a Mg

-water complex bound. Mg

constitutes the

cofactor for the Cnx1E catalyzed reaction

. (B) Conformation of MPT-AMP bound in

the active site of Cnx1G

. (C) First state of the catalysis with the substrate molybdate

bound to the entry site. The presence of molybdate in the entry site induces an unfavorable

(tense) backbone conformation in Cnx1E

. (D) The conformation of MPT-AMP found in

Cnx1G is not compatible with the Cnx1E active site

. Hence, the MPT-AMP pterin moiety

must re-orient upon transfer from Cnx1G to Cnx1E, mostly involving two bond rotations.

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(E) Second state of the catalytic cycle with molybdate bound to the entry site and active site

bound MPT-AMP. The orientation of the pterin moiety was derived from active site bound

Moco-AMP identified in this work. (F) The transfer of molybdate from the entry to the

insertion site is assisted by the relaxation of the tense backbone conformation

. According

to our model of molybdate insertion, one molybdate oxygen ligand is two-fold protonated by

the MPT dithiolene function and released as water. Simultaneously, the sulfur to

molybdenum bonds are formed: This mechanism begins with a proton transfer from the

diacid form of the MPT dithiolene side chain to a molybdate oxygen. A second, less

activated protonation on the same oxygen atom would then yield a [(MPT)MoO

(OH

)]

2−

transition state. This [(MPT)MoO

(OH

)]

2−

species is expected to possess a highly labile

aqua ligand, which would subsequently dissociate from Mo along the reaction coordinate to

yield a more stable trioxo [(MPT)MoO

]

2−

intermediate. A final protonation of basic

[(MPT)MoO

]

2−

by a suitable Cnx1E active site residue (most likely residue Arg369) yields

our XAS derived [(MPT)MoO

(OH)]

1−

structure for Cnx1E Moco-AMP (for details see

Figure 5 and supplementary Figure 11). The protonated oxygen atom is marked by an

asterisk. (G) The Cnx1E active site with bound Moco-AMP.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

ACKNOWLEDGEMENTS

Correspondence and requests for materials should be addressed to M.L.K and T.K. We thank Adelina Calean (TU

Braunschweig) for excellent support with ICP-MS analysis. M.L.K. gratefully acknowledges support of this

research by the National Institutes of Health (Grant No. GM-057378 to MLK). M.L.K. and J.Y. thank the UNM

Center for Advanced Research Computing, supported in part by the National Science Foundation, for providing

computing resources used in this work. Work at Caltech was supported by the National Institutes of Health (NIH)

grant GM045162. We acknowledge the Gordon and Betty Moore Foundation and the Beckman Institute at Caltech

for their support of the Molecular Observatory at Caltech and the staff at Beamline 12-2 and 7-3. M.L.K. and J.Y.

acknowledge the Stanford Synchrotron Radiation Lightsource, which is supported by the US Department of Energy,

Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. R.R.M. and T.K.

gratefully acknowledge the support of this research by the Deutsche Forschungsgemeinschaft (GRK 2223/1).

DATA AVAILABILITY STATEMENT

The protein structure data that support the findings of this study are publicly available from

the Worldwide Protein Data Bank (

https://www.rcsb.org/

) with accession number 6Q32. We

deposited the protein structure along with the structure factor data file that allows for the re-

computing and re-evaluation of the structure. Figures 2, 3, S3, S4, and S12 are associated

with these raw data. Manuscript datasets are available as Supplementary Data files, which

are freely accessible on

nature.com

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