Structural and Functional Characterization of a Short-Chain

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Structural and Functional Characterization of a Short-Chain Flavodoxin Associated with a Non-Canonical PDU Bacterial Microcompartment Jefferson S. Plegaria, Markus Sutter, Bryan Ferlez, Clément Aussignargues, Jens Niklas, Oleg G. Poluektov, Ciara Fromwiller, Michaela TerAvest, Lisa Marie Utschig, David Michael Tiede, and Cheryl A. Kerfeld Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00682 • Publication Date (Web): 28 Sep 2017 Downloaded from http://pubs.acs.org on October 4, 2017

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Biochemistry

Structural and Functional Characterization of a Short-Chain Flavodoxin Associated with a NonCanonical PDU Bacterial Microcompartment Jefferson S. Plegaria1, Markus Sutter1,2, Bryan Ferlez1, Clément Aussignargues1, Jens Niklas3, Oleg G. Poluektov3, Ciara Fromwiller1, Michaela TerAvest4, Lisa M. Utschig3, David M. Tiede3, and Cheryl A. Kerfeld*1,2,4,5 1

MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824, United States 2

Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States

3

Solar Energy Conversion Group, Argonne National Lab, Argonne, Illinois 60439, United States 4

Department of Biochemistry & Molecular Biology, Michigan State University, East Lansing, Michigan 48824, United States 5

Berkeley Synthetic Biology Institute, Berkeley, California 94720, United States

CORRESPONDING AUTHOR *E-mail: [email protected]. Phone: 517-432-4371.

ACS Paragon Plus Environment

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ABBREVIATIONS BMCs, bacterial microcompartments; PD, propanediol; ox, oxidized; sq, semiquinone; hq, hydroquinone; SHE, standard hydrogen electrode; AldDH, aldehyde dehydrogenase; AlcDH, alcohol dehydrogenase; PTAC, phosphotransacylase; Fld, flavodoxin; FAD, flavin dinucleotide; FMN, flavin mononucleotide; ATR, adenosyltransferase; SC, short-chain; LC, long-chain; UVVis, Ultraviolet-Visible; EPR, electron paramagnetic resonance; Fmr, flavin reductase


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Biochemistry

ABSTRACT Bacterial microcompartments (BMCs) are proteinaceous organelles that encapsulate enzymes involved

in

CO2

fixation

(carboxysomes)

or

carbon

catabolism

(metabolosomes).

Metabolosomes share a common core of enzymes and a distinct signature enzyme for substrate degradation that defines the function of the BMC (e.g. propanediol or ethanolamine utilization BMCs, or glycyl-radical enzyme microcompartments). Loci encoding metabolosomes also typically contain genes for proteins that support organelle function, such as regulation, transport of substrate, and co-factor (e.g., vitamin B12) synthesis/recycling. Flavoproteins are frequently among these ancillary gene products, suggesting that these redox active proteins play an as-yet undetermined function in many metabolosomes. Here, we report the first characterization of a BMC-associated flavodoxin (Fld1C), a small flavoprotein, derived from the non-canonical 1,2propanediol utilization BMC locus (PDU1C) of Lactobacillus reuteri. The 2.0 Å X-ray structure of Fld1C displays the α/β flavodoxin fold, which non-covalently binds a single flavin mononucleotide molecule. Fld1C is a short-chain flavodoxin with redox potentials of –240 ± 3 mV oxidized/semiquinone (ox/sq) and –344 ± 1 mV semiquinone/hydroquinone (sq/hq) versus the standard hydrogen electrode (SHE) at pH 7.5. It can participate in an ET reaction with a photoreductant to form a stable sq species. Collectively, our structural and functional results suggest that PDU1C BMCs encapsulates Fld1C to store and transfer electrons for the reactivation/recycling of the B12 cofactor utilized by the signature enzyme.

KEYWORDS Metabolosomes; Flavoproteins; Electron Transfer; Vitamin B12 Reduction


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Bacterial Microcompartments (BMCs) are bioinformatically predicted to be present across at least 23 different bacterial phyla,1 underscoring their prevalence in microbial metabolism. The core enzymes of BMCs are encapsulated in a shell comprised of homologous proteins that form hexamers (BMC-H)2 and trimers (BMC-T)3-5 and pentamers (BMC-P).6,

7

Confinement of

sequential reactions by the BMC shell presumably enables substrate channeling, promoting the catalytic efficiency of the encapsulated enzymes.8-11 Many heterotrophs contain functionally diverse BMCs (metabolosomes) that catabolize a range of carbon compounds in niche environments. The metabolosome core includes a signature enzyme [e.g., propanediol dehydratase,12 ethanolamine-ammonia lyase,13 or glycyl-radical enzyme (GRE)14, generate an aldehyde. They also include four conserved core enzymes:1, aldehyde

dehydrogenase

(AldDH),17

an

alcohol

dehydrogenase

11, 16

15

] that

an acylating

(AlcDH),18

and

a

phosphotransacylase (PTAC).19, 20 In addition, bioinformatic studies have shown that BMC loci typically also encode ancillary enzymes/proteins1, 14 that play as-yet uncharacterized roles in the metabolosome function.

Among these ancillary proteins are several flavoproteins including a flavodoxin (Fld) (pfam 00258). These small flavoproteins are found in the loci of 1,2-propanediol (PD) utilization (PDU), ethanolamine utilization, and GRE-containing BMCs. Flavoproteins21-23 bind a redox active nucleic acid derivative of riboflavin, either a flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) and participate in one- or two-electron transfer (ET) reactions. For example, the canonical PDU BMC (PDU1A) of Salmonella enterica12 (Figure 1a) and Citrobacter freundii,24 encapsulates a vitamin B12-dependent dehydratase (PduCDE) to degrade 1,2-PD (Figure 1b). A flavoprotein (PduS) reduces the B12 cofactor, allowing the


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adenosyltransferase (ATR, PduO) to generate the active form of this cofactor. PduS contains an NADH-binding site and binds an FMN molecule and two iron-sulfur clusters.25-27 To-date, PduS is the only characterized BMC-associated flavoprotein. Because the processes by which electrons are transported into and stored within BMCs are not well-understood, the several other types of flavoproteins found in BMC loci may be playing an unrecognized and important role in BMCassociated redox reactions. Accordingly, further characterization of these BMC-associated redox active proteins provides a first step towards understanding the flux of electrons in BMCs and other native or synthetic encapsulated systems.

In this work, we report the first structural and functional characterization of an ancillary shortchain (SC) Fld (Fld1C) conserved in the understudied PDU1C BMC locus (Figure 1). Metabolomic studies on L. reuteri that encode the PDU1C locus showed that PduCDE breaks down 1,2-PD28 and glycerol to produce an antimicrobial compound, reuterin29,

30

(Figure 1b).

Notably, the PDU1C loci are missing the pduS gene. To-date only two PDU1C BMC components have been structurally characterized. This includes PduO/ATR31, 32 and a trimeric shell protein PduB.33 We show that Fld1C has the characteristic flavodoxin fold with five αhelices that surround a five-stranded β sheet, a single FMN cofactor that is non-covalently bound, and a negatively charged patch that surrounds the FMN binding site. The sq/hq second redox couple (E1) of Fld1C has a redox potential that is within the range of SC Flds, while the ox/sq first redox couple (E2) has a value that is more comparable to long-chain (LC) Flds. This similarity of the E2 value with LC Flds can be attributed to a negatively charged residue in a loop region that is typically occupied by a Gly in SC Flds. Our results suggest that Fld1C supplies electrons to the B12 regeneration/reactivation reaction in PDU1C BMCs.


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(a)

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PDU1A: Salmonella enterica pduA B

C

D

E

G

H

J K

L M N O

P

Q

S

T U V

PDU1C: Lactobacillus reuteri pduA

B

C

BMC-H BMC-T BMC-P AldDH

D

AlcDH PTAC Signature

(b)

E

phosphoglycerate

G

H K

J

L M N O O

P

Q

U mutase

W

PDU1C 1,2-propanediol

propanol NADH

NAD+

glycerol

PduQ

B-hydroxypropionaldehyde /reuterin

propionaldehyde

PduP

NAD+ HS-CoA

ATP

NADH

PduGH

NAD+

NADH

ADP Pi

Adenosylcobalamin propionyl-CoA Cob(III)alamin

PduL

NADH

PduS NAD+

PPPi

PduO

propanol NADH

NAD+

PduCDE

NAD(P)H-dependent FMN reductase

O tyrosine CadD V phosphatase short-chain flavodoxin (Fld1C)

Re-activating Conserved and shared with other locus type Conserved and specific to locus type

PDU1A 1,2-propanediol

X

W

propionyl-PO4

1,3-propanediol

2-

propionaldehyde

PduP

NAD+ HS-CoA

ATP

AlcDH

PduGH NADH

ADP Pi

Adenosylcobalamin propionyl-CoA Cob(III)alamin

PduL

?

Pi

HS-CoA ATP Cob(I)alamin

PduQ PduCDE

PPPi

PduO

PduW propionate

Pi

HS-CoA ATP Cob(I)alamin

propionyl-PO42-

PduW propionate

Figure 1. Comparison of 1,2-PD utilization (PDU) BMCs. (a) Cartoon representation of the PDU1A loci of Salmonella enterica, the canonical PDU BMC, (upper) and the PDU1C of Lactobacillus reuteri (DSM 20016) (lower). Genes are colored according to their annotation: blue, BMC-H (pduAJKU); cyan, BMC-T (pduBT), yellow, BMC-P (pduN); dark purple, vitamin B12-dependent PD dehydratase subunits (pduCDE); light purple, diol dehydratase reactivation factor subunits (pduGH); pink, phosphotransacylase (PTAC) (pduL); orange, adenosyltransferase (pduO); red, aldehyde dehydrogenase (AldDH) (pduP); green, alcohol dehydrogenase (AlcDH) (pduQ); dark gray, genes present in other PDU loci (e.g., pduMVW); genes present in specific PDU loci (e.g., pduSX in PDU1A and Fld1C in PDU1C). (b) Schematics of the function of PDU1A (left) and PDU1C (right) BMCs.


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Biochemistry

MATERIALS AND METHODS Plasmids and Protein Purification. The PDU1C-associated flavodoxin gene of L. reuteri (Fld1C) [Integrated Microbial Genomes (IMG) ID 2674092797] was designed to include an encapsulation peptide (EP)34 from the AldDH of the Haliangium ochraceum BMC locus35 (IMG ID 646390480), and a (5x) GlySer linker at the N-terminus. The gene for this fusion construct was synthesized by Integrated DNA Technologies (Supplementary Table 1) using E.coli codon optimization and cloned into the pACYCDuet-1 vector (Novagen) using NcoI and AvrII sites. A poly-histidine tag (GlySer-6xHis) was added to the C-terminus of the EP-Fld1C construct using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent) (Supplementary Table 2 for the primers). The EP-Fld1C-6xHis construct was then used to construct a variant without the Nterminal EP (Supplementary Table 1). This gene was created via PCR amplification (Supplementary Table 2 for primers) and cloned into the pACYCDuet-1 vector using NcoI and AvrII sites. Two nucleotides were added after the 5’-NcoI site, resulting in an additional Ala at the second position of Fld1C.

Escherichia coli BL21(DE3) cells was transformed with the Fld1C (6xHis) plasmid. Cells were grown in LB-chloramphenicol (50 mg/L) media at 37 °C until an OD600 of 0.6, induced with 1 mM Isopropyl β-D-1-thiogalactopyranoside, and incubated overnight at 22 °C. Cells were harvested by centrifugation at 8,000 x g for 15 min, and the pellet was re-suspended (cell wet weight to buffer volume ratio 1:2) in buffer A (50 mM Tris/HCl pH 7.5 and 100 mM NaCl) containing DNase. The suspension was lysed by two passages through a French press at a pressure of 137 mPa. The lysate was then centrifuged at 64,000 x g for 45 min, and the supernatant was clarified by passing it through a 0.22 µm filter. The supernatant was applied to a


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Ni-agarose resin column (HisTrap HP, GE Healthcare, Inc) equilibrated in buffer A with 25 mM imidazole. Fld1C proteins were eluted using buffer A containing 500 mM imidazole. The EPFld1C construct (without the GlySer-6xHi) was transformed, expressed and extracted using the same methods. EP-Fld1C was purified on Sepharose Q column (HiTrap Q, GE Healthcare, Inc), equilibrated in 50 mM Tris/HCl pH 7.5 and 75 mM NaCl, and eluted using a gradient from 75 to 500 mM NaCl in 10 column volumes. The yellow fractions collected from the HisTrap HP or HiTrap Q purification were combined, buffer exchanged in buffer A, and reconstituted in a buffer A solution containing 1-5 mM FMN to obtain ~100% holo protein. These proteins were further purified using a gel filtration column (HiLoad 16/600 Superdex 75 pg, GE Healthcare). Pure Fld1C was buffer-exchanged using a PD 10 desalting column (GE Healthcare, Inc) in 50 mM Tris/HCl with 100 mM NaCl or 50 mM potassium phosphate buffer all at pH 7.5 for physical characterization. EP-Fld1C was buffer exchanged using a PD 10 column in 5 mM Tris/HCl pH 7.5 and used in protein crystallography.

Bioinformatic Analysis. The amino acid of sequence of Fld1C was queried using BLAST against the Uniprot reference proteomes sequence database, applying the phmmer algorithm on the EMBL-EBI webserver (https://www.ebi.ac.uk/Tools/hmmer/search/phmmer); the BLAST analysis was restricted to bacteria. The first 500 protein sequences were extracted and redundant sequences (>90% similarity) were removed, which reduced the number of sequences to 463. To construct a phylogenetic tree (Supplementary Figure 1), the sequences were aligned using the ClustalW multiple sequence alignment method on the EMBL-EBI Muscle webserver and then trimmed

via

TrimAl

(gappyout

method)

on

the

Phylemon2

webserver

(http://phylemon.bioinfo.cipf.es). Hidden Markov model (HMM) sequence logos were created


8

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Biochemistry

on Skylign (http://skylign.org) with the following parameters: alignment processing via observed counts, aligned sequences are full length, and letter height is above background.

Ultraviolet-Visible (UV-Vis) and Fluorescence Measurements. Fld1C samples were prepared in 50 mM Tris/HCl pH 7.5 with 100 mM NaCl. UV-Vis spectra were recorded at room temperature using an Agilent Technologies Cary60 UV-Vis spectrophotometer. The molar extinction coefficient of the FMN cofactor of Fld1C was determined using a previously reported method.36 Emission spectra were recorded at room temperature using a SpectraMax M2 (Molecular Devices) fluorimeter. Fld1C was excited at 455 nm and fluorescence emission spectra were recorded from 490 to 690 nm.

Structure Determination. EP-Fld1C was crystallized via sitting drop vapor diffusion by combining 2 µL of 3 mg/mL protein in 5mM Tris/HCl pH 7.8 with 2 µL of a reservoir solution containing 25% (w/v) PEG 3,350, 200 mM calcium acetate, 100 mM MES buffer pH 5.5, and 6 mM cadmium acetate in sitting drop trays. The crystals were cyroprotected using 25% (v/v) PEG 400 and flash cooled in liquid nitrogen until data collection. X-ray diffraction data were collected at beamline 5.0.2 (100 K, 1.0 Å wavelength) of the Advanced Light Source at Lawrence Berkeley National Laboratory. Diffraction data were integrated with XDS,37 and scaled and merged with SCALA (CCP4).38 The structure of Fld1C was solved via molecular replacement with phenix.phaser39 using PDB entry 5LJL as the search model. Autobuilding was performed using phenix.autobuild, with multiple cycles of manual rebuilding in COOT40 and refinement with phenix.refine.39 There were three molecules in the asymmetric unit. All residues are visible in the density besides the C-terminal K178 in two of the three chains and those of the N-terminal


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EP (which had been included for shell targeting experiments, which were unsuccessful) in all three chains. The 3 chains in the asymmetric unit have a moderate surface area according to a PISA analysis,341 with a low ∆Gdiss of -1.3 kcal/mol, indicating that the interactions are likely due to crystal packing. The quality of the electron density allowed complete building of the three chains. Statistics for diffraction data collection and refinement are listed in Table 1. The omit map (Supplementary Figure 2) was calculated using output from phenix.refine after setting the occupancy of the FMN to 0 and running three cycles of refinement with a Cartesian simulated annealing step (5000 K-300 K) in the second cycle.

Electrochemical Measurements. Cyclic voltammetry measurements were obtained on a VMP3 potentiostat (Bio-Logic USA, Knoxville, TN). The electrochemical apparatus contained a glassy carbon working electrode (7.1 mm2), a titanium wire counter electrode, and an Ag/AgCl (saturated KCl) reference electrode. The carbon surface was polished with an alumina slurry (BASi, Inc.). The carbon electrode was then conditioned in an electrochemical cell (under N2g), which contained 100 mM phosphate buffer at pH 7.5 with 100 mM sodium sulfate, by scanning 20 times from -700 mV to 0 mV (vs. saturated KCl) at 500 mV/s, until the cyclic voltammograms (CVs) overlaid well, indicating a homogeneous surface. CVs were collected at varying scan rates (100, 75, 50, 25 and 10 mV/s) (~22 °C) for a buffer only solution and for 0.5 mM Fld1C reconstituted in the electrochemical cell buffer.

Redox Potentiometry. Fld1C was brought to a final concentration of 86 µM in 50 mM Tris/HCl/100 mM NaCl pH 7.5 containing 5 µM methyl viologen, 5 µM benzyl viologen, and 5 µM anthroquinone-2-sulphonate. Anoxic conditions were maintained throughout the titration by


10

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Biochemistry

continuous sparging with N2g in a custom-built glass titration cell. Potentiometric redox titrations were carried out as described by Dutton.42 The redox potential of the Fld1C solution was poised by small additions of concentrated sodium dithionite or duroquinone stock solutions prepared in 1 M Tris/HCl pH 7.5 or DMSO, respectively. The solution potential was monitored using a digital voltmeter connected to a platinum working electrode and an Ag/AgCl reference electrode. An absorbance spectrum (path length = 1 cm) was recorded (on the spectrophotometer listed above) once the potential remained constant (< 1 mV/min drift). The Ag/AgCl electrode was calibrated using a saturated solution of quinhydrone at pH 7.0 (1M Tris/HCl). At solution potentials between –139 and –282 mV (for the ox/sq couple) and –289 and –428 mV (for the sq/hq couple), the absorbance of the semiquinone form of Fld1C at 575 nm43 was fit to the Nernst equation for two subsequent one-electron processes using a non-linear least squares algorithm in Igor Pro (Igor Pro, Lake Oswego, OR).

Electron Paramagnetic Resonance (EPR) Measurements. Samples for EPR spectroscopy contained 0.3 mM Fld1C in 50 mM phosphate buffer with 50 mM NaCl and were prepared under an N2 atmosphere. The control sample consisted of Fld1C and buffer, and the experimental sample contained five-fold molar excess of ruthenium trisbipyridine (1.5 mM) and 10 mM sodium ascorbate. The EPR tubes were purged with N2g (~5 min) and 150 – 200 µL of protein sample was added, which were purged further with N2g. The tubes were septa-capped and frozen in liquid N2. Continuous wave X-band (9 GHz) EPR spectra were collected on a Bruker ELEXSYS E500 II EPR spectrometer (Bruker Biospin, Inc.), equipped with a Bruker ER4102ST resonator a helium gas-flow cryostat (ICE Oxford, Inc.), and an ITC temperature controller (Oxford Instruments, Inc.). Samples were illuminated with a 300 W Lamp (PE300F; Atlas


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Specialty Lightning, Inc.). A 15 cm water filter and a KG2 filter (Schott) was utilized to minimize IR irradiation of the sample, as well as a 400 nm long pass filter (Schott) to block UV light. All measurements were performed at 10 K. Data were processed using the MatlabTM 7.11.2 (MathWorks) environment. The magnetic parameters were obtained from theoretical simulation of the EPR spectra, which was performed using the EasySpin software package (version 5.1.10).44

RESULTS Bioinformatic Analysis of Fld1C/Pfam00258 of the PDU1C Loci. The PDU1A and PDU1C loci share 16 homologous pdu genes that encode for components of the PDU shell (PduABJKNU), conserved core enzymes (PduCDEQPL), and ancillary proteins involved in B12 recycling/regeneration (PduGHO) (Figure 1a and Supplementary Table 3 and 4). Both loci contain part of the B12 synthesis operon.1 The PDU1C loci is, however, distinctive in three ways. First, PDU1C is missing the gene for pduT, a BMC-T shell protein that has been shown to bind an iron-sulfur cluster in its pore when heterologously expressed.24, 45 Second, the PDU1C locus contains three additional genes not found in PDU1A. This includes genes that putatively encode for a tyrosine phosphatase (pfam 13350), phosphoglycerate mutase (pfam 00300), and permease CadD (pfam 03596). Lastly, PDU1C lacks the flavoprotein gene, pduS. Instead, PDU1C contains a flavodoxin gene (Fld1C) that is adjacent to a second flavoprotein gene, a putative NAD(P)Hdependent FMN reductase (Fmr). The Fld1C and Fmr genes are sandwiched by one of three atr/pduO (pfam 01923) genes and a pduV (pfam 14542) gene; PduV is proposed to function in the spatial organization of PDU BMCs in the cytosol.46


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Biochemistry

The sequences of 463 Fld1C homologs obtained from a BLAST search on the Uniprot Reference Proteomes database were used to build a phylogenetic tree (Supplementary Figure 1). Twenty-three clusters were identified, and the Fld1C-containing cluster was assigned as cluster 1. Because of their disparate locations in the tree, clusters, 2, 5, and 12, were compared to cluster 1; cluster 2 branches adjacent to cluster 1, cluster 5 is in the center of the tree, and cluster 12 is on the opposite end of the tree. Clusters 1, 2, and 5 contain Flds from Lactobacillales, while cluster 12 contains Flds from the Bacillales Order (Supplementary Table 5). Cluster 1 includes three genomes with a PDU1C-type BMC locus (Supplementary Table 6). Like the Fld1C in the PDU1C locus of L. reuteri (Figure 1a), the Fld gene in L. mellifer and L. collinoides is adjacent to an Fmr gene. Moreover, ten additional genomes in cluster 1 contain a PDU-type locus (Supplementary Table 6). These loci also lack the gene for PduS but encode a flavoprotein pfam domain 00724 or 03358 (Fmr) proximal to the PDU genes. The gene for these flavoproteins is often next to a BMC-H gene, pduU.

Sequence alignment and structural comparisons have been used to distinguish two groups of Flds: SC (~140 residues), which includes Fld1C, and LC (~180 residues) Flds.23 The amino acid sequence of Fld1C was compared to structurally characterized SC and LC Flds (Figure 2). The sequence identity of Fld1C with these Fld counterparts is low (18% – 27%), except with the SC Fld of Streptococcus pneumoniae (~49%). Notably, in the loop 4 region, SC Flds typically have a Gly at position 60 (Figure 2), which has been shown to modulate the redox potential of the ox/sq redox couple.47 In contrast, Fld1C contains an Asp at position 60. A branched residue (Asn or Tyr) is also observed in LC Flds at this corresponding position.


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*

C. beijerinckii C. braakii L. reuteri (FldC1) S. pneumoniae D. desulfuricans D. gigas D. vulgaris E. coli Nostoc S. elongatus C. beijerinckii C. braakii L. reuteri (FldC1) S. pneumoniae D. desulfuricans D. gigas D. vulgaris E. coli Nostoc S. elongatus

Figure 2. Sequence conservation among structurally characterized flavodoxins. Aligned SC (black) and LC (gray) Fld sequences. SC Flds from Clostridium beijerinckii (PDB 5nll),48 Citrobacter braakii (PDB 4oxx),49 Streptococcus pneumoniae (PDB 5lji),50 Desulfovibrio desulfuricans (PDB 3kap),51 Desulfovibrio gigas (PDB 4heq),52 and Desulfovibrio vulgaris (PDB 1j8q)53 share 17.8, 24.0, 48.7, 20.0, 27.3, and 22.1 percent identity with Fld1C (PDB 5veg), respectively. The Fld sequence of S. pneumoniae and D. gigas are located in cluster 18 and 6, respectively (Supplementary Figure 1). LC Flds from Escherichia coli (PDB 2mok), Nostoc sp PCC 7120 (PDB 1flv),54 and Synechococcus elongatus PCC 6301 (PDB 1ofv)55 share 25.2, 22.5, and 21.0 percent identify with Fld1C, respectively. The sequence of Fld1C was used as the reference. Blue coloration indicates conservation strength according to the BLOSUM62 matrix. The red asterisk marks the residue at the ~60 position of loop 4.

Table 1. Data collection and refinement statistics. Fld1C Data collection Space group

P 212121

Unit cell dimensions a, b, c (Å)

47.3, 95.8, 102.4


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Biochemistry

α, β, γ (°)

90, 90, 90

Resolution (Å)

48-1.99 (2.04-1.99)

Rmerge

0.091 (0.903)

CC1/2

0.999 (0.790)

I / σI

16.3 (2.3)

Completeness (%)

100.0 (100.0)

Redundancy

12.4 (10.3)

Refinement Resolution (Å)

48-1.99 (2.06-1.99)

Number of reflections 32,687 (3236) Rwork / Rfree

20.1 (26.1) / 25.0 (35.0)

Number of atoms Protein

3421

Ligand/ion

102

Water

179

B-factors Protein

39.2

Ligand/ion

32.8

Water

39.3

R.m.s. deviations Bond lengths (Å)

0.005

Bond angles (deg)

0.71

Ramachandran plot favored (%)

96.8

allowed (%)

3.2

outliers (%)

0


15

Biochemistry

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Page 16 of 37

Structural Characterization of Fld1C. Fld1C was heterologously expressed in and purified from E. coli. Fld1C crystallized in the orthorhombic space group P212121 (Table 1), and the crystals diffracted to a resolution of 2.0 Å. Fld1C has the classic α/ β sandwich flavodoxin fold, composed of five α-helices that pack against five parallel β-strand sheets (Figure 3a). Fld1C displays a striking charge asymmetry, with a large negative patch that surrounds the flavin cofactor (Figure 3b). It non-covalently binds a single FMN cofactor (Figure 3c). The identification of FMN in the flavin binding site is unambiguous and appears fully occupied (for omit map see Supplementary Figure 2). The FMN is located in a cavity formed by loops 1 (residues 9-14), 4 (residues 56-69), 6 (residues 91-101), and 8 (residues 125-131); the phosphate and ribityl groups are buried in the apolar core and the isoalloxazine ring is partly exposed to solvent. It is bound to these loop regions via hydrogen bonding and nonbonding/van der Waals interactions (Figure 3c).

Hidden Markov model (HMM) sequence logos were generated for the 50 sequences in cluster 1 (Figure 3d), as well as for the sequences in clusters 2 (38 seqs), 5 (6 seqs), and 12 (47 seqs) (Supplementary Figure 3). The HMM logo of cluster 1 (which includes Fld1C) shows a pronounced conservation of residues in the first coordination sphere of the FMN in Fld1C (Supplementary Figure 4a). Except for Tyr98, 10 of the 11 residues that form both H-bond and nonbonding interactions in the FMN center of Fld1C are well conserved in cluster 1 (Supplementary Figure 4b). Eight of the nine residues that only form nonbonding interactions with the FMN cofactor are also well conserved in cluster 1. These nonbonding residues include Tyr57, 59, and 95 that pack the isoalloxazine ring of FMN (Figure 3c). Tyrosine 95 is coplanar with the isoalloxazine ring, forming stabilizing π-π stacking interactions. Furthermore, the


16

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

negatively charged residues that surround the FMN center are well conserved in cluster 1 (Supplementary Figure 5). These acidic residues, which contribute to the surface charge asymmetry of Flds, were shown to modulate the sq/hq redox couple.56

(a)

(c)

Y98

loop 4

E61

β5

F99

α4

β3

Y95

α3 α5

D92 G62

β4

N100

β2

D60

T58 G91

α1

L126

α2

β1

Y59 Y57

(b)

N15

S90 N14

T12

M11

180°

T10 −5 kT/e

(d)

β1

1

5

+5 kT/e

α1

10

15

20

β2

25

85

30

35

α2

40

90

95

100

105

β3

45

α4

β4

80

G13

50

α3

55

60

115

120

125

70

75

α5

β5

110

65

130

135

140

145

150

Figure 3. Structural characterization of a BMC-associated SC Fld. (a) The ribbon structure of Fld1C. (b) Charge distribution map of Fld1C with the surface colored by electrostatic potential


17

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Page 18 of 37

from -5 kT/e (red) to +5 kT/e (blue). Fld1C is oriented as in (a). (c) Close-up view of the Hbonding (light blue) and nonbonding (purple) residues at the FMN center. Dashed-lines indicate H-bond interactions (see Supplementary Figure 4 for bond distances). The figures of the structural models were prepared using PyMOL (www.pymol.org). (d) Hidden Markov model (HMM) sequence logo for the Fld1C cluster from the phylogenetic tree (Supplementary Figure 1). The height of the stack corresponds to conservation at that position, and the height of each individual letter represents the frequency of that amino acid within the distribution. Filled-in light blue and purple circles denote residues that form hydrogen bonds and nonpolar interactions with FMN in the Fld1C structure, respectively.

Solution-State Characterization of Fld1C. The UV-VIS spectrum of purified Fld1C exhibits the absorption profile of an oxidized flavodoxin with a maximum absorption (λmax) band at 455 nm with a shoulder at 475 nm and a second prominent band at 373 nm (Figure 4a). Mass spectrometry confirms further that Fld1C binds FMN (Supplementary Figure 6). At 455 nm, the FMN cofactor of Fld1C has a molar extinction coefficient of 13,768 M-1 cm-1, which is slightly larger than previously reported values (10,000 – 11,000 M-1 cm-1).57 The fluorescence emission spectrum of Fld1C was obtained by exciting the FMN at 455 nm (Figure 4a). Fld1C has an emission maximum band at 530 nm, an emission fluorescence profile that is comparable to its flavodoxin homologs.57

The oligomeric state of Fld1C was examined using analytical size-exclusion chromatography (Figure 4b) and dynamic light scattering (DLS). The majority of Fld1C eluted at a volume corresponding to a calculated molecular weight of 23 kDa, which is higher than the


18

Page 19 of 37

theoretical MW of a monomeric holo form (with GlySer-6xHis tag) (17.8 kDa). DLS analysis on Fld1C confirmed a monomer as the most dominant form in solution (Supplementary Figure 7). The DLS histogram fit of Fld1C displays a single species with a radius of 2.0 nm and MW of 19 kDa.

(a)

(b) Absorption Emission

100

12000 80

10000 8000

60

6000

40

4000 20

2000 0 300

350

400

450

500

550

600

650

0 700

6.0

FldC1 1.0 0.8

5.5

158 kDa

5.0

66 kDa 44 kDa

0.6

4.5

17 kDa

4.0 0.4

3.5

0.2 0.0 10.0

Log(MW)

ε (M-1 cm-1)

14000

1.2

120

Normalized Abs (A.U.)

16000

Emission Intensity (A. U.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

3.0 12.5

Wavelength (nm)

15.0

17.5

20.0

22.5

2.5 25.0

Elution Volume (mL)

Figure 4. Characterization of the FMN and oligomeric state of Fld1C. (a) Absorption (blue line) and fluorescence emission (red line) spectra of holo Fld1C. (b) Size-exclusion chromatography analysis of holo Fld1C, using a protein standard that contains proteins with varying molecular weights (red closed circles).

Redox Characterization of Fld1C. Cyclic voltammetry was used to determine the midpoint redox potential of the ox/sq (E2) and sq/hq (E1) couples of Fld1C (Supplementary Figure 8). The cyclic voltammogram of Fld1C at 10 mV/s displays the ox/sq couple of liberated FMN at –222 mV (vs. SHE) (pH 7.5), which is a typical redox potential for this couple.58, 59 The sq/hq couple of Fld1C is at –326 mV (vs. SHE). Because the ox/sq redox potential could not be measured via CV, we used optical redox potentiometry to determine the potential of this redox couple. The absorbance of the sq form of Fld1C was measured at 575 nm as a function of solution potential (Figure 5a, Supplementary Figure 9), and the E2 and E1 values were calculated to be –240 ± 3


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Page 20 of 37

mV and –344 ± 3 mV (vs. SHE) at pH 7.5, respectively (Table 2). The E1 value is comparable to the value determined from CV. Interestingly, the gap between the E2 and E1 (∆E) values of Fld1C is smaller (+104 mV at pH 7.5 and +74 mV at pH 7.0) than the corresponding value for its Fld counterparts (∆E = +192 – +329 mV) (Table 2).

Table 2. Comparison of Redox Potential of Short-Chain and Long-Chain Flavodoxins. E2 (mV) Source ox/sq

E1 (mV)

Em

∆E ox/hq (mV)

pH

Function

–344 (3)

–292

104

7.5

–344a

–307

74

7.0

PDU1C-BMC B12 reduction?

sq/hq

Ref

SC Fld –240 (3) L. reuteri –270

a

associated this work

C. beijerinckiib

–92

–399

–246

307

7.0

48

D. vulgarisb

–143

–440

–292

267

7.0

60

D. desulfuricansb

–58

–387

–329

329

7.0

Nostocb

–196

–425

–310

229

7.0

S. elongatusb

–221

–447

–334

226

7.0

E. colib

–260

–452

–356

192

7.0

–238

–172

–205

–66

7.0

58

–314

–124

–219

–190

7.0

65

Sulfate-reducing pathway

61

LC Fld Reduction of ferredoxinNADP+ reductase in photosynthesis B12 reduction methionine synthesis

in

62 63

64

Other Free FMN

S. entericaPduS

– 150c

7.5

PDU1A-BMC-associated B12 reduction

26


20

Page 21 of 37

– 260d Reduction potential values are versus SHE. Em is the average of the E1 and E2 values. ∆E is the difference between the E2 and E1 values. aA –59 mV/pH was applied to estimate the E2 value at pH 7.0 and the E1 value is not expected to change above pH 7.0.66, 67 bThe E2 values for these Flds were either measured at or were adjusted to pH 7.0.47 Measured cwith and dwithout Fe-S cluster bound.

The sq species of Fld1C was photochemically generated and characterized by EPR spectroscopy (Figure 5B and Supplementary Figure 10). Three EPR spectra were collected: Fld1C only sample, an EPR sample incubated in the dark, and the same EPR sample that was illuminated for 10 seconds prior to freezing. No EPR signal was observed for the Fld1C sample (data not shown), and the dark spectrum (Fld1C + [Ru(bpy)3]) showed no signal because the ox state is EPR silent. Following the illumination of Fld1C in the presence of excess [Ru(bpy)3], a radical species with a peak linewidth of 2.3 mT at g = 2.005 was observed, which is consistent with a singly reduced FMN sq species.68

(a)

(b)

1 cw X-band EPR, T = 10 K Fld1C+Ru(bpy)3+asc, dark Fld1C+Ru(bpy)3+asc, 10 s illuminated

cw EPR signal (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

0

320

330

340

350

360

Magnetic Field (mT)

Figure 5. Redox characterization of Fld1C. (a) Optical redox titration at pH 7.5. The absorbance of the sq species at 575 nm is plotted as a function of solution potential. These data are best fit as


21

Biochemistry

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Page 22 of 37

two separate one-electron processes using the Nernst equation (dashed black lines). (b) X-band continuous wave EPR spectra of Fld1C samples containing [Ru(bpy)3] and ascorbate (asc). A spectrum was collected in the dark (black line) and after 10 s illumination with visible light (red line). All measurements were performed at 10 K.

DISCUSSION Fld1C is a short-chain flavodoxin derived from the PDU1C locus of L. reuteri. The crystal structure confirms that Fld1C possesses the canonical α/β flavodoxin fold, with a single FMN molecule non-covalently bound (Figure 3). A large number of acidic residues encompassing the FMN center form a negative patch (Figure 3b), which is a structural feature that is conserved across both SC and LC Flds (Supplementary Figure 11). The acidic residues comprise 29 Glu and Asp residues, 15 of which are highly conserved (Supplementary Figure 5). Previous studies have shown that the negative patch surrounding the FMN of Flds serves as a recognition site for its redox partner.69, 70 Our photophysical work shows that Fld1C can participate in an ET reaction with a positively charged compound ([Ru(bpy)3]+), which is consistent with a role in recognition by the negatively charged surface patch. This suggests that electrostatic interactions are important in facilitating the interaction of Fld1C with its redox partner(s).

While electrostatics orient the protein-protein interactions, the stability of the FMN-Fld complex was shown to be largely driven by the nonbonding/van der Waals interactions with the isoalloxazine ring.71-73 The phosphate group contributes via H-bonds with Thr/Ser/Asn sidechains and main chain groups, while the ribityl group forms minimal interactions with the protein fold. A closer analysis of the FMN center of Fld1C shows that the predominant mode of


22

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Biochemistry

ligand-FMN interactions also involves nonbonding interactions (Figure 3c and Supplementary Figure 4). About half of the nonbonding interactions with the isoalloxazine ring are mediated by aromatic residues. Tyr57, 59, and 95 in loop regions 4 and 6 are well conserved; Tyr59 and 95 sandwiches the isoalloxazine ring moiety of FMN (Figure 3c). In the Fld of Nostoc71 and D. vulgaris,72 the aromatic residues in these structurally conserved positions were shown to be critical in FMN binding and in stabilizing the three oxidation states of the FMN.74, 75 Therefore, the high conservation of aromatic residues, specifically Tyr residues, and their frequent participation in nonbonding interactions in Fld1C suggest that they serve an important role in stabilizing the coordination environment of the FMN cofactor.

FMN can exist in three oxidation states: an oxidized quinone, a one-electron reduced sq, and a two-electron reduced hq. The redox property of FMN is significantly tuned by the Fld fold, allowing Flds to participate in either one or two ET reactions. The first reduction of Fld1C, E2 couple, which forms a neutral sq (Figure S9), has a redox potential of –270 mV (pH 7.0) (Table 2). This value is within the range of the values reported for LC Flds but is 130 – 210 mV more negative than its SC flavodoxin counterparts, indicating that the sq state is less stable in the SC Fld fold of Fld1C. Loop 4 is known to modulate the E2 redox potential through a structural rearrangement of an O carbonyl in the ~60 position in both SC48,

76, 77

and LC Flds47

(Supplementary Figure 12). SC Flds typically contain a Gly residue at the ~60 position, while LCs possess a polar/branched residue (Asn or Tyr) at that respective position (Figure 6). Fld1C also contains a charged/branched residue, Asp, (Tyr59-Asp-Glu-Gly) (Figure 6). Mutation of the Gly residue to a polar side-chain containing residue (e.g. Asp, Asn, or Thr) in the SC Fld of C. beijerinckii48 resulted in 60 –180 mV decrease in the E2 potential, which reflected a +1.3 – 4.0


23

Biochemistry

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Page 24 of 37

kcal/mol penalty associated with the loop rearrangement. The opposite change (Asn60Gly) in the LC Fld of S. elongatus (PCC 7942),47 resulted in a +46 mV increase of the ox/sq potential. Fld1C also has a Tyr residue at the 98 position that packs against the residues in loop 4 (Figure 6), which may further destabilize the ox/sq redox couple. The SC Flds of D. vulgaris and C. beijenrinckii lack this secondary interaction, in contrast to the LC Flds of Nostoc, S. elongatus, and E. coli; the LC Fld of E. coli also has a Tyr residue at that position. In the context of these mutational analyses and based on our structural and redox characterization, Fld1C, like LC Flds, may therefore undergo a similar energetically unfavorable rearrangement during the ox/sq redox couple, which could explain the lower E2 value.

The second reduction step, E1, was shown to be modulated by a backbone amide interaction (~90 position) with the N1 atom of FMN which prevents a second protonation step, generating an anionic hq species.78, 79 In Fld1C, the backbone amide of Asp92 forms a hydrogen bond with N1 atom of FMN (Figure 3c and FigureS4). Aspartate 92 is highly conserved in the Fld1C cluster, suggesting that this interaction is characteristic of the clade that includes Fld1C. Furthermore, charge repulsion around and solvent exposure of the FMN cofactor creates an unfavorable environment for the anionic hq, resulting in low potential values of –380 – –450 mV56 (Table 2). Fld1C has an E1 value of –344 mV, which is 40 – 110 mV more positive than the values determined for SC and LC Flds (Table 2). Removal of the acidic residues near the FMN increased the sq/hq potential by ~15 mV per residue,56 and since Fld1C has a higher E1 value, this suggests that Fld1C should have fewer negatively charged residues near its FMN center. Fld1C has nine acidic residues (within a 15 Å distance) that surrounds its FMN, but the Fld of C. beijenrinckii (Supplementary Figure 13) has six; this Fld has an E1 value of –399 mV.


24

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Biochemistry

Therefore, acidic residues alone do not account for this more positive E1 value. Moreover, the E1 value of LC Flds tends to be more negative than the values for SC Flds (Table 2), which was attributed to the lower solvent accessibility of the FMN center.47 The SC Fld isoalloxazine is more solvent exposed. Considering the E1 value of Fld1C, when compared to SC Flds, one would predict that the FMN of Fld1C is more solvent exposed. However, our structure shows that the FMN center is less solvent exposed (112 Å2) than the cofactors of D. vulgaris (127 Å2) and C. beijenrinckii (159 Å2) SC Flds (Supplementary Figure 14); they have E1 values that range from –400 to –440 mV. Accordingly, solvent accessibility also does not fully account for the high E1 value of Fld1C. Short Chain Flavodoxins W90

Y98

Y100

H100 Y59, D, E, G

M56, G, E, E

W60, G, M, E

L. reuteri E2 = −270 mV

D. desulfuricans E2 = −58 mV

G61

G57

G61

D60

W60, G, D, D

C. beijerinckii E2 = −92 mV

D. vulgaris E2 = −143 mV

Long Chain Flavodoxins

W57, N, V, G N97 N58

Nostoc sp PCC 7120 E2 = −196 mV

W57, Y, Y, G

W57, N, I, G N97 N58

S. elongatus PCC 6301 E2 = −221 mV

Y97 Y58

E. coli E2 = −260 mV

Figure 6. Comparison of loop 4 residues among Flds in the oxidized state. Residues in bold correspond to those that undergo O-down to O-up rearrangement during ox/sq couple. The


25

Biochemistry

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Page 26 of 37

dashed lines show where a new H-bond interaction is formed between these atoms upon reduction/protonation to the sq state.

Putative Role of Fld1C in the PDU1C Microcompartment. The role of flavoproteins in PDU BMCs remains largely enigmatic. The cob(II)/cob(I) couple in B12 regeneration/recycling is a thermodynamically unfavorable process (Em = –610 mV).80 The interaction of 4-coordinate cob(II) with PduO/ATR81, 82 was predicted to raise this potential to ~–360 mV,83 a value that is within the physiological range of flavoproteins. The sq/hq couple of Fld1C is 16 mV more positive than the cob(II)/cob(I) couple bound to PduO, and perhaps, does not provide the proper driving force (–∆G) to achieve this reduction. PduS has an even higher thermodynamic barrier to overcome, possessing Em values of –150 and –262 mV with and without the iron-sulfur clusters,26 respectively. However, in both the PDU1A and PDU1C microcompartments, in vivo B12 reduction may involve a flavoprotein-B12-PduO84 complex that properly tunes and enhances the reduction of the B12 cofactor. Furthermore, Fld1C has a smaller ∆E value (74 mV at pH 7.0) compared to its SC and LC Fld counterparts (∆E = of +190 – +330 mV) (Table 2). The Fld (Nrdl) involved in the reduction of ribonucleotide reductase in E. coli was reported to have a ∆E value of ~10 mV, which allows Nrdl to transfer two successive electrons.85 It is unlikely for Fld1C transfers electrons in a manner similar to Nrdl because the ∆E is greater than 59 mV, disfavoring a two-electron process. The low ∆E value of Fld1C may have functional significance in B12 reduction, however a structural explanation is not apparent. Future work on BMCassociated flavoproteins/flavodoxins is necessary to determine if the unique redox properties (e.g., E2, E1 and ∆E) of Fld1C are conserved and serve a functional role.


26

Page 27 of 37

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Biochemistry

Nevertheless, based on our structural and redox characterization, we propose that Fld1C takes on the role of PduS in PDU1C microcompartments in providing the electrons for the maintenance of the B12 cofactor (Figure 7). As proposed for PduS,26,

27

Fld1C is likely to be

encapsulated by “piggy-backing”86 on another encapsulated enzyme. Fld1C could be reduced by Fmr, which oxidizes the NADH cofactors generated within the BMC (Figure 7). Alternatively, Fld1C may receive electrons from the exterior of the shell via the putative redox centers of PduK, much like the PduS and PduT pair in PDU1A BMCs.26 Then reduced Fld1Cs, either in the sq or hq state, (or Fmr independently) first reduces 4-coordinate cob(III) species. The hq state of Fld1C transfers an electron to the cob(II)-ATR (PduO) complex to generate cob(I) species for adenosylation by PduO.

The PDU1C locus is suggested to have been acquired by specific Lactobacillus species via horizontal gene transfer (HGT).29, 87 Our bioinformatic analysis of cluster 1 in the phylogenetic tree supports (Supplementary Figure 1) this conclusion; only 3 of the 40 Lactobacillus genomes (e.g., L. reuteri, L. mellifer, and L. collinoides) contain a PDU1C locus (Supplementary Table 6). This analysis also suggests a single assimilation of Fld1C/pfam00258 into the PDU1C locus. Metabolomics studies on L. reuteri that encode the PDU1C locus demonstrate that the encapsulated B12-dependent dehydratase breaks down both 1,2-PD28 and glycerol.29,

30

Overexpression of a transcription factor in the PDU1C locus in L. reuteri resulted in upregulation of both B12 biosynthesis and the PDU BMC genes,30 including the Fld1C gene (Lreu_1727) and PduK (Lreu_1742) (see Table S1 of ref 26). The PDU1C also locus lacks two iron-requiring proteins, PduS and PduT, suggesting that PDU1C may be a PDU microcompartment that is adapted to low iron conditions. Interestingly, our bioinformatic


27

Biochemistry

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Page 28 of 37

analysis revealed that ten additional genomes in cluster 1 (Supplementary Table 6) possess a PDU-type locus that encode other flavoprotein genes. These flavoprotein genes are regularly adjacent to a PDU gene, such as the shell protein PduU and PduV. The latter is involved in the spatial organization of PDU BMCs.46 This suggests that there may be alternative roles for flavoproteins other than electron transfer in PDU microcompartments, perhaps in shell assembly.

Adenosylcobalamin Cob(III)alamin Fld1C (NADH + Fmr)

NADP+

NAD(P)H X

Fmr-FMN

Fmr-FMN–

Fld1C-FMNH– Fld1C-FMNH

PPP i

PduO ATP

Cob(I)alamin

Fld1C-FMN/Fld1C-FMNH

PduK

Fld1C-FMNH/Fld1C-FMNH–

PduK-

Co(I)

Co(II)

Co(III) Fld1C-FMNH Fld1C-FMN

ATP, ATR

Fld1C-FMNH–

Fld1C-FMNH

Ado

Co(III) PPPi

Figure 7. Proposed function of Fld1C in PDU1C BMCs. Fld1C provides the electrons in the reactivation/recycling of the B12 cofactor. Fmr: NAD(P)H-dependent FMN reductase.

CONCLUSIONS Functionally diverse BMC loci, including the PDU subtypes, the ethanolamine-utilization BMCs, and glycyl radical enzyme-associated BMCs, encode many different types of uncharacterized flavoproteins, implying that these redox active proteins serve an unrecognized and important role in redox reactions associated with the organelle. Here, we report the first characterization of a BMC-associated short-chain flavodoxin (Fld1C) encoded in the PDU1C


28

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Biochemistry

BMC of L. reuteri. Recombinant Fld1C exhibits the α/β fold of flavodoxins with the redox property to serve as a low-potential redox-active protein. Given that the genes that encode Fld1C and the PDU1C metabolosomes are co-transcribed,30 the PDU1C operon lacks a B12 reductase (PduS), and Flds are native reductants of B12 cofactors, our results suggest a functional role for Flds in the reactivation/recycling reaction of a BMC-encapsulated B12 cofactor. ASSOCIATED CONTENT Accession codes Atomic coordinates and structure factors were deposited in the Protein Data Bank (http://www.pdb.org/) with accession ID 5VEG.

Supporting Information. The following files are available free of charge. Experimental procedures (for liquid chromatography-mass spectrometry, circular dichroism, dynamic light scattering), bioinformatic analysis (Figure S1 & S3), FMN omit map (Figure S2), homology structures (Figure S4 & S5), mass profile (Figure S6), histogram (Figure S7a), circular dichroism spectrum (Figure S7b), cyclic voltammograms (Figure S8), UV-Vis spectra of redox potentiometry experiment (Figure S9), EPR-photophysical scheme (Figure S10), electrostatic potential map of flavodoxins (Figure S11 & S14), loop 4 region of C. beijerinckii (Figure S12), ribbon structures of flavodoxins with negatively charged residues shown as sticks (Figure S13), Fld1C sequences and primers (Table S1 & S2) gene content of PDU1A and PDU1C loci (Table S3 & S4), classification of the species in clusters 1, 2, 5, and 12 (Table S5), species that encode a PDU-type BMC locus (Table S6), and genes that are adjacent to flavoprotein genes in cluster 1 (Table S7).


29

Biochemistry

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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Phone: 517-432-4371. ORCID

Cheryl A. Kerfeld: 0000-0002-9977-8482 Funding Sources This material is based upon work supported by the U.S. Department of Energy, Basic Energy Sciences, contract DE-FG02-91ER20021 with infrastructure support from MSU AgBio Research (JSP, MS, BF, CA, and CAK) and by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences, and Biosciences, under contract number DE-AC02-06CH11357 at Argonne National Laboratory (J.N., O.G.P., L.M.U., D.M.T.). This research used resources of the Advanced Light Source, which is a DOE Office of Science User Facility under contract no. DE-AC02-05CH11231 Notes The authors declare no competing financial interest. REFERENCES [1] Axen, S. D., Erbilgin, O., and Kerfeld, C. A. (2014) A taxonomy of bacterial microcompartment loci constructed by a novel scoring method, PLoS Comput Biol 10, e1003898. [2] Kerfeld, C. A., Sawaya, M. R., Tanaka, S., Nguyen, C. V., Phillips, M., Beeby, M., and Yeates, T. O. (2005) Protein structures forming the shell of primitive bacterial organelles, Science 309, 936-938.


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Biochemistry

FOR TABLE OF CONTENTS USE ONLY

propanol

1,2-propanediol

AlcDH dioldehydratase

propionaldehyde

AldDH reactivase Adenosylcobalamin propionyl-CoA Cob(III)alamin

PTAC Fld1C

ATR Cob(I)alamin propionyl-PO 24

Fld1C e, H+

Catabolic BMC e

FMN FMNH FMNH– –240 mV –344 mV


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Structural and Functional Characterization of a Short-Chain

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