disease, recombinant protein expression, tetrahydrofolate (THF), X-ray crystallography Keywords: Database: methosulfate; THF, tetrahydrofolate; WT, wild-type. human medium-chain acyl-CoA dehydrogenase;mean MRE, residue ellipticity; phenazine PMS, hMCAD, ; ETF-ubiquinone ETF-QO, flavoprotein; transferring electron human hETF, dehydrogenase; dimethylglycine human hDMGDH, dimethylglycine; DMG, dichlorophenolindophenol; Abbreviations: title: Running FAX: +43(0)316-873 6952; E-mail:[email protected] Technology, Instituteof Biochemistry,Petersgasse 12/II, A-8010 Graz, Telephone: +43-(0)316-873 6450; To whom correspondence should be addressed: Prof. Dr. Peter Macheroux, Graz Universityof 3 Austrian Centre ofIndustrial Biotechnology, Graz, Austria Austria Graz, Graz, of University Biosciences, of Molecular Institute 2 1 Institute of Biochemistry, Graz Universityof Technology, Graz, Austria This articlehas beenaccepted for publicationundergone and full peer review buthas not been Conflict ofinterests: :Article type Original Article This article is This protectedAll rights byreserved. copyright. 10.1111/febs.13828 Version the and version this between differences throughcopyediting, the typesetting, pagination and proofreading process, which may leadto

Accepted ArticleStructureand BiochemicalProperties RecombinantHuman of Dimethylglycine Peter Augustin Peter Dehydrogenase and Comparison to the Disease-Related H109R Variant Disease-RelatedDehydrogenase H109R tothe and Comparison Structural data are available in the PDB database under the accession number 5L46. number accession the under database PDB the in available are data Structural Choline,dehydrogenase, electron transfer, flavin adeninedinucleotide (FAD),genetic Human Dimethylglycine Dehydrogenase Dehydrogenase Dimethylglycine Human Ag DMGO, 1 The authors The declare noconflict interests. of , Altijana, Hromic Arthrobacter globiformis Arthrobacter 2 , Tea Pavkov-Keller Tea ,

of Record. Please cite this article as doi: asdoi: article this cite Please Record. of dimethylglycine oxidase; DCPIP, 2,6- 3 , Karl Gruber , Karl 2 , and Peter Macheroux

1

predominantly found in individuals ofAfrican descent. bodyfluids causing muscle fatigueand afish like odor (OMIM: 605850). Interestingly,mutation the is human in dimethylglycine of levels higher significantly with deficiency dehydrogenase dimethylglycine suffers from lower stability andenzymatic activity [9,10]. The observed phenotype was described as 78351682-T-C) results in the exchange of histidine 109 toarginine (H109R). This variant reportedly sequences (Exome Aggregation Consortium online browser; http://exac.broadinstitute.org/variant/5- properties,thermostability and theredox potential ofhDMGDH. Moreover,we have successfully oxidoreductase (ETF-QO) forfurther utilizationin the mitochondrial respiratory chain [7,8]. flavoprotein (hETF), which in turn transfers the electrons to the membrane-anchored ETF-ubiquinone Regeneration of oxidized FAD is achieved by electron transfer to the human electron transferring N thus prevents releasethe of cell-toxic duringcatalysis [3,6]. Incourse the ofthis reaction the oxidationof DMG. On the other hand, THF is used as the acceptor of the incipientmethyl group and covalently attached via its 8 key- of this pathway, requires two cofactors: FAD and tetrahydrofolate (THF). The FAD is aldehyde, betaine,dimethylglycine andsarcosine to the amino acid glycine (Figure 1)[5]. hDMGDH, a neurotransmitteracetylcholine [4].Degradation of choline proceeds by consecutive oxidations via betaine variety ofvital biomolecules suchasmembrane the phospholipid phosphatidylcholineand the part of choline degradation (Figure 1) [2,3]. Choline isan essential nutrient and building block in a dimethylglycine(DMG)to sarcosine,and also to alesser extentthe conversion ofsarcosine to glycine as dimethylglycine dehydrogenase (hDMGDH, EC: 1.5.8.4) catalyzes the oxidative demethylation of emphasizing their importance in human metabolism [1]. The human mitochondrialmatrix flavoprotein characteristics. Sixty of percent theknownflavoproteins involved are inhuman diseases and disorders INTRODUCTION that the variant suffersfrom decreased protein stability, saturation and affinity. particular with respect to reactivity. A comparative studywith the H109R variantdemonstrated analysis of our biochemical data provided new insights into the kinetic properties of the enzyme in crystallized the wild-type enzyme and determinedthe structure3.1-Å to resolution. The structure-based known as as known development of an intracellular, heterologous expression system in hampered by insufficient heterologous expression of theprotein. In thepresent study we thereport concentrations.detailed A biochemical andstructural characterization ofhDMGDH wasthus far dimethylglycine dehydrogenase deficiency leading to increased blood and urinary dimethylglycine carbon metabolism and electron transfer to the respiratorychain. The naturalrare variantH109R causes tetrahydrofolate (THF) dependent,mitochondrial matrix enzymetaking choline partin degradation, one- ABSTRACT This article is This protectedAll rights byreserved. copyright. -5,10-methylene tetrahydrofolate isformed, which plays animportant role in one-carbon metabolism. The human flavoproteome comprises 90 with versatile functions, structures and protein The human dimethylglycine dehydrogenase (hDMGDH) is a flavin adenine dinucleotide (FAD) and Accepted A rare naturallyoccurring pointmutation found in 58 out of 118 Article Pichia pastoris Pichia ) providing the opportunity to determine kinetic parameters, spectroscopic spectroscopic parameters, kinetic determine to opportunity the providing ) α -position theto N3of ahistidyl residueand servesaselectron the acceptor in

656 (0.049%) analyzed human gene Komagataella phaffii (formerly protein. On the other hand, heterologous expression in the methanotrophic yeast yeast methanotrophic the in expression heterologous hand, other the On protein. This article is This protectedAll rights byreserved. copyright. Escherichia coli from rat[2,3,16]pig and liverwell astherecombinant [8] as [17,18] rat and human enzyme[19] using several publications havedealt with the characterization of mammaliandimethylglycine dehydrogenases development of diabetes, further emphasizing the importanceof theenzyme [13]. epidemiological study revealed a possible connection ofdimethylglycine dehydrogenasedeficiency tothe sarcosine levels and the enzymes involved are in the focus of research. Furthermore, a recent as abiomarker for aggressive prostate cancer [11,12]. Therefore, investigations on the regulation of (Figure 2B, lanes3) was approximately70 and25 mg and ofWT H109R the variant, respectively (40 or WT (Figure 2A). After cell disruption and Ni-NTA affinity chromatography,yield the of proteins higher amountsvariant thanthe underidentical fermentation conditions thusyielda higherto leading of in expressed was WT the that showed intensities signal of Acomparison pellet. cell wet kgof 1.9 to with methanol. Typically, fermentations werestopped after 96 h after methanol induction resulting in1.6 of as known (formerly dimethylglycine dehydrogenase (hDMGDH) in – Purification and Expression Enzyme RESULTS [25,26]. respectively oxidases, and dehydrogenases reactivity inthevanillyl familyoxidase applicableis tothefamily ofsarcosine and dimethylglycine isoalloxazinering system. Invein, this we show that a previously proposed model to rationalizeoxygen oxygen reactivity and supports concepts that arebased on gatekeeper residues inthevicinity of the and the H109R variant in structure of the humanwild-type enzyme. concerningkinetics, redox behavior andspectral properties. Furthermore, we have elucidatedthe crystal deficiencies we present a profound analysis of therecombinant hDMGDH and the H109R variant information on thehDMGDH appears insufficient and partly contradictory.In order to overcome these ( (hDMGDH) andanoxidase ( oxygenreactivity in flavoenzymes still is controversial a topic. Thedirect comparison ofa dehydrogenase to substrate binding and oxygen reactivity. The latter issue is especially interesting as the control of subsequent crystallographic analysis provided us with the opportunity to analyze the structure with regard function that leadsto DMGDH-deficiencyhumans. in Moreover,successful crystallizationofand theWT of loss putative the address to variant H109R the generate to system expression our employed also have parameters as well as physical and spectroscopic properties of wild-type DMGDH. Consequently, we pastoris Ag K. phaffiiK. DMGDH wasfirst identified andpurified fromliver ratin the 1950sand early [14,15]. 60s Since then Inyears, recent also the productof the reaction catalyzed by hDMGDH, sarcosine, attracted attention In order to characterize hDMGDH we have established a recombinant expression system forwild-type

Accepted ArticleDMGO)characterized was biochemicalin andstructural detail[6,20–23]. However,available the [24]) and a subsequent purification protocol. This approach enabled us to determine keykinetic cell lysates at different time points indicates a stable expression of the protein after induction as expression host.Furthermore, dimethylglycineoxidase from Pichia pastoris Komagataella phaffii Ag DMGO) allowed new insights into the structural elements involved in in involved elements structural the into new insights allowed DMGO) [24]) was successful. As shown inFigure 2A, Western blot analysis

Initial attempts toexpress the geneencoding human Escherichia coli Escherichia (formerlyknownas

(BL21 DE3) yielded largely insoluble insoluble largely yielded DE3) (BL21 Pichia pastoris Pichia Arthrobacter globiformis Komagataella phaffi or or Komagataella Komagataella

second, much higher DCPIPwas usedas electron acceptor data linearization using themethod of Eadie and Hofstee revealed a anin approximately 10-fold lower catalytic efficiencyof theH109R variant (Table 1). Notably, when was strongly increased bya factor of16 23and inthe ferrocene and DCPIPassay, respectively, resulting DMG (Figure 4A). dissociation constant of 280 ± 10mM clearly indicating that sarcosine isa much poorer substratethan the dissociation constant of 4.9 ±mMfor 0.3 DMG. Thus, the reductive rate is 5to times20faster than k of reoxidation of 0.006 ± 0.001 s rate observed an with slowly very proceeds buffer air-saturated with hDMGDH of Reoxidation kinetics. reduced FAD to the artificial electron acceptor is rate limiting inthe assaysusedevaluate to steady-state the from transfer electron the (Table assay that DCPIP and indicating 1) ferrocene the in determined DCPIP as electron acceptor. oxidized by ferrocene iscloser to the rate of flavin reduction, it appears that ferrocene is superior to from 370 to 350 nm and a bathochromic shift from 445 to 460 nm (Figure 2C). absorptionVis maxima ofFAD bound to either WT or the H109R variantexhibits a hypsochromicshift spectral properties with identical absorption maxima. In comparison withfree FADsolution, in theUV- enzyme bound FAD and free FAD (Figure 2C) whereashDMGDH-WT and the variant featured similar WT and variant protein, respectively. The UV-Vis absorption spectra show aclear difference between reaction measurements with sarcosineyielded a limiting rate of reduction of 0.7 ± 0.1 s electron acceptor the k the acceptor electron variant variant exhibited a two-fold higher activity than the WT in both assays. At the same time, the Determination of Kinetic Parameters for hDMGDH-WT and H109R Variant – Variant H109R and hDMGDH-WT for Parameters of Kinetic Determination purity. asimilar give not did but explored were chromatography, other chromatographic methods, like size-exclusion chromatography orhydrophobic interaction chromatography. Although weexperienced hDMGDH that tends toprecipitate atlow salt concentrations, concentrations after Ni-NTA affinity chromatography required for the subsequent anion exchange respectively. protein The loss mainly during occured the necessary bufferchangelower salt to proteinyields of 30 enzyme/gand wet2 mg cell weight) (17or1 of µg WT and H109R variant, were further purified using anionexchange chromatography (Figure 2B, lanes 4) resulting in lower enzyme/g wet cell 15 µg weight). In order to achieve higher purityfor crystallization trials, the proteins This article is This protectedAll rights byreserved. copyright. DMG concentrationDMG fitted tohyperbolic a equation yielding a limiting reductiverate of 17 ± s 0.3 solelymeasuredwith the wild-type enzyme. shown As inFigure 4A, the rate ofreduction as afunction of terms ofthe k cofactor. The obtained parameters show significant differences between the two assay systemsboth in ferrocene or2,6-dichlorophenolindophenol (DCPIP)electron as acceptoroxidize to reduced the FAD steady-state kinetic parameters forhDMGDH-WT H109R and variant wasconductedusing either

The A Accepted Due to much lower enzyme amountsavailable of the H109R variant pre-steady-state kinetics were Article K M was significantly higher (1.4 ± 0.1 versus 0.3 ± 0.002 mM, Table 1). Interestingly, the H109R H109R the Interestingly, 1). mM, Table ± 0.002 0.3 versus ±0.1 (1.4 higher significantly was 280 /A cat 450 450 as well as the ratios of the highly protein pure fractions were usuallybetween 14-16and 20-25 for the K cat M was 5-fold greater than with DCPIP (213 ± 4 versus 44 ± 1 44 min versus ± 4 (213 DCPIP with than greater 5-fold was value of of value K M for DMG (Figure 3, Table 1). When ferrocene was used as the artificial artificial as the used was ferrocene When 1). Table 3, (Figure DMG for ≈ -1 30 mM for DMG (inset Figure 3A). Since the rate at which FAD is FAD which at rate the Since 3A). Figure (inset DMG for mM 30 atanoxygen (inset Figure concentration 4A). ofRapid 135 µM

The evaluation of the -1 , Table and 1) Table , K M of DMG of DMG -1 anda -1 and a cat

confirm that the variantexhibits reduced thermal stability. 53 °C and 47°C, for WT and the variant, respectively (Figure 7B). Thus, both independent methods C similar to each other with an rmsd of 0.22 Å calculated after the superposition of 810 out of 828 dehydrogenase molecules in theasymmetric unit. The two molecules inthe asymmetric unit are very 25-95 °C showed that the WT and the H109R variant have melting points (T respectively (Figure7A). similar A result was obtained bymeans ofCD-spectroscopy yielding aT resolution (Table 2). crystalThe belongs tothe monoclinicspace group ( Protein Structure Analysis – determined using Thermofluor Thermal Stability ofhDMGDH-WTand hDMGDH-H109R – calculatedfrom six independent measurementsto -93 ±mV. 1 redox titration (Figure 6D). The redox potential for thereduction of FAD bound tohDMGDH was number of electrons ( of the double logarithmic plot was close to unityindicating that the dye andthe flavin receive anequal method described by Massey [27] using indigotrisulfonic acid potassiumsalt asreference dye.slope The In ordertodetermine the redox potential of hDMGDH we employedxanthine the oxidase/xanthine hDMGDH was reacted with air-saturated buffer in thestopped flow device (data not shown). semiquinone upon reaction with molecular dioxygen (Figure 6C). This was also seen when reduced dihydroquinone (Figure 6B), which is directly converted tooxidized the state without theoccurrence of a absorption maximumat 372 nm (Figure 6A). Further photoreduction eventually leadsto the fully reduced reduction, photoreduction ofhDMGDH leadsto the anionic (red)flavin semiquinonewith a typical state (Figure 4B) without any transient appearance of asemiquinone radical. In contrast to substrate showsDMG amonophasicconversion ofoxidized the to the FAD two-electron reduced dihydroquinone Photoreductionand Determination ofthe Redox PotentialhDMGDH of – This article is This protectedAll rights byreserved. copyright. surrounded by the rat enzyme [18]. Subdomain 1 (residues 467-537 and 626-728) contains a Greek-key motif divided into three subdomains positioned “cloverleaf”-likeain arrangement as previously described for group of theisoalloxazine moietyas confirmed by thecrystal structure. The folate bindingdomain canbe cofactoris covalently attached tothe protein through alinkage between of N3 His91 and the 8 466) and the folate binding domain (C-terminal domain with residues 467-855, Figure 8A). The FAD human enzymeconsists of two domains:FAD the binding domain(N-terminal domain with residues46- after superposition with the rat enzyme (rmsd of 0.37 Åcalculated for 739 out of 809 C THF.As showninFigure 5, theactivity ofdecreases WT asfunction a of the THF concentration. 0.1DMG,1 mM mM EDTA and0.2freshly mM preparedferroceneaconcentration in range of 0-75 µM describedas above25°C at andpH 7.8in 50 mM HEPES/NaOH with 150mM NaCl, 100 nM DMGDH, ferrocene assay (Figure 5). The activity measurements were done similarly to the ferrocene activity assay α ‑ Finally, we also studied the effect of tetrahydrofolate (THF) on the activity ofhDMGDH using the Accepted Article atomsusing the program PyMOL(DeLano Scientific). As previously observedin rDMGDH and also α -helices. Subdomain 2 (residues 538-625 and 729-770) is defined byfive-stranded a i.e. two) althoughanionic the semiquinone radical isclearly observableduring the

The X-ray crystal structure of hDMGDH was determined at3.1 Å ® and CD-spectroscopy. Thermofluor

The thermalThe stability ofbothenzymeswas ® temperature scans from from scans temperature

Reduction of hDMGDH by m P ) of 52 °C and 47 °C, 47 and °C 52 of ) 2 1 ) and contains two α -atoms), the α -methyl- m of solution(compare spectra shown in Figure 9). fact, theonly publishedspectrum ofhDMGDH expressed in very poor quality orapparently lack thespectral features that are typically found in these enzymes.In absorption spectrafor heterologously expressedrat DMGDH [17]and human DMGDH [19] areeither of that could be conducted. beconducted. could that Overall,thisled tosubstantiallyreducedyields of thevariant protein and limitedthe scopeof experiments ofapo-protein may beresponsiblethe recurring for protein precipitation during purification ofvariant. the the WT (ca.to 40-50%apoprotein), similarto previously reported observations [19]. Thehigher fraction FAD. Inofcase thevariant, the ratio was much higher indicatinga muchlower saturation compared H109R variant respectively, revealed that the WT is almost fully loaded with the covalently attached calculatedto 14. observedThe A isolated from rat and pig liver aswellrecombinant asfor DMGDH for earlier reported those to similar very are characteristics spectral These 2C). (Figure solution characteristic UV-Vis absorption properties that clearly distinguish the bound FAD from FAD free in Spectral Characteristics and Steady-state Kinetics of hDMGDH – (Figure 2A). Westernblot analysisshowed that theH109Rvariant isexpressed inloweramountscompared the to WT [18],this C-terminal tag should not interfere with the native fold of theprotein or catalytic activity. means of affinity chromatography. According to the previously published structure of the rat enzyme blotting. A C-terminal nona-histidine-tag was added to the gene in order to facilitate purification by before [19] was unsuccessful and failed yieldto detectable amounts of protein as judged by Western enzyme resulted in improved protein yields. The expression of hDMGDH in targeting sequence [28].intracellular The co-expression of the enzymewasexpressed successfullyrepresenting the mature form ofthe enzyme lacking its mitochondrial version aswellas secretory expression were unsuccessful. However, an N-terminal biochemicalstructuraland characterization.The expression offull-length the protein anda expression of hDMGDH in DISCUSSION enzymes, it is still very likely thatfolate binds in the sameposition (Figure8B). enough tounequivocally position a folate molecule. Based on the structural similarity of the two the diffraction data and/or an incomplete occupancyof theligand, however, this densitywas notclear differenceelectron density in the same area in the folate binding domain. Dueto the lower resolution of structure of the rDMGDH wasdetermined in complex with tetrahydrofolate [18].We also observed some antiparallel This article is This protectedAll rights byreserved. copyright. ferroceniumhexafluorophosphate electronas acceptor andontheotherhand combination the of These values were obtained using two different enzymatic activity assays, on theone hand with Heterologous Enzyme ExpressionPurificationand – AcceptedA summary of ourdata and the previously published kinetic steady-state data is given in Table 3. A theoretical A Article β -sheet with flanking flanking with -sheet 280 /A 450 ratio for hDMGDH fully saturated with FAD cofactor can be roughly K. phaffii K. 280 α -helices. Subdomain 3 (residues 771-850) adopts a jellyroll fold. The /A 450 is feasible and provides enough hDMGDH toperform a detailed ratios of the highly pure proteins, 14-16 and 20-24 for WT and WT for 20-24 and 14-16 proteins, pure highly of the ratios

In studywe this demonstrated intracellularthat Ag

Saccharomyces cerevisiae DMGO[8,16,20]. Incontrast, theUV-Vis E. coli

Recombinant hDMGDH features is very similar to free FAD in Δ 28 truncation of the E. coli protein disulfide disulfide protein Δ 50 truncated truncated 50 as reported reported as or H componentDCPIP activityassay,with PMS aselectron mediator, butnot the in onecomponentferrocene be inspected further. further. inspected be not could protein the to binding FAD of analysis therefore and protein insoluble solely produced system variant is clearly more similar to that of free FAD (Figure 9). In hands, our the bacterial expression non-covalentas binding. Infact, the absorption spectrum reported forwild-type DMGDH and its H109R recombinant DMGDH generated inbacterial host cells suffersfrom analteredFAD binding mode, such enzyme areunwarranted [16,17]. Moreover, we haveshown that the ofrate enzyme reduction isfaster than k speculations that the presence of two observed for DMGDH isolated from natural sources ( sources natural from isolated DMGDH for observed preparationsobtained from the significantly decreased catalytic activity of the H109R variant. Since the spectral properties ofDMGDH Hence,our results areclearly in contradiction with previously published data [19] that claimed experiments. to hDMGDH is more reliably expressed as the dissociation constant determined in pre-steady-state K stability.Interestingly, the variant showedelevated turnoverrates bothin ofour assay systems although Further characterization of the variant protein demonstrated lower cofactor saturation and thermal K. phaffii a mild phenotype including muscle fatigue and fish like odor [9,10]. During heterologous expression in 605850).defect The results inanaccumulationof DMGin blood serumand urineandpatients suffer from variant H109R causes a non-fatal disease called dimethylglycine dehydrogenase deficiency (OMIM: Effectsofa Naturally Occurring H109REnzyme Variation– apartfrom theresults reported by McAndrew Michaelis-Mentenconstants similarto previously published data forthe rat enzyme[16,17]. Interestingly, bacterialthe DMGO [22]. However, analysisof the data obtainedwith thePMS/DCPIPyieldedassay two ferrocenethe assay, wehave obtained comparable results as reportedfor the rat enzyme[18] and also for interact with the electrontransferring flavoprotein (hETF)for reoxidation of the reduced cofactor. With DMGDHs mammalian all as irrelevant physiologically are assays Both acceptor. electron terminal phenazinemethosulfate (PMS)electron as mediator and2,6-dichlorophenolindophenol (DCPIP) as This article is This protectedAll rights byreserved. copyright. . turnover rate (Figure 5) asthe substrate DMG competeswith THFforentrance into the cavitythe THF at accordance with this interpretation, increasing THF concentrations exhibited a negative effect on the structurallythe related DMGO[6] andalso confirmed by cavity analysis ofourstructure (Figure10A). In binding siteof THF is the only entry-exit point ofthe protein, which wasrevealed by MD simulations for active substrate site, conversion, aswell asintermediate channeling toTHFandproduct formation. The thein protein [6,18]. This cavity serves“reaction as chamber” responsible for substrate delivery tothe M values were 15-25times higher and thecatalytic efficiency 10-fold lower than for the WT (Table 1). Accepted Previous reports on thestructure of bacterial DMGO andrat DMGDHidentified a large internal cavity Article 2 O cat 2 indicating that reoxidation is rate-limiting for turnover inboth assays. Thus, theaffinity DMGof assay suggesting that it is an experimental artifact. In view ofthis apparent inconsistency, wenoticed decreased expression levels resultingin much yieldslower ofvariant the protein. E. coli E. K M values reflect the presence ofactivating regulatory or sites in the expression system lacks the characteristic features typically et al. [19]two e.g. rat and pigliver) it isconceivable that

K M values were only observed thetwo for

The rare naturally occurring enzyme extensively discussed by Luka discussed extensively [6]. THFThe binding sites of mammalian and bacterialenzymes againare very similar and were already intermediate,most likely acycliclactone ischanneled throughthe protein cavityto the THF binding site of hDMGDH (Figure 11A). After binding and conversion of the substrate at the , an (Figure 11). Furthermore, residues interacting with DMG (andsarcosine) were identified inthe structure deprotonation of the substrate amine [21]. This catalytic dyad isalsopresent inthehuman enzyme relative turnover of sarcosine by these three FAD-dependentenzymes must await further studies. dehydrogenase (SARDH) [16] andalso by peroxisomal (PIPOX) [29]. Therefore, the Ag physiologicallyrelevant substratefor theenzyme already as shown previously for thepigenzyme [3]and obtained forthe bacterial DMGO (Table 4)[22]. Therefore, sarcosine is apoorbutpossibly is more permissible in transferring electrons from the reduced FAD to the artificial electron acceptors. acceptors. electron artificial the to FAD reduced the from electrons transferring in permissible more is (Figure 10B). theother On handthevariant H109R possessed higher k suggests that replacement ofhistidine bybulkier the arginine impedes access of DMG tothe binding site the substrate and amore than 50-fold higher sarcosine as substrate for hDMGDH-WT show a25-fold decrease of the reductiverate for sarcosine as in the crystal structure of DMGO (Figure 11C). Pre-steady-state measurements comparing DMG and differencesobserved inaffinityand catalytic activity.This binding similar mode is acetate tothe moiety results in a similar positioning of both substrates (Figure 11A, B) and thus do not rationalize the hDMGDH (Figure 11). Docking of DMG and sarcosine to the active site of the hDMGDH structure active site of ion that mimics thenegatively charged carboxylate group of DMG ( could be crystallized with a bound folic acid ( similar to the rat enzyme ( the active site of hDMGDH with the covalently FAD bound as well as the THF bindingare site very Substrate Specificity, Redox Behavior and Active Site Composition – This article is This protectedAll rights byreserved. copyright. overall reduced DMGDH activitymanifested in affected human subjects. mayberelevant also for homologous expression ofvariant the in human cells and thuscontribute to the exchange.acid diminished The protein expression and cofactor saturation foundin ourexpression system and anapproximately 10-foldlower catalytic efficiency 1) (Table as major effects of the single amino as described in detail elsewhere [6]. [6]. elsewhere detail in described as measurementof theintermediatecyclic lactone couldobserved,be not most likely short lifetimedue toits reduction of hDMGDH by DMGwas adequatelyfit by a single exponential equation. The actual DMGO [22]. On the other hand, sarcosine is apparently oxidized byahomologous sarcosine In the bacterial enzyme, acatalytic dyad,consisting of ahistidine and atyrosine, isresponsible for In conclusion,ouranalysis ofdisease the related H109Rvariant has revealed a lower thermal stability higher 23-fold) to (16 consistently The Accepted Basran Article et al. et Ag reported amultistepkinetic model for the reduction ofDMGO DMG. by In contrast, the DMGO described to play arolein and substrate binding are also present in PDB ID: 4PAA ID: PDB et al . [18]. . [18]. ; [18]), and the structure of K PDB ID: 1PJ6 ID:PDB M K observed in both steady-state assayswith the variant D . These results are in verygood agreement with data ) ) but additionallyalso with abound acetate

PDB ID: 1PJ5 Ag cat

The overall protein structure and and structure protein overall The values indicatingthe that variant DMGO [21]. In2003, ). Mostresidues inthe Ag DMGO DMGO Pichia pastoris or Komagataella pastoris pastoris or Komagataella pastoris Pichia designed for intracellular enzyme expression in hDMGDH ExpressionStrain Generation– (Karlsruhe, Germany) and Sigma-Aldrich (St.Louis, MO, USA)and were of the highest grade available. TCIN.V.Europe (Zwijndrecht, Belgium). All other chemicals and media were Roth from Carl GmbH redox dye for the determination of the redox potential, indigotrisulfonicacid potassium salt, was from The UK). (Cambridge, Technology Signaling Cell from were blot Western for used Antibodies Austria). Giles, andUK) salt free purified oligos for site directed mutagenesis from VBC-Biotech (Vienna, Scientific (Waltham, MA, USA), purification columns andmaterials from GE Healthcare (Chalfont St This article is This protectedAll rights byreserved. copyright. expression in ETF-bound FAD. oxidized/semiquinone couple, [33]) ensures thatelectrons from hDMGDH are readilytransferred to the potential [32]. In anycase, the much higher redox potential of hETF (+37 mV for the [30,31]) in agreement with the fact that covalent linkages tothe8-methylgroup increase the redox hDMGDH. obtainedThe value of -93 ± 1mV is40 mVpositive more than that ofhMCAD (-135 mV determined[30,31]. promptedThis us todeterminethe redox potential for thecovalently bound FADin electronreduction of human medium-chain acyl-CoA dehydrogenases (hMCAD) waspreviously two- the for potential redox the only clients these Among (ETF). flavoprotein transferring electron the to and isoleucine,whereas that hDMGDH features residues typical fordehydrogenasessuppressing oxygenreactivity, belong to the VAO-,comparison of the hDMGDH to the prolinestructurally at a conserved position nearthe isoalloxazine ring [26]. AlthoughhDMGDH does not Leferink valinewas foundgrant to access to the whereas isoleucine deniesaccess [25]. Similarly, reaction intermediates during oxidation of the reduced isoalloxazine ring by dioxygen. In this family, allergenPhl p 4 [25]. This gate keeperappears to control accessan tooxyanion hole essential to stabilize oxidase (VAO)family, discovereda new “gatekeeper” residue inberberine bridge enzyme andthe pollen hETF proceeds over 30 times faster (unpublishedresults). 300 timeslower and apparently physiologically irrelevant asreoxidation by its cognate partner protein than more is hDMGDH reduced of reactivity oxygen the that, to 4).Incontrast (Table DMGO bacterial Enzymesand Reagents – PROCEDURES EXPERIMENTAL concerned residues has tobe conducted. (DAO) family of FAD-dependent (pfam: 01266), a detailed mutagenesis study ofthe oxidase acid D-amino the of member a DMGDH, to applicable is flavoproteins in controlled is reactivity and valine (Figure 12). Nevertheless, in order furtherto support, if the emergingconcept of howoxygen The The rates of reduction determined for hDMGDH are in good agreement with those observed for twoThe dehydrogenases in choline degradation belongfamilyto a ofenzymes delivers that electrons vanillyl-alcohol the of enzymes in reduction dioxygen of control steric concerning study a Recently, Accepted Article et al. K. phaffii discovered that a large numberof oxidases in the VAOfamily contain either aglycine or a using GeneOptimizer Ag

Restrictionenzymes andPhusion DNA polymerasefrom were Thermo Fisher DMGO possesses residues compatible with high oxygen reactivity, hDMGDH and hDMGDH-H109R expression plasmids were were plasmids expression hDMGDH-H109R and hDMGDH [24]). hDMGDHThe sequence was codonoptimized for ® (ThermoFisher Scientific). Inaccordance to Binzak Komagataella phaffii

KM71H strain (formerly known as as known (formerly strain KM71H Ag DMGO structure reveals i.e. analanine i.e. proline et al ., KOH – 18.2 g/L; NaCl – 4.13 g/L; glycerol – 40 g/L; 85% H 85% 40 g/L; – – 4.13 NaCl g/L; glycerol g/L; – 18.2 KOH 5’-CTTGATGGAGTCGTA TCTGATCTTCTTCAAGTT-3’ as reverse primer (alternated codon codon (alternated primer reverse as TCTGATCTTCTTCAAGTT-3’ 5’-CTTGATGGAGTCGTA and forward as 5’-AACTTGAAGAAGATCAGATACGACTCCATCAAG-3’ and polymerase DNA Phusion employing method mutagenesis directed site by two-step a constructed was variant Scientific). Positive clones wereselected byZeocin resistanceverified and by Western blot. The H109R cerevisiae from isomerase protein-disulfide the for gene the containing vector pPICK-PDI recombinant plasmid was transformed to into apPICZB expression vector (Thermo Fisher Scientific) and verifiedby automated sequencing. The startthe codon (AAAA) and aC-terminal nona-histidine tag were added. The designed gene was cloned cycles. Then, after a 2h Afterwards, the twoPCR reactionswere combinedand the sameprogram was employedfor another 20 underlined).For the first reaction step,two separatePCR reactions using either the forwardor the reverse were cleared by centrifugation(18 lysates The disruption. cell before added were of FAD tip a spatula and 7.8) pH PMSF, mM 1 imidazole, everygharvested 1 wet cell pellet, 2mL cell lysis buffermM (50 HEPES/NaOH, 150mM NaCl, 35 mM (4 taken for Western blot analysis atdifferent time points.cell The pellet was collected by centrifugation 400-500 g methanolwere fed tothecultures before harvest after 96hof inducedgrowth. Samples were g/hover6 2h andamixed MeOH/glycerolwas feed maintaineduntil the endof thefermentation. In total, stepwise increase of methanolfeed from 0to6g/h over 2h.Glycerol feed wasreduced from 15g/h to methanolinduction was by started injection of5 g MeOH directlythe fermenter into andafterwards by a (50 mM HEPES/NaOH,150 mMpH NaCl, 7.8) using Amicon ultracentrifugal units filter (50 kDa cut- SDS-PAGE and fractions containing hDMGDH were concentrated and rebuffered tostorage buffer by monitored was purification The 7.8). pH 300 imidazole, mM NaCl, mM 150 HEPES/NaOH, mM (50 buffer elution with stripped enzyme the and pH7.8) mMimidazole, 75 NaCl, mM 150 HEPES/NaOH, columns (GE Healthcare) at4°C. Afterwards, the material waswashed with washing buffer (50 mM affinitychromatography was donebyapplying the lysates onto self-packednickel-Sepharose 6Fast Flow photometric studiesofthe enzymes. Ifhigher enzyme a puritywas especially needed, for protein off, Merck-Milipore, Darmstadt, Germany). The obtained enzyme puritywas sufficient for kinetic and Melsungen, Germany) glassbeads cell homogenization under CO Enzyme Purification fermentation medium was aminimal basalsalt medium (MgSO by Schrittwieser as described out carried were fed-batch and batch fermenter, the fermenter BBI CT5-2 system (Sartorius, Göttingen, Germany). Preparation of inoculum, preparation of Enzyme Expression done as described above. cells for amplification before transformation to primer were run (98 °C (2 min)–[98 °C (50 sec) – (2060 °C sec) –68°C (16.5 min)] x 5–4 °C with sequencethe of themature form ofhDMGDH (lackingthe first 28amino acids, [28]) wasflanked used, This article is This protectedAll rights byreserved. copyright. crystallization, the eluted fractions of the affinity chromatographywere rebuffered to buffer(25 A mM

Accepted °C. at -20 30 min, 4 °C) and rpm, stored 000 Article

Xho I and I and following the guidelines ofInvitrogen the EasySelect Not I restriction sites.For enzyme expression and purification,a Kozak sequence infront of – – hDMGDH and hDMGDH-H109R expression was carried out in a 7 L glass Cell lysates were prepared by Merckenschlager (Braun Biotech International, Dpn I digestion step, the plasmid was transformed to

000rpm, 30min, 4 °C) andfiltration throughapaperfilter. Nickel ion K. phaffii K. phaffii KM71H strain (Thermo Fisher Scientific), harboring a a harboring Scientific), Fisher (Thermo strain KM71H KM71H [pPICK-PDI]. Strain selection was

3 PO TM 4

2  4 Pichia cooling (3 min disruption steps). For steps). disruption min (3 cooling 7 H – 27mL/L). After the fed-batch, the 2 O – 1.2 g/L; K g/L; 1.2 – O Expression(Thermo Kit Fisher Escherichia coli 2 et al. et SO Saccharomyces 4 – 14.9 14.9 – g/L; [34]. The TOP10 ∞ ). 300 nm overmin.3 For each concentrationat leasttriplicate a measurementperformed, was the initial (Fc indophenol (DCPIP) according to Okamura-Ikeda (Thermo Fisher Scientific) and detection in a G:BOX (Syngene, Cambridge, UK). UK). Cambridge, (Syngene, inaG:BOX detection and Scientific) Fisher (Thermo Immunoreactivesignals were obtained using SuperSignal West Pico Chemiluminescent Substrate used. As loading control, a rabbit anti-GAPDH antibody was used in a 1:1000 dilution overnight. linked goat anti-rabbit IgG antibody 1:5000 at for 1 h atroom temperature as secondaryantibody were antibodies,As a rabbit anti-histidine IgGantibody at1:2000 overnight at 4°C as primary andan HRP- following theGeneral Protocolfor Western blotting from Bio-Rad (Hercules, CA, USA, Bulletin6376). (p.42, Thermo Fisher Scientific). Western blot analysis was doneon nitrocellulosemembranes essentially Steady-state Kinetics Macheroux [36] to 11 450 nm. molarThe extinction coefficient for hDMGDH was calculated using themethod described by purified hDM Enzyme Quantification and Calculation of the Extinction Coefficient – were recordedwith a Specord210 spectrophotometer (Analytik Jena, Jena, Germany). purity, qualitycofactor and saturationas well foras steady-state kineticmeasurements and photoreduction UV-Vis AbsorptionSpectroscopy prepared by glass bead disruptionfollowing the Invitrogen EasySelect Western Blot thevariant enzyme. rebuffered to storagebuffer as described above. Purification was done identicallyfor thewild-type and and concentrated were hDMGDH containing fractions and by SDS-PAGE controlled was graphy elutedwithagradient from 0-50% buffer (25 B mM HEPES/NaOH, 1MNaCl, pH7.8). The chromato- column (GE Healthcare) wasdone on an FPLCÄKTA system(GE Healthcare) at4°C. proteinThe was HEPES/NaOH, 25 mM NaCl, pH 7.8) and anion exchange chromatographywith aMonoQ 5/50 GL This article is This protectedAll rights byreserved. copyright. standard a PageRuler Prestained protein ladder (Thermo Fisher Scientific) was employed. employed. was Scientific) Fisher (Thermo ladder protein Prestained PageRuler a standard Western blot analysisstained or with Coomassie BrilliantBlue forR-250 purification control.As protein Western blot 10%), under reducing conditions as described byLaemmli [35]. Gels were either used for hDMGDH,0-100 mM andfreshly DMG prepared 0.2 mM Fc nM 150 100 25 at EDTA, mM mM HEPES/NaOH, 0.1 50 in mM NaCl, 7.8 performed pH at °C were following thechange of absorption spectrophotometrically 600 at nm over 3 min. Ferrocenium assays phenazinemethosulfate (PMS) as intermediateelectron mediatorand 0-100 mM dimethylglycine(DMG) HEPES/NaOH, 150 mMDCPIP NaCl, 135 and100 µM nM hDMGDH with freshly prepared 3 mM 50 in 7.8 pH and °C 25 at performed were DCPIP-assays mM In short, spectrophotometer. the

Accepted+ Article SDS-PAGE –

PF 6 - ) as described by Lehman – GDH enzymes were GDH enzymes K. phaffii Enzyme samples were separated by SDS-PAGE, with 12.5% separation gels (for

– 600 M 600 Steady-state kinetic parameters were determined using 2,6-dichlorophenol- cell lysates for Western blot screening and control of fermentation were -1 cm -1 – determined according the characteristic absorption of bound FAD at . et al. UV-Vis absorption spectraassess to protein concentration, activity, [38] asterminal electron acceptor and measurable dimension in et al. et [37] or ferrocenium hexafluorophosphate

+ PF 6 - following thechange of absorption at TM

Pichia

Protein concentrations of Expression Kit manual manual Kit Expression (k was determined by ahyperbolic fittingcurve employing(OriginLab 8.6 Origin Corp.). oxidativeThe rate pH 7.8). 7.8). pH 150 NaCl, mM HEPES/NaOH, mM (50 buffer equilibrated air with hDMGDH reduced substrate mixing WA, USA). according to Minnaert [39]. A linear least-squares fit was donewith Excel 2010 (Microsoft, Redm calculated using double logarithmic plots, log(ox/red) of the enzyme versus log(ox/red) of the dye, dye indigotrisulfonic acid dipotassium (E salt ofxanthine oxidase (approx. 200 nM, frombovine milk, Grade III purity, Sigma-Aldrich) andthe redox benzylviologen andappropriate an 5 µM amount of enzymewith asolutioncontaining catalytic amounts xanthine, µM 300 containing solution a bymixing started was reaction The Limited). Scientific TgK (MG-6560, detector array diode KinetaScanT a with monitored was dye redox the and FAD of reduction (Belle Technology) at 25 °C in 50 mM HEPES/NaOH, 150 mM NaCl, pH 7.0. The simultaneous Scientific61DX2, TgK Limited).measurements The tookunderanaerobicplace glove conditionsa box in maximawere in the same range. The reactions were performed with a Hi-Tech stopped-flow device(SF- Massey [27]. Theconcentrationsof enzyme andredox dyewere chosen in a way thattheir absorption measured by thedye-equilibration method using the xanthine/xanthine oxidase system asdescribed by fittingfunction inthe Kinetic Studiosoftware (TgK Scientific Limited). The dissociation constant ( were recorded until nofurther changes wereobserved. observed. For reoxidation ofthe enzyme, the cuvettes were exposed airto oxygenand absorption spectra Germany) andcooling of the cuvette to15 °C. Spectra were recorded until no further changes were Technology). Photoirradiation was carried out with a10 W LED flood light (Luminea, Buggingen, viologen were rendered anaerobic by 2h incubation insealable quartz cuvettes in aglove box (Belle methyl µM 2 and 5-diaza-FMN 1µM 150 EDTA, 1 mM containing mM NaCl, 7.8 pH HEPES/NaOH, procedure reported by Massey and Hemmerich purified [40]. 20enzyme µM sample in 50 mM Anaerobic Photoreductionand Reoxidation – Determination of the Redox Potential– observed rate constants (k detector (MG-6560, Scientific TgK Limited). Each concentration was measured intriplicates and the mM HEPES/NaOH, 150 mM NaCl 7.8 pH and monitored at 460nm with a KinetaScanT diode array shot againstdifferent concentrations of (0.25-250DMG or sarcosine mM) (0-500dissolved mM) in 50 bound FAD, purified 20 hDMGDH50 µM in mM HEPES/NaOH, pH 7.8 containing 150mM NaCl was box (Belle Technology, Weymouth, UK) at 25 °C. For determination of the reductive rates of enzyme aglove in UK) Bradford-on-Avon, Limited, Scientific TgK (SF-61DX2, instrument flow stopped Tech Pre-steady-state Kinetics – Corp., Northampton, MA, USA). velocities determined and This article is This protectedAll rights byreserved. copyright.

Accepted Articleox ) of theenzyme wasmeasured three times at an oxygen concentration of 10.5% O

obs K

) for) different substrate concentrations were calculated using anexponential Pre-steady-state reaction kinetics were measured anaerobicallywith aHi- M andk cat assessed by non-linear hyperbolic fit in Origin 8.6 (OriginLab

The redox potential (E potential redox The

0 Photoreduction of hDMGDH was done according to the , pH 7.0, 25 °C = -81 mV). The redox potential was was potential redox The mV). =-81 25°C 7.0, pH ,

0 ) of the enzyme bound FAD was 2 (135 µM O (135 µM 2 ), by by ), ond, K D ) group C ellipticity at208nm. melting temperaturewas estimated as the point ofinflection ofthe temperature dependent mean residue concentration used was in50 approximately 6µM mM HEPES/NaOH, 50 mM NaCl, pH 7.8. The with°C aheating rate of 1°C/min, a response of 1 sec and a resolution of 0.1°C. The protein F25, Seelbach, Germany). Thermal denaturation data were recorded inatemperature range from 25–95 jacketedcylindrical cell thermostatically controlledan external by computer-controlled water(Julabo bath were recordedonPS-150J a spectropolarimeter (Jasco, Groß-Umstadt, Germany) using a0.02 water-cm using CFX Manager 3.0 software (Bio-Rad). 3.1Å onbeam lineID29 ( a Thermofluor monitoringthe change fluorescence in using the solvatochromic dyeSYPRO Orange (Sigma-Aldrich) in Fluorescence datawere collected using theFRET channel. Melting temperatures (T sampleswere pre-heated to 25°C and then the temperature was increased in 0.5 °C/min steps to 95°C. ofof a500 enzyme x of dilution aSYPRONaCl, 2.5 µL 10 and µM Orange Enzyme Gelstain. The Connect real time PCR system of 50mM(Bio-Rad) HEPES/NaOH, in 25 µL 7.8, pH containing150 mM Data Collection and Processing – PEG MME550, 0.02 M of each aminoacid and0.1 M MOPS/HEPES-Na pH 7.5. v/v 20% 20000, PEG w/v 10% of consisting Screen Morpheus the of H5 condition original the of dilution HEPES,mM 25mM NaCl,mMDTT, 2 pH 7.5. Diffracting hDMGDH crystals were obtained with a1:3 month in various conditions.Further optimization involved dilutions oforiginal the condition using 25 siliconand oil trays the incubated at20°C. Firstcrystal clusters wereobserved after approximately one robot (DouglasInstruments, Hungerford, UK). drops The were sealed with a 3:1 mixture of paraffin and ORYX an using 8 pipetting liquor mother of volume an equal with 2 and 7.5) pH NaCl mM DTT, mM prepared by mixing 0.5 (Aliso Viejo, CA, USA)and Molecular Dimensions(Newmarket, UK),respectively). Dropswere using different commercial crystallization screens (Index andMorpheus Screen from Hampton Research – hDMGDH of Crystallization – Stability Temperature This article is This protectedAll rights byreserved. copyright. observed for the majority of theamino acids except of the first 18 N electrondifference electron densitypeaks before the last cycle ofrefinement. Clear electron densitywas were applied. A small number of water moleculeswere added manually only into the most significant model contained one B-factor per amino acid residue. Non-crystallographic-symmetry (NCS) restraints Structurerebuilding andrefinementwere performedusing the programs Coot[43] and PHENIX [44]. The structure of the rat dimethylglycine dehydrogenase ( processed using the XDS package [42] andthe structurewas solved bymolecular replacement using the accession number 5L46. atomicThe coordinates and structure factors have been deposited in the Protein Data Bank under the [45]. Detailed statistics pertainingdata to processing andstructure refinement are summarized in Table 2. flavin adenine dinucleotide (FAD) inboth chains. The final structure was validated using MolProbity ‑

Acceptedterminal residues inbothchains present thein asymmetric unit. Residualdensity was interpreted as Article

P 2 1 ) with unit-cell parameters a=83.38 Å, b=119.87 c=86.47Å, Å and ® assay.Thermofluor μ 1) Thermofluor 1) L of the protein solution (at concentrationa of4mg/ml mM in 25 HEPES, 25 λ =0.972 Å)atthe ESRF Grenoble, France.crystals The were monoclinic (space

Crystallization experiments were performed with the microbatchmethod ®

measurementswere carried out asreviewed elsewhere [41] in an FX X ‑ ray diffraction datawere collected toa maximum resolution of ® – The temperatureThe stability ofthe enzymeswas assessed by 2) Circular Dichroism (CD) Spectroscopy – PDB ID: 4PAA

, 90%sequenceidentity), [18]. ‑ terminal residuesand the13 β =92.6°. The data were m ) were determined CD spectra 11 Sreekumar A, Poisson LM, Rajendiran TM, Khan AP, Cao Q, Yu J, Laxman B, Mehra R, Lonigro RJ, RJ, Lonigro R, B,Mehra Laxman Yu J, Q, Cao AP, TM,Khan LM, Rajendiran Poisson A, Sreekumar 11 10 Binzak B a, Vockley JG, Jenkins RB & Vockley J(2000) Structure andanalysis of the human 6 Tralau T,Lafite P,Levy C, Combe JP, Scrutton NS & LeysD (2009) An internal reaction chamber in 5 Wittwer AJ (1980) Identificationof Folate Binding Protein of Mitochondriaas Dimethylglycine disease. and health in betaine and Choline PM (2011) Ueland 4 3 Wittwer AJ & Wagner C (1981) Identificationof the folate-binding proteins of ratliver mitochondria as 2 Wittwer AJ & Wagner C (1981) Identificationof the folate-binding proteins of ratliver mitochondria as 1 LienhartWD, Gudipati V & MacherouxP (2013) The humanflavoproteome. REFERENCES the manuscript. andPA PM designed biochemical experiments and interpreted data;the PA, AH, TPK, KG and PM wrote variant; AH, TPKand KG crystallizedproteins and determined the crystal structure of WThDMGDH; Author contributions: listof contributinggroups can be found athttp://exac.broadinstitute.org/about. Consortium Aggregation Exome to KG and PM (Doctoral program “Molecular Enzymology” W908). The authors would like to thank the Acknowledgements: (CASoX plugin ofPyMOL, [48]). YASARA. Cavity analyses after and before mutagenesis was performed with the LIGSITE algorithm DMG and sarcosine into the hDMGDH structurewas doneusing Autodock/VINA [47] asimplemented in energyminimization using YASARA (Yasara Biosciences, Vienna, Austria). Docking experiments of with the mutagenesis wizard of PyMOL (DeLano Scientific, San Carlos, CA, USA) and subsequent [46]). In Silico Procedures – This article is This protectedAll rights byreserved. copyright. Accepted Binzak JGN, A,De Jong Heerschap JMB, Corstiaensen UFH, J, Engelke Poggi-Bach SH, Moolenaar 9 8 Steenkamp DJ & Husain M (1982) The effect of tetrahydrofolate on the reduction of electron transfer 7 Ruzicka FJ & Beinert H (1977) newA iron-sulfur flavoprotein of the respiratory chain. Article

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131 , isnBfco 47.09 97.7 (93.81) 30495 (2897) 86727 (7935) 58.65-3.09 (3.2-3.09) B-factor Wilson

Slet 15.2 30.1 6 38.2 38.1 1617 106 12782 lengths Bond ( R.m.s. deviations Solvent Ligands Macromolecules Average B-factor Protein residues Water Ligands Macromolecules Number non-hydrogen of atoms CC* 0.989 (0.868) CC ID29, ESRF,Grenoble, R France R R Wavelength ( X-ray source Table 2. a ferrocene WT WT DCPIP mM µM ferrocene). and (DCPIP assays activity different two with variant hDMGDH-H109R Table 1. TABLES min This article is This protectedAll rights byreserved. copyright. H109R ferrocene ferrocene H109R Temperature (K) 100 100 Cell dimensions group Space Temperature (K) odage ° 1.37 94 0.31 PDB 5L46 Clashscore 9.40 Ramachandran favored (%) (%) outliers Ramachandran Bond angles (°) (Figure 3). H109R DCPIP DCPIP H109R Two work meas mer β a σ 1/2 , (°) 92.59 92.59 (°) g

AcceptedI> 4.81 Article(1.53) e b / 0.2531 0.958 (0.604) 0.2058 (0.6573) , K R c M free (Å) 83.38, 119.87, 86.47 119.87, 86.47 83.38, (Å) and v and Data collectionandrefinement statistics. 17.90/26.93 Summary of kinetic parameters obtained with steady-state kinetics for hDMGDH-WT and Å ) 0.972 ) max Å values were obtained with the DCPIP assay, the higherwas roughly estimated from Eadie-Hofstee plots ) 0.010 1.4 ± 0.1 ~30 0.3 ± 0.002 K 32.2 ± 5.6 ~90 4.7 ± 0.4 M v a a

~22 7.4 ±0.3 ~11 4.7 ±0.1 22.1 ±0.3 44.1 ±3.2 max a a k -1 min 4±1 0.5 ± 0.01 91 ± 4 44 ± 1 1 20±00 137± 16 2.0 ± 0.04 213 ± 4 544 ± 40 P ca 2 t -1 A 1 µmol mg

1 ±0.04 6 ±0.4 s p ec k -1

min

-1 mM 20 ± 2 146 ± 12 11 ± 1 ca t -1 / K min M -1

LEGENDS FIGURE A. globiformis enzyme. bacterial the for pH8.5 and °C and 25 hDMGDH °C for 25 rat - the physiologically relevant is displayed. Ag a hDMGDH mM results from Table 4. nd… Not determined or stated in the publication. human - human - human - Enzyme e sapiens Table 3. This article is This protectedAll rights byreserved. copyright. for the substrate reduced enzyme. enzyme. reduced substrate the for pig - rat - rat - Ag Retrieved fromTable 2 in[23] DMGO Accepted Article- [22] [17] [16] [18] [8] a

(human), a a a [19] this study this study

Comparison of steady-state kinetic parameters of published results for the DMGDH from from DMGDH the for results published of parameters kinetic steady-state of Comparison Summary andcomparison of transient kinetic data of hDMGDH (this study) with published

A. globiformisA. ( K . . 1 . 20±1 . . .4±00 0.006 ± 0.001 0.04 ± 0.01 0.7 ± 0.1 280 ± 10 17± 0.3 4.9 ± 0.3 ~ 10

Ag D , DMG

Rattus norvegicus ). ). ercn p .,2 C 0.04 ±0.01 pH7.5,25 °C ferrocene ercn H75 5° 0610n 270 nd 160 0.05 pH7.0,30 °C 0.6 7.5, pH 25 °C DCPIP ferrocene O DCPIP DCPIP CI pH7.8,25 °C ferrocene DCPIP a - 5±02 5 0 01 . 21 26 201 0.16 ± 0.1 850 ± 100 15± 0.2 -Acceptor Conditions Conditions -Acceptor 2 k

b Measured at an oxygen concentration of 135 µM with p with µM 135 of concentration oxygen an at Measured DMGO [22,23]. Measurements were done in a stopped flow at pH 7.8 and 7.8 pH at flow a stopped in done were Measurements [22,23]. DMGO c Estimation from Figure 8 in [22]. in 8 Figure from Estimation s -1 red H70 5° n n 017 nd 0.157 nd nd 0.05 pH 7.0,25 °C pH 7.0,25 °C H78 5° 1.4 ±0.1 pH 7.8,25 °C H85 5° 24±0260±2 nd ± 640 20 2.4 ±0.2 pH 8.5,25 °C

, DMG DMG (rat),

a No standard deviations available.

Sus scrofa mM K 0.3 ±0.002 mM K D M , SAR

k b b

(pig) aswell as the dimethylglycine oxidase from b b

1 . .4 137± 16 2.0 ± 0.04 213 ± 4 min 4±1 . .1 146± 12 0.165 8.4 0.14 nd nd 12.1 0.5 0.24± 0.01 18 ± 2 44 ± 1 s k -1 cat red

-1 A , SAR b Two O2 O2 = 0.105 bar,K µmol mg K mM s ox K p ec ec M -1 s values were observed but only only but observed were values a 450 ± 110 -1

-1 min -1 mM H,pc H,pc = 791 bar= M 270 ± 32 k s k -1 ca obs

c t -1

/ , K ox min M

-1

Homo -1 [49] b

betainealdehyde dehydrogenase (BAD) and betaine homocysteine Choline degradation todimethylglycine iscatalyzed by enzymes, three choline dehydrogenase (CHD), Figure 1. Mammalian choline degradation pathway with hDMGDH as metabolic branch point. This article is This protectedAll rights byreserved. copyright. mitochondrial respiratorychain. interactionthe with hETF and the derived electrons arefurther channeled by ETF-QO to the group is donated to THF resultingFADH in Conversion ofdimethylglycine (DMG) to sarcosine by reduces DMGDH the FAD cofactor and a methyl Accepted Article sarcosine oxidase (PIPOX). peroxisomal and dehydrogenase dimethylglycine for asubstrate also is but (SARDH), dehydrogenase glycine only enzyme convertingdimethylglycine tosarcosine and the only otherknown route sarcosineto isvia methylene-THF reductase (MTHFR)and serine hydroxymethyltransferase(SHMT). hDMGDH is the N -methyl (GNMT). Sarcosine isfurther converted to glycine, mostly sarcosine by N -5,10-methylene-THF is consumed in further reactions catalyzed by 2 and N

-5,10-methylene-THF. The enzyme reoxidized is by

S -methyltransferase (BHMT).

velocities (v velocities experimentswere performed with DCPIPand (A)ferrocene activity assays by (B) initial plotting reaction ferrocene assaya producedsingle DMG) and oneat very high substrate concentrations (>50mM DMG, inset panel A).In contrast, the n=3. deviations; standard as shown are bars Error B). the data in Eadie-Hofstee diagrams reveal two two reveal diagrams Eadie-Hofstee in data the represented black in andred, respectively. Kineticparameters aresummarized inTable Linearization 1. of Figure 3. line. blue dotted a as shown is solution FAD in Free nm, respectively). 450 and exchange chromatography. spectra The werenormalized tothelong-wavelength absorption maxima (445 absorptionspectraof DMGDHwild-type (blackline) andtheH109R variant (red line) afteranion chromatography; affinity NTA chromatography. exchange anion MonoQ and affinity Ni-NTA with purification enzyme efficient shows variant variant 2. Figure This article is This protectedAll rights byreserved. copyright. Accepted Articleloading control, against Antibodies are directed against a C-terminal nona-histidine tagfusedto the recombinant enzymes and, as . (A) Western blot of blot Western (A) .

Heterologous expression and purification of hDMGDH-WT and hDMGDH-H109R and hDMGDH-H109R of hDMGDH-WT purification and expression Heterologous Steady-state kineticshDMGDH-WT of and H109R variant. 0 ) against the substrate concentration. Data obtained for WT and H109R variant are Lane

1 K. phaffii , fermentation pellet fraction; K. phaffiiK. 4 GAPDH.SDS-PAGE(B) ofthe different purificationsteps of and WT , enzyme, fraction after Mono Q; K M value when measured in thesame concentration range (insetpanel cell lysates takendifferent at timepoints after methanol induction.

K M values for the DCPIP assay, one at low (0-10 mM mM (0-10 low at one assay, DCPIP the for values 2 , K. phaffii

lysate; 5 , protein standard. (C) UV-Vis UV-Vis (C) standard. protein , 3 , enzyme fraction after Ni- Steady-state kinetic sarcosine. mM observedby mixingwith WT 125mM DMG, the bottomtheinset change 460nm at mixing with WT 125 change in absorption is depicted by arrows. The top inset shows theabsorption change at460 nm spectra of the hDMGDH-WT anoxic reduction with of 125mM DMG. 10µM The direction of the measurements were performed, error bars are shown asstandard deviations. Selected(B) absorption function of time at an oxygen concentration ofO 10.5% (135 µM as a FAD reduced of reoxidation the shows inset The axis). right (red, sarcosine and axis) left (black, under anoxic conditions with the stoppedflow device. (A) Reductive rates were determined for DMG hDMGDH-WT. of kinetics Pre-steady-state 4. Figure This article is This protectedAll rights byreserved. copyright. Accepted Article

Reductive and oxidative rates were measured

2 ) at 460 nm. 460) at At least three independent Figure 5 Figure This article is This protectedAll rights byreserved. copyright. Accepted Article measurementsfor each THF concentration. theusing ferrocene activityassay. error Thebars showthe standard deviationof atleast three .

hDMGDH-WT activity as a function of the THF concentration. THF of the afunction as activity hDMGDH-WT

The activitywasmeasured Figure 6. Figure This article is This protectedAll rights byreserved. copyright. Accepted Article changein absorption is depicted by arrows. spectra are shown asdashed lines. Only selected absorption spectra areshown. directionThe of the 600at nm. Initial absorption spectra in all panels are depicted in bold lines, whereas final absorption 480 nm where the dyedoes not showcontributiona tothe absorption. Valuesfor the dye weremeasured overmin 120 with indigotrisulfonicacid potassium salt and the evaluation forthe enzymewas done at evaluation ofthe data according to Minnaert hDMGDH[39] in wereinset. the 20 reduced µM at 25 °C [27]. The plotshows thesimultaneous reduction ofthe enzyme with andthedye thedouble logarithmic potential for theWT was determinedwith the xanthine/xanthine oxidase system described by Massey not proceed throughsemiquinonea redox stateand directlyyields the oxidized state (D) (C). redox The reduced hydroquinone species(B). The admissionof oxygen tothe enzyme shows reoxidation that does reduced to the red semiquinone radical state before(A) proceeding tothe typical spectrum of afully the presence of 1 methylviologen. 5-deazamM FMN andEDTA, First, 2µM 2µM theenzyme is spectra of the anoxic photoreduction of the wild-type of enzyme. hDMGDH 20 were µM photoreduced in

Photoreduction and redoxpotential of hDMGDH-WT.

(A) and (B) show selectedabsorption spectroscopy (B). Thermofluor wild-type and the variant enzyme differ significantlywhen measured with Thermofluor representation. sticks binding domains of DMGDHfrom human(raspberry) (cyan). andrat The THF cofactoris shown blue in depicted with an arrow and the FAD-cofactor shownyellow in (B) sticks. Superposition of thefolate domain is shown in deep blue and the folate binding domain in raspberry. The THF binding site is Figure 8. Crystal structure of hDMGDH. measurements. both in line) (black wild-type the as temperature derivative curves of thesigmoidal fits. variantThe (red line) exhibits aninflection point at alower The data were fitted with asigmoidal curve in Origin. The inset in panel B shows the–d(MRE)/dT every 0.1°C at 208 nm which is shown byblack and red filled circles for WT and variant, respectively. spectroscopywas enzyme. donewithresidue Mean approximately ellipticity 6µM (MRE) wasmeasured respectively.inset The panel in showsA the–d(fluorescence)/dTderivative curves of the raw data. CD- change in fluorescence during heating is shown for WT and H109R variant in black and red lines, Figure 7. This article is This protectedAll rights byreserved. copyright. Accepted Article

Thermal stability of hDMGDH-WT and theH109R variant. ® was done with 2 µM enzymewas done andtheaddition with 2µM ofSYPRO Orange. The (A) The overall structure of hDMGDH with the FAD binding binding FAD the with hDMGDH of structure overall The (A)

The meltingtemperatures of ® (A) or CD- or (A)

amino acidreplacement ( green surface view andtheTHF binding site indicated by anarrow. Close-up (B) view ofthesite hDMGDH donewith CASoX in PyMOLwithout a bound THF cofactor. The proteincavity isshown in variation. H109R of effect and analysis cavity internal hDMGDH 10. Figure insolution and not ourspectral findings. (grey line, [19]) and free FAD in solution (dotted blue line) yellow, residues the in shown reduces the size of the substrate tunnel, substratethus deliverythe FAD to binding site. FAD cofactor is bulky, charged arginine extends into the cavity calculated for wild-typethe protein and potentially done inthewild-type enzyme (His-109,mesh) blue and the H109R variant(Arg-109, green surface). The 9 Figure This article is This protectedAll rights byreserved. copyright. Accepted Article .

Protein spectrum of this study (black line)compared with the published hDMGDH spectrum i.e. H109R) near theactiveofsite the protein. A cavityanalysisthis at site was at position 109 in green and blue sticks, respectively. sticks, blue inrespectively. and 109 green at position

. The published The spectrum resemblesfree FAD

(A) Cavity analysis of

hydrogen bonds are shown with blackdotted lines. yellow and allresidues are depicted in stick representation colored according theatom type. Key dockedThe substrates and thebound acetateare shownin green, magenta and black.FAD isshown in Autodock/VINA [47]. The activesite ofDMGO with bound acetatewas retrievedfrom DMGO structure. in acetate bound with comparison in of hDMGDH composition site active and Docking 11. Figure This article is This protectedAll rights byreserved. copyright. Accepted Article DMG (A) and sarcosine (B) were docked into the active site of hDMGDH with

PDB ID: 1PJ5 ID: PDB .

Figure 12. residuesare shown in magenta. Fora better orientation, the catalytic active histidine onthe flavin isoalloxazine ring shown ingreensticks. The bacterial structure is shown inpanel B, important human structure is shown in panel A with the most significant alternated residues on the This article is This protectedAll rights byreserved. copyright. Accepted are depicted in stick representation, FAD in yellow and colored according the atom type. keeper residues shown corresponding to the reactiveC(4a) carbon of the isoalloxazine ring. All residues displayed.Panel shows C thealignmentof thebacterialthe and human enzymewith thesupposed gate- Article

Active site composition of the oxygen binding site of hDMGDH and

Ag re- DMGO si- face is also is also face face of the of the face

. The