Structure-Guided Expansion of the Substrate Range of Methylmalonyl Synthetase (MatB) of Rhodopseudomonas palustris

Heidi A. Crosby,a Katherine C. Rank,b Ivan Rayment,b and Jorge C. Escalante-Semerenaa Departments of Bacteriologya and Biochemistry,b University of Wisconsin, Madison, Wisconsin, USA

Malonyl coenzyme A (malonyl-CoA) and methylmalonyl-CoA are two of the most commonly used extender units for polyketide biosynthesis and are utilized to synthesize a vast array of pharmaceutically relevant products with antibacterial, antiparasitic, anticholesterol, anticancer, antifungal, and immunosuppressive properties. Heterologous hosts used for polyketide production such as Escherichia coli often do not produce significant amounts of methylmalonyl-CoA, however, requiring the introduction of other pathways for the generation of this important building block. Recently, the bacterial malonyl-CoA synthetase class of has been utilized to generate malonyl-CoA and methylmalonyl-CoA directly from malonate and methylmalonate. We demonstrate that in the purple photosynthetic bacterium Rhodopseudomonas palustris, MatB (RpMatB) acts as a methylmalo- nyl-CoA synthetase and is required for growth on methylmalonate. We report the apo (1.7-Å resolution) and ATP-bound (2.0-Å resolution) structure and kinetic analysis of RpMatB, which shows similar activities for both malonate and methylmalonate, making it an ideal for heterologous polyketide biosynthesis. Additionally, rational, structure-based mutagenesis of the of RpMatB led to substantially higher activity with ethylmalonate and butylmalonate, demonstrating that this enzyme is a prime target for expanded substrate specificity.

alonyl-coenzyme A (malonyl-CoA) synthetase (MatB) be- boxylate transporter , matC, suggesting that it is involved in Mlongs to the AMP-forming acyl-CoA synthetase protein malonate metabolism to acetyl-CoA (3). family (PFAM 00501). These enzymes catalyze the conversion of It has been suggested that growth on malonate is important organic acids to acyl-CoA thioesters via a ping-pong mechanism, inside nitrogen-fixing nodules on plant roots. This idea is sup- in which ATP and the organic acid are first converted to acyl-AMP ported by the fact that a ⌬matB strain of R. leguminosarum dis- with the release of pyrophosphate, followed by coenzyme A bind- plays impaired nodule formation (4). In contrast, matB from Si- ing, displacement of AMP, and release of the acyl-CoA product norhizobium meliloti 1021 is required for growth on malonate in (Fig. 1). This class of enzymes activates a wide range of com- liquid medium, but a ⌬matB strain did not affect nodulation of pounds, including short-, medium-, and long-chain fatty acids, alfalfa or nitrogen fixation (13). While matB homologues are dicarboxylic acids, and aromatic acids, which are then oxidized as found in a number of other nonsymbiotic soil-dwelling and ma- a carbon and energy source or used in biosynthetic reactions. rine bacteria, their functions in the cell have not been character- AMP-forming acyl-CoA synthetases also share sequence and ized. structural homology with firefly luciferase and the adenylation MatB is of commercial interest for its utility in generating ma- domains of nonribosomal peptide synthetases (NRPSs), and all lonyl-CoA and methylmalonyl-CoA starter units for polyketide belong to the family of enzymes that include acyl-CoA synthe- synthesis (for a review, see reference 12). Polyketides are pharma- tases, NRPS adenylation domains, and luciferase enzymes, also ceutically relevant natural products that are utilized as antibiotics, known as the ANL superfamily (5, 23, 43, 61). antiparasitics, antifungals, cancer treatments, and immunosup- pressives, and malonyl-CoA and methylmalonyl-CoA are the two The active site of acyl-CoA synthetases is located at the interface of most common extender units in the synthesis of these products. the ϳ400-amino-acid N-terminal domain and ϳ100-amino-acid Malonyl-CoA can be produced either from acetyl-CoA by acetyl- C-terminal domain. The C-terminal domain rotates ϳ140° dur- CoA carboxylase or directly from malonate by malonyl-CoA syn- ing the two-step reaction such that the enzyme exhibits one con- thetase (MatB) (32, 60). Synthesis of polyketide antibiotics in het- formation (termed the adenylation conformation) during the re- erologous hosts like Escherichia coli often requires introduction of action of ATP with the organic acid and a second conformation a pathway for production of methylmalonyl-CoA, through either (thioester-forming conformation) during formation of the acyl- carboxylation of propionyl-CoA, conversion from succinyl-CoA CoA product (25, 36, 53, 54; reviewed in reference 23). A short by methylmalonyl-CoA mutase, or production directly from linker region connects the two domains, and most of the rotation during the conformational change occurs at a conserved hinge residue, typically either an aspartic acid or lysine (64). Received 29 May 2012 Accepted 2 July 2012 MatB activates the C3 dicarboxylic acid malonate to malonyl- Published ahead of print 6 July 2012 CoA (Fig. 1) and has been isolated from a variety of soil bacteria Address correspondence to Jorge C. Escalante-Semerena, and from mammalian mitochondria (27, 33, 34, 63). The best- [email protected]. studied member of this group is MatB from the nitrogen-fixing H.A.C. and K.C.R. contributed equally to this article. legume symbiont Rhizobium leguminosarum bv. trifolii (RlMatB). Copyright © 2012, American Society for Microbiology. All Rights Reserved. The gene encoding RlMatB, matB, is located in an operon with a doi:10.1128/AEM.01733-12 malonyl-CoA decarboxylase gene, matA, and a putative dicar-

September 2012 Volume 78 Number 18 Applied and Environmental Microbiology p. 6619–6629 aem.asm.org 6619 Crosby et al.

FIG 1 MatB converts malonate to malonyl-CoA in two steps via a malonyl-AMP intermediate. methylmalonate using MatB (20, 47). Total enzymatic synthesis of cally on photosynthetic medium containing succinate and kanamycin in several polyketide natural products has also been reconstituted in Balch tubes (7) flushed with O2-free N2. Strains were diluted 1:16 in fresh vitro, and in these cases MatB serves as an affordable means of medium supplemented with the appropriate carbon source and kanamy- generating malonyl-CoA and methylmalonyl-CoA in situ (14, 41). cin. Tubes were incubated at 30°C in light without shaking, and growth RlMatB is often utilized for polyketide synthesis, as it has been was monitored at 660 nm using a Spectronic Spec20D. Three biological kinetically characterized and can use methylmalonate and other replicates of each strain were monitored, and each experiment was re- peated three times. malonate derivatives. However, RlMatB activity with methylma- ϳ Genetic and recombinant DNA techniques. An in-frame deletion of lonate is only 20% of its activity with malonate (3, 41, 50). It was matB (locus tag rpa0221) was constructed using the method of Schäfer et reported that Streptomyces coelicolor MatB (ScMatB) could syn- al. (56), as described elsewhere (17). Primers used to amplify matB-flank- thesize 15 different polyketide extender units, encompassing all of ing regions for cloning into pK18mobsacB are listed in Table 2. the combinations of five different malonate derivatives and three To construct complementation vectors, the matB gene was amplified different thiol acceptors (CoA, pantetheine, and N-acetylcysteam- ine), which could be used for in vitro polyketide synthesis (27), although the kinetics of this enzyme were not reported. This TABLE 1 Strains and plasmids used in this work opened up the possibility that characterization of MatB homo- Reference or logues from different organisms may lead to expanded substrate Strain or plasmid Genotype and description sourcea specificity and improved activity for different malonate deriva- Strains tives. Additionally, detailed kinetic and structural studies would R. palustris CGA009 43 provide a template for rational mutagenesis to further expand the Derivatives of R. palustris JE13366 ⌬matB substrate specificity of MatB. JE11529 WTb/pBBR1MCS-2 In this study, we have characterized the MatB protein from the JE16618 WT/pBBR1MCS-5 purple photosynthetic bacterium Rhodopseudomonas palustris JE13568 ⌬matB/pBBR1MCS-2 (RpMatB), and we show that it is most likely a methylmalonyl- JE13569 ⌬matB/pRpMatB23 JE13911 ⌬matB/pRlMatB1 CoA synthetase in vivo. In support of its biological role, RpMatB Rhizobium leguminosarum bv. ATCC can efficiently use methylmalonate and has significant activity trifolii ATCC 14479 with ethylmalonate. To further expand the substrate specificity of E. coli RpMatB, we determined both the apo and ATP-bound crystal JE8125 S17-1 recA pro hsdR 62 RP4-2-Tc::Mu-kanϩ::Tn7 structures of RpMatB and utilized these two structures combined JE6090 C41␭(DE3) with a thioester-forming conformation structure of ScMatB (27) JE9314 C41␭(DE3) pka12::kanϩ Lab collection as a homology model to identify residue T208 in the binding pocket that influences the binding affinity of malonate derivatives. Plasmids T208A T208G pK18mobsacB Deletion construction in R. 60 Two RpMatB variants, RpMatB and RpMatB , had in- palustris, kanϩ creased activity with the larger malonate derivatives ethylma- pBBR1MCS-2 Broad host range 40 lonate and butylmalonate. The data presented here suggest that complementation, kanϩ RpMatB is a good template for future mutagenesis studies to gen- pTEV5 Overproduction vector, N- 59 terminal His6 tag erate unique polyketide extender units. pRpMatB4 R. palustris matB deletion construct in pK18mobsacB MATERIALS AND METHODS pRpMatB23 R. palustris matB in pBBR1MCS- 2, kanϩ Strains, culture media, and growth conditions. Bacterial plasmids and pRpMatB1 R. palustris matB in pTEV5, blaϩ strains used in this work are listed in Table 1. E. coli strains were grown at pRpMatB39 R. palustris matB K488A in 37°C on LB medium (8, 9). Rhizobium leguminosarum bv. trifolii ATCC pTEV5, blaϩ 14479 was obtained from the American Type Culture Collection and cul- pRpMatB60 R. palustris matB T208A in ϩ tured at 30°C on TY medium (5 g literϪ1 tryptone peptone, 3 g literϪ1 pTEV5, bla pRpMatB61 R. palustris matB T208G in yeast extract, 10 mM CaCl ). Rhodopseudomonas palustris CGA009 was ϩ 2 pTEV5, bla grown at 30°C on defined basal photosynthetic medium (30) supple- pRlMatB1 R. leguminosarum matB in ϩ mented with 150 nM vitamin B12 and either succinate (10 mM), malonate pBBR1MCS-2, kan (25 mM), or methylmalonate (10 mM). When necessary, growth media pRlMatB3 R. leguminosarum matB in were supplemented with ampicillin (100 ␮gmlϪ1) or kanamycin (50 ␮g pTEV5, blaϩ Ϫ1 Ϫ1 ml for E. coli and 75 ␮gml for R. palustris). For growth curves, R. a Strains were constructed for this study unless otherwise indicated. palustris starter cultures were grown for 3 to 4 days photoheterotrophi- b WT, wild type.

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TABLE 2 Primer sequences used in this work Primer name Sequencea MatB_delA_5=EcoRI GAT GAA TTC CGA TTT CGT TAC TGC CGA AT MatB_delB TTA CTT GTA GAT GTC CTT GTA GGT GGC GAA CAG GTT GGC GTT CAT GTC MatB_delC GAC ATG AAC GCC AAC CTG TTC GCC ACC TAC AAG GAC ATC TAC AAG TAA MatB_delD_3=HindIII GTA AAG CTT AGC GGC ATT CGG TTA CTT C MatB_5=HindIII GTA AAG CTT ATG AAC GCC AAC CTG TTC GC MatB_3=BamHI CCT GGA TCC TTA CTT GTA GAT GTC CTT GTA GGT MatB_5=NheI GTA GAC GCT AGC ATG AAC GCC AAC CTG TTC GC MatB_K488A_fwd GCG CAA CAC CAT GGG TGC GGT CCA GAA GAA CGT C MatB_K488A_rev GAC GTT CTT CTG GAC CGC ACC CAT GGT GTT GCG C MatB_T208A_fwd GCC GAT CTA TCA CGC CCA TGG ATT GTT C MatB_T208A_rev GAA CAA TCC ATG GGC GTG ATA GAT CGG C MatB_T208G_fwd CTG CCG ATC TAT CAC GGC CAT GGA TTG TTC G MatB_T208G_rev CGA ACA ATC CAT GGC CGT GAT AGA TCG GCA G Trifolii_MatB_5=NdeI GTA CAT ATG AGC AAC CAT CTT TTC GA Trifolii_MatB_5=HindIII GTA AAG CTT ATG AGC AAC CAT CTT TTC GA Trifolii_MatB_3=BamHI CAT GGA TCC TTA CGT CCT GGT ATA AAG AT a Underlined sequences identify restriction sites.

from R. palustris genomic DNA using primers MatB_5=HindIII and the column was washed with wash buffer (sodium phosphate buffer [50 MatB_3=BamHI (Table 2), cut with HindIII and BamHI (Fermentas), and mM, pH 8.0], NaCl [300 mM], imidazole [20 mM]), and MatB was eluted ligated into plasmid pBBR1MCS-2 (37) to generate pRpMatB23. The R. with elution buffer (sodium phosphate buffer [50 mM, pH 8.0], NaCl leguminosarum matB gene was amplified with primers Trifolii_ [300 mM], imidazole [250 mM]).

MatB_5=HindIII and Trifolii_MatB_3=BamHI (Table 2) from an R. legu- Fractions containing His6-MatB were pooled, and the tag was cleaved minosarum colony and ligated into the HindIII and BamHI sites of with rTEV protease (1:50 to 1:100 rTEV/H6-MatB ratio) for3hatroom pBBR1MCS-2. Gene sequences were confirmed using BigDye (ABI temperature. The cleaved protein was dialyzed against buffer 1 (sodium PRISM) protocols, and sequencing reactions were resolved and analyzed phosphate buffer [50 mM, pH 8.0], NaCl [300 mM], EDTA [0.5 mM]), at the University of Wisconsin Biotechnology Center. The R. legumino- then buffer 2 (sodium phosphate buffer [50 mM, pH 8.0], NaCl [300 sarum matB sequence matched the published sequence (40) deposited in mM]), and finally binding buffer. The protein was passed over an Ni

the NCBI database under locus AF117694. Plasmids were introduced into column again to remove the tag and His7-tagged rTEV protease, using the R. palustris by electroporation or conjugation, using E. coli S17-1 as a buffers employed in the first purification step. Untagged proteins eluted donor strain (28, 58). in the flowthrough and were pooled and dialyzed against storage buffer 1 To construct overexpression vectors, the R. palustris matB gene was (Tris-HCl [50 mM, pH 7.5], NaCl [100 mM], EDTA [0.5 mM], glycerol amplified from genomic DNA using primers MatB_5=NheI and [20%, vol/vol]) and then storage buffer 2 (Tris-HCl [50 mM, pH 7.5], MatB_3=BamHI (Table 2), cut with NheI and BamHI, and ligated into NaCl [100 mM], glycerol [20%, vol/vol]) before being drop frozen into Ϫ plasmid pTEV5 (55). The R. leguminosarum matB gene was amplified liquid N2 and stored at 80°C until used. with primers Trifolii_MatB_5=NdeI and Trifolii_MatB_3=BamHI (Table Proteins were purified for crystallography in the same manner, except 2) using pRlMatB1 as a template, and the PCR product was ligated into the that prior to freezing they were dialyzed against storage buffer 3 (Tris-HCl NdeI and BamHI sites of pTEV5. Both plasmids directed the synthesis of [10 mM, pH 7.5], NaCl [100 mM], EDTA [0.5 mM]), then storage buffer

RpMatB or RlMatB with an N-terminal His6 tag that was removed using 4 (Tris-HCl [10 mM, pH 7.5], NaCl [20 mM]), and finally storage buffer recombinant tobacco etch virus (rTEV) protease (10). Site-directed mu- 5 [Tris-HCl (10 mM, pH 7.5), tris(2-carboxyethyl)phosphine (TCEP; 1 tants were constructed using the QuikChange protocol (Stratagene) using mM)]. Proteins were concentrated to 10 to 12 mg mlϪ1 using Ultracel primers listed in Table 2 and pRpMatB1 as a template. centrifugal filters with a molecular weight cutoff (MWCO) of 10,000

Protein expression and purification. Plasmids encoding wild-type (Amicon Ultra) and drop frozen into liquid N2. Protein concentrations and variant MatB proteins were transformed into strain JE9314, a Pka were determined using a NanoDrop 1000 spectrophotometer (Fisher) ␭ (formerly YfiQ)-deficient derivative of E. coli C41 (DE3) (46). The result- and A280 molar extinction coefficients for each protein, which were ob- ing strains were grown to early stationary phase and subcultured 1:100 tained from the Integrated Microbial Genomes (IMG) database (42). (vol/vol) in 2 liters of lysogenic broth supplemented with ampicillin (100 Selenomethionine (Se-Met)-labeled MatB was prepared as follows. ␮gmlϪ1). Cultures were grown with shaking to an optical density at 600 Plasmid pRpMatB1 was transformed into strain JE9314 [C41(␭DE3) pka:: ϳ nm (OD600)of 0.6, and gene expression was induced by the addition of kan], and a 60-ml culture of the resulting strain was grown overnight in LB isopropyl-1-thio-␤-D-galactopyranoside (IPTG; 0.5 mM). Cultures were medium. The culture was harvested by centrifugation and washed twice grown overnight at 30°C, cells were harvested by centrifugation at 8,000 ϫ with M9 minimal medium. The washed cells were used to inoculate 2 liters ϳ g for 12 min in a Beckman Coulter Avanti J-20 XOI refrigerated centrifuge of M9 glucose medium and grown to an OD600 of 1. The culture was with a JLA-8.1000 rotor, and cell pellets were frozen at Ϫ80°C until used. cooled on ice to ϳ16°C for 10 min, and a defined amino acid mixture To purify MatB, the pellet was resuspended in 30 ml of binding buffer containing selenomethionine (50 mg literϪ1) was added to suppress me- (sodium phosphate buffer [50 mM, pH 8.0], NaCl [300 mM], imidazole thionine biosynthesis (62). The culture was grown at 30°C for 30 min [10 mM]) containing lysozyme (1 mg mlϪ1). Cells were lysed using a sonic before the addition of IPTG (1 mM) to induce matB expression. The dismembrator (Fisher Scientific) at power level 9, with 2-s pulses sepa- culture was grown overnight at 30°C, and MatB protein was purified as rated by 5-s breaks, for 1.5 min on ice. Debris was removed by centrifu- described above. gation at 39,000 ϫ g for 30 min at 4°C, and the soluble fraction was passed Variant protein MatBK488A was purified as described above and frozen ␮ Ϫ ϳ through a 0.45- m filter (Thermo Scientific). His6-MatB was purified by at 80°C. A sample containing 40 mg of protein was thawed and dia- Ni affinity purification using a 1.5-ml bed volume of Ni-nitrilotriacetic lyzed against HEPES buffer (50 mM, pH 7.5) to remove any traces of acid (Ni-NTA) Superflow resin (Qiagen). After binding to the column, Tris-HCl buffer. The lysine side chains of MatB were reductively methyl-

September 2012 Volume 78 Number 18 aem.asm.org 6621 Crosby et al.

ated using formaldehyde and dimethylamine borane complex as de- TABLE 3 Crystal structure data collection and refinement statistics scribed previously (51). The reaction was quenched by addition of Tris- Statistic(s) MatB MatB-ATP HCl buffer (pH 7.5) to a final concentration of 500 mM, and the methylated MatB was dialyzed overnight against 2 liters of Tris-HCl buf- Data collection fer (50 mM, pH 7.5) containing dithiothreitol (DTT; 2 mM), before dial- Space group P21 P61 ysis into storage buffer 5. Cell dimensions Size exclusion chromatography was performed using an A¨ KTA fast- a, b, c (Å) 133.9, 58.8, 139.0 173.1, 173.1, 47.7 protein liquid chromatography Explorer system equipped with a Super- a, b, g (°) 90, 91.7, 90 90, 90, 120 Wavelength 0.98 0.98 dex 200 column (GE Healthcare). The running buffer contained Tris-HCl Resolution (Å)a 25–1.7 (1.74–1.7) 25–2.0 (2.03–2.0) (50 mM, pH 7.5) and NaCl (100 mM). RpMatB (0.6 to 2.3 mg) was a Rmerge 7.6 (48.3) 4.7 (45.4) applied onto the column, and the column was developed isocratically at a a Ϫ I/␴I 16.8 (3.5) 31.4 (7.3) rate of 0.5 ml min 1. A plot of the log of the molecular mass versus the Completeness (%)a 97.1 (95.5) 99.5 (100) retention time of thyroglobulin (670 kDa), gamma globulin (158 kDa), Redundancya 4.2 (3.8) 12.5 (10.8) ovalbumin (44 kDa), and myoglobin (17 kDa) (R2 ϭ 0.999) was used to estimate the apparent oligomeric state of RpMatB. Refinement RpMatB activity assays and kinetic parameters. MatB specific activ- Resolution (Å)a 25–1.7 (1.74–1.7) 25–2.0 (2.03–2.0) ity was quantified using an NADH consumption assay (53). All chemicals No. of reflectionsb 225,397 (11,309) 53,454 (2,718) were purchased from Sigma. Reaction mixtures contained HEPES buffer c Rwork/Rfree 0.16/0.20 0.16/0.20 (50 mM, pH 7.5), TCEP (1 mM), ATP (2.5 mM), free CoA (CoASH; 0.5 No. of atoms mM), MgCl2 (5 mM), phosphoenolpyruvate (3 mM), NADH (0.1 mM), Protein 15,413 3,927 pyruvate kinase (1 U), myokinase (5 U), lactate dehydrogenase (1.5 U), Water 2,607 619 and organic acid substrate (2 mM). All reactions were started by the ad- Average B-factor (Å2) 17.6 26.7 ␮ dition of MatB (3 pmol per 100- l reaction mixture), and the change in Ramachandran (%) the absorbance at 340 nm was monitored for 8 min in a 96-well plate Most favored 98.9 99.2 format using a Spectramax Plus UV-visible spectrophotometer (Molecu- Allowed 1.1 0.8 lar Devices). For kinetic measurements, the same assay was used, except Disallowed 0 0 that the concentration of one substrate was varied while the others were RMSDs held constant at a saturating concentration. Organic-acid concentrations Bond lengths (Å) 0.006 0.007 ranged from 0.01 to 2 mM (malonate and methylmalonate), 0.01 to 10 Bond angles (°) 1.008 1.052 mM (ethylmalonate), and 0.2 to 20 mM (butylmalonate), while ATP was PDB accession no. 4FUQ 4FUT held constant at 2.5 mM. Kinetic parameters for ATP were determined a Data in parentheses represent highest resolution shell. using a concentration range of 0.01 to 1 mM, while malonate (RpMatB b Data in parentheses represent the number of reflections used during refinement. T208A T208G c ϭ Α Ϫ Α and RlMatB) or ethylmalonate (RpMatB and RpMatB ) was Rfactor is determined as Rfactor |Fobs Fcalc|/ |Fobs|, where Rwork refers to the held constant at 3 mM. Pseudo-first order kinetic parameters were deter- Rfactor for the data utilized in the refinement, Rfree refers to the Rfactor for 5% of the data mined using Prism version 4.0 software (GraphPad Software), fitting the that were excluded from the refinement, Fobs refers to the observed structure factor data to the Michaelis-Menten equation. Each point was measured in trip- amplitude, and Fcalc refers to the calculated structure amplitude from the licate, and the entire experiment was repeated on a separate day. Repre- corresponding model. sentative results from one experiment are reported. Isothermal titration calorimetry. Proteins were dialyzed into Tris- HCl buffer (50 mM, pH 7.5, 22°C) containing MgCl2 (10 mM). MatB was solution with the addition of 1 mM CoA, 2.5 mM AMP, and 2.5 mM ␮ K488A ␮ used at a concentration of 68 M, and MatB was diluted to 21 M sodium methylmalonate and then transferred stepwise to a solution of ␮ (for ATP binding) or 81 M (ADP binding). ATP (0.4 mM for 20% (wt/vol) polyethylene glycol 8000, 20% (wt/vol) glycerol, 100 mM RpMatBK488A or1mMforRpMatB) and ADP (1 to 1.5 mM) were dis- bis-Tris (pH 6.5), 40 mM MgSO4, 2 mM CoA, 2.5 mM AMP, and 2.5 mM solved in spent dialysis buffer. Experiments were conducted at 22°C and sodium methylmalonate. Crystals were flash-frozen in liquid nitrogen. ␮ consisted of 28 10- l injections into a 1.45-ml sample cell containing Crystals of methylated RpMatBK488A in complex with ATP crystals K488A K488A RpMatB or RpMatB ; the titration of ATP into RpMatB con- were grown by mixing 2 ␮l of 10-mg/ml protein in Tris buffer (10 mM, pH ␮ sisted of 34 5- l injections. Injections were made over 20 s (10 s for ATP 7.5) containing TCEP (1 mM) and MgATP (10 mM) with 2 ␮l of reservoir K488A titration into RpMatB ) and were separated by 3 min. The sample cell solution (bis-Tris buffer [100 mM, pH 6.5]) containing trimethylammo- was stirred at 307 rpm. Data were acquired on a MicroCal VP-ITC micro- nium chloride (25 mM), polyethylene glycol 8000 (19.5%, wt/vol), and calorimeter and analyzed using ORIGIN, version 7.0383, by fitting to the glycerol (5%, wt/vol) and hanging the resulting mixture over 500 ␮lof single-set-of-sites model. The change in free energy (⌬G) was calculated reservoir solution in Linbro plates at 25°C. After 24 h, crystals were nu- ⌬ ϭϪ using the free-energy equation G RTlnKa, where R is the ideal gas cleated by streak seeding with previously grown crystals and allowed to constant, T is temperature (Kelvin), and Ka is the association constant. grow for an additional week. For freezing, crystals were transferred to 100 Each binding isotherm was measured in duplicate, and values for the ␮l of reservoir solution with the addition of MgATP (10 mM) and then number of binding sites, association constant, enthalpy, entropy, and free transferred stepwise to a solution of polyethylene glycol 8000 (25%, wt/ ⌬ ⌬ ⌬ energy changes (N, Ka, H, S, and G, respectively) were averaged. vol), glycerol (20%, wt/vol), bis-Tris buffer (100 mM, pH 6.5), trimeth- X-ray crystallography. Selenomethionine and native apo RpMatB ylammonium chloride (100 mM), and MgATP (10 mM). Crystals were crystals were grown by mixing 2 ␮l of 10-mg/ml protein in 10 mM Tris flash-frozen in liquid nitrogen. (pH 7.5), 1 mM TCEP, 4 mM CoA, 5 mM AMP, and 5 mM sodium X-ray diffraction data for all crystals were collected at the SBC 19-BM methylmalonate with 2 ␮l of reservoir solution (100 mM bis-Tris [pH beam line (Advanced Photon Source, Argonne, IL). The data sets were inte- 6.5], 84 mM MgSO4, 19.5% [wt/vol] monomethyl polyethylene glycol grated and scaled with the program HKL2000 (47a). X-ray data collection 2000) and hanging the resulting mixture over 500 ␮l of reservoir solution statistics are given in Table 3. in Linbro plates at 25°C. After 24 h, crystals were nucleated by streak The structure of apo MatB was solved by single-wavelength anoma- seeding with previously grown crystals and allowed to grow for an addi- lous dispersion. The positions of the 52 selenium atoms were determined tional week. For freezing, crystals were transferred to 100 ␮l of reservoir using the Hybrid Substructure Search submodule of the Phenix package

6622 aem.asm.org Applied and Environmental Microbiology Expanded Substrate Range of Methylmalonate-CoA Ligase

FIG 2 matB function is needed for R. palustris growth on methylmalonate. Wild-type and ⌬matB strains of R. palustris were grown photoheterotrophically on the indicated carbon sources: malonate (25 mM) (A), methylmalonate (10 mM) (B), and succinate (10 mM) (C). The symbols identify wild-type cells carrying the cloning vector (empty squares), the ⌬matB strain carrying the cloning vector (empty triangles), the vector containing R. palustris matBϩ (filled inverted triangles), and the vector containing R. leguminosarum matBϩ (filled diamonds). Absorbance was monitored at 660 nm.

(2, 22) and refined using the programs BP3 (48, 49) and Solomon (1). The effect of the absence of MatB appeared to be limited to Parrot (16) was used for density modification, and these phases were used malonate and methylmalonate, since growth of the R. palustris to build an initial model in Buccaneer (15). This was followed by iterative wild type was indistinguishable from that of the ⌬matB strain on cycles of manual model building in Coot (19) and restrained and transla- succinate (Fig. 2C, empty triangles and empty squares, 13 h each), tion, libration, and screw motion (TLS) refinement in Refmac 5.6 and and full density was already reached at 40 h of incubation. Phenix.refine:1.6_289 (2, 59). Data processing and refinement statistics Substrate specificity of RpMatB. We purified RpMatB and are presented in Table 3. The structure of methylated MatBK488A with ATP was solved by mo- used a coupled spectrophotometric assay to test its activity with a lecular replacement using the program Phaser (11, 44) and the apo MatB variety of short-chain mono- and dicarboxylic acids (Table 4). structure presented here as the search model. This was followed by itera- The highest specific activity was seen with malonate (17.7 Ϯ 1.8 Ϫ1 Ϫ1 tive cycles of manual model building in Coot (19) and restrained and ␮mol AMP min mg ), although RpMatB also used methylma- refinement in Refmac 5.6 and Phenix.refine:1.6_289 (2, 59). Data process- lonate and ethylmalonate well (specific activities, 11.3 Ϯ 1.4 and ing and refinement statistics are presented in Table 3. All structural align- 7.2 Ϯ 0.5 ␮mol AMP minϪ1 mgϪ1, respectively). Since RpMatB ments were done with the program Superpose (38). could use malonate derivatives with one- and two-carbon substi- PDB accession numbers. Structures have been deposited with the tutions in the C-2 position, we tested its ability to use butylma- Protein Data Bank (PDB) under accession numbers 4FUQ and 4FUT for lonate, a malonyl moiety with a four-carbon chain at the C-2 apo MatB and ATP-MatB, respectively. position; surprisingly, it retained 4% of the activity measured with Ϯ ␮ Ϫ1 Ϫ1 RESULTS malonate (specific activity, 0.8 0.1 mol AMP min mg ). In contrast, RpMatB had very low activity with the C dicarboxylic matB is required for growth of R. palustris on methylmalonate. 4 acid succinate and all of the monocarboxylic acids tested, indicat- In the soil alphaproteobacterium Rhizobium leguminosarum bv. ing that the enzyme was specific for dicarboxylic acids with a trifolii, MatB catalyzes the conversion of malonate to malonyl- three-carbon backbone. RpMatB also had low activity with mono- CoA, which is subsequently decarboxylated to yield acetyl-CoA carboxylic acids such as acetoacetate (C ), ␤-hydroxybutyrate (3). To test whether matB function was also required for growth of 4 (C ), and isovalerate (C ). the purple photosynthetic alphaproteobacterium R. palustris on 4 5 The conserved lysine residue K488 is known to be required for malonate, we constructed an in-frame deletion of matB. The catalyzing the adenylation half-reaction in many acyl-CoA syn- ⌬matB strain showed a modest but reproducible growth defect on malonate (25 mM) (Fig. 2A, empty triangles, 214-h doubling time), which was corrected by ectopic expression of R. palustris ϩ TABLE 4 R. palustris MatB specific activity with a variety of substrates matB (Fig. 2A, filled inverted triangles, 128-h doubling time) or a R. leguminosarum matB (Fig. 2A, filled diamonds, 118-h doubling Enzyme Substrate Sp act % activity time); the doubling time of the wild-type strain carrying the empty RpMatB Malonate 18 Ϯ 2 100 expression vector on malonate was 116 h. Notably, the doubling Methylmalonate 11 Ϯ 161 Ϯ time of the wild-type strain on methylmalonate was half of that on Ethylmalonate 7 0.5 39 Ϯ malonate (64 h), but the R. palustris ⌬matB strain struggled to Butylmalonate 0.8 0.1 4.4 RpMatBK488A Malonate 0.04 Ϯ 0.01 0.2 grow on methylmalonate (426-h doubling time). Collectively, Methylmalonate 0.03 Ϯ 0.01 Ͻ0.2 these data indicated that matB function was required for growth of a Specific activity is reported as ␮mol AMP minϪ1 mgϪ1. Each compound was tested at R. palustris on methylmalonate but not on malonate. These data a concentration of 2 mM. The specific activity of the enzyme when succinate, suggested that MatB most likely functions as a methylmalonyl- propionate, acetate, butyrate, acetoacetate, isobutyrate, isovalerate, ␤-hydroxybutyrate, CoA synthetase in R. palustris. or 2-methylsuccinate was used as substrate was Ͻ0.03 ␮mol AMP minϪ1 mgϪ1.

September 2012 Volume 78 Number 18 aem.asm.org 6623 Crosby et al.

Ϫ Ϫ TABLE 5 MatB kinetic parameters with different substrates 0.03 Ϯ 0.01 ␮mol AMP min 1 mg 1 for methylmalonate), repre- senting an ϳ400-fold reduction in activity compared to that of the kcat/Km Ϫ1 kcat (M wild-type enzyme (Table 4). ␮ Ϫ1 Ϫ1 Enzyme Substrate Km ( M) (s ) s ) We investigated further the use of substituted derivatives of RpMatB Malonate 110 Ϯ 818Ϯ 0.3 2 ϫ 105 malonate by RpMatB. We determined kinetic parameters for ma- Methylmalonate 81 Ϯ 612Ϯ 0.2 1 ϫ 105 lonate, methylmalonate, ethylmalonate, butylmalonate, and ATP Ϯ Ϯ ϫ 3 Ethylmalonate 1,250 55 11 0.2 9 10 (Table 5). RpMatB had similar Km values for malonate and meth- Butylmalonate 19,400 Ϯ 1,400 7 Ϯ 0.3 4 ϫ 102 ylmalonate (110 Ϯ 8 ␮M and 81 Ϯ 6 ␮M, respectively), and al- ATP 242 Ϯ 19 24 Ϯ 0.7 1 ϫ 105 ϳ Ϯ Ϫ1 though the kcat for malonate was 50% higher (18 0.3 s for malonate and 12 Ϯ 0.2 sϪ1 for methylmalonate), the catalytic RpMatBT208A Malonate 391 Ϯ 15 19 Ϯ 0.3 5 ϫ 104 Methylmalonate 108 Ϯ 618Ϯ 0.2 2 ϫ 105 efficiencies of the enzyme (kcat/Km) were similar for malonate and 5 Ϫ1 Ϫ1 5 Ϫ1 Ϫ1 Ethylmalonate 144 Ϯ 715Ϯ 0.2 1 ϫ 105 methylmalonate (1.64 ϫ 10 M s and 1.48 ϫ 10 M s , 4 Butylmalonate 349 Ϯ 18 11 Ϯ 0.2 3 ϫ 10 respectively). Notably, the Km values for ethylmalonate and butyl- ATP 195 Ϯ 14 20 Ϯ 0.5 1 ϫ 105 malonate were much higher, at 1,250 Ϯ 55 ␮M and 19,460 Ϯ 1,400 ␮M, respectively (Table 5). Unsurprisingly, the enzyme was T208G Ϯ Ϯ ϫ 4 RpMatB Malonate 1,140 30 14 0.2 1 10 very inefficient; i.e., the k /K values were 5% and 0.2% (respec- Methylmalonate 425 Ϯ 13 15 Ϯ 0.2 4 ϫ 104 cat m tively) relative to the efficiency of the reaction when malonate was Ethylmalonate 370 Ϯ 911Ϯ 0.1 3 ϫ 104 Butylmalonate 282 Ϯ 15 11 Ϯ 0.2 4 ϫ 104 the substrate. ATP 93 Ϯ 811Ϯ 0.3 1 ϫ 105 For comparison, we determined the kinetic parameters of Ϯ ␮ Ϯ Ϫ1 RlMatB. The Km (118 12 M) and kcat (14 0.4 s ) values for RlMatB Malonate 118 Ϯ 12 14 Ϯ 0.4 1 ϫ 105 RlMatB when malonate was the substrate were similar to those for Methylmalonate 464 Ϯ 30 9 Ϯ 0.2 2 ϫ 104 RpMatB, but the catalytic efficiencies of RlMatB with methylma- Ϯ Ϯ ϫ 2 Ϫ Ϫ Ϫ Ethlymalonate 5,600 200 2 0.04 3 10 lonate (1.98 ϫ 104 M 1 s 1) and ethylmalonate (3.21 ϫ 102 M 1 Ϯ Ϯ ϫ 5 ATP 199 15 21 0.6 1 10 sϪ1) were ϳ7-fold and 27-fold lower than that of RpMatB. For Ϯ ␮ RlMatB, the Km values for methylmalonate (464 30 M) and ethylmalonate (5,600 Ϯ 200 ␮M) were Ͼ5-fold higher than the K488A thetases (26). Hence, we constructed an RpMatB variant to corresponding Km values of RpMatB (Table 5). test whether this residue was important for RpMatB activity. As RpMatB substrate binding affinities. We used isothermal expected, activity of the RpMatBK488A enzyme was affected titration calorimetry to measure the affinities of RpMatB and strongly (0.04 Ϯ 0.01 ␮mol AMP minϪ1 mgϪ1 for malonate and RpMatBK488A for various substrates. No binding was observed

FIG 3 Representative isothermal titration calorimetry binding curves. (A) Binding of ATP to RpMatB. ATP (1 mM) was used as the titrant. (B) Binding of ATP to RpMatBK488A. ATP (400 ␮M) was used as the titrant. Top panels show the heat released after each injection, and bottom panels show the resultant binding curves versus the ratio of ATP to enzyme obtained by integrating the heats of injection in the top panels. Data were fit using a single-set-of-sites model, and ⌬ ⌬ ⌬ average values of N, Kd, H, S, and G can be found in Table 6.

6624 aem.asm.org Applied and Environmental Microbiology Expanded Substrate Range of Methylmalonate-CoA Ligase

TABLE 6 Binding of ATP and ADP to MatB and MatBK488A ␮ ⌬ Ϫ1 ⌬ Ϫ1 Ϫ1 ⌬ Ϫ1 Protein Substrate N Kd ( M) H (kcal mol ) S (cal mol K ) G (kcal mol ) MatB ATP 0.96 Ϯ 0.05 5.6 Ϯ 1.6 Ϫ14.1 Ϯ 0.62 Ϫ23.7 Ϯ 2.69 Ϫ7.10 Ϯ 0.17 MatB ADP 1.06 Ϯ 0.06 10.4 Ϯ 2.0 Ϫ6.30 Ϯ 0.60 1.49 Ϯ 2.42 Ϫ6.74 Ϯ 0.12 MatBK488A ATP 0.90 Ϯ 0.01 0.31 Ϯ 0.09 Ϫ8.94 Ϯ 1.12 Ϫ0.48 Ϯ 3.19 Ϫ8.80 Ϯ 0.18 MatBK488A ADP 0.98 Ϯ 0.11 40.5 Ϯ 17.5 Ϫ6.17 Ϯ 1.12 Ϫ0.71 Ϯ 4.69 Ϫ5.96 Ϯ 0.26 when methylmalonate was titrated into a sample cell containing ture is presented. The crystal structure of Se-Met-labeled RpMatB RpMatB alone. In contrast, ATP bound to RpMatB with a disso- was determined to a 1.7-Å resolution using multiwavelength Ϯ ␮ ciation constant (Kd)of5.6 1.6 M(Fig. 3; Table 6). This sug- anomalous dispersion (MAD) phases and includes density for all gested to us that RpMatB likely binds ATP before binding its or- residues except T166 (Fig. 4). Although AMP, CoA, and methyl- Ϯ ␮ ganic acid substrate. The Kd for ADP was 10.4 2.0 M, which is malonate were added prior to crystallization, no electron density close to the Kd of ATP, suggesting that under some physiological for any of these ligands was observed. There were four RpMatB conditions ADP could inhibit RpMatB activity. Surprisingly, the monomers in the asymmetric unit that were essentially identical, RpMatBK488A protein bound ATP approximately 10-fold more with a root mean square deviation (RMSD) between monomers of Ϯ ␮ Ͻ tightly than wild-type RpMatB, with a Kd of 0.31 0.09 M(Fig. 1.03 Å for 3,776 structurally equivalent atoms. These monomers Ϯ ␮ 3), and the affinity of the protein for ADP (Kd, 40.5 17.5 M) were arranged as a dimer of dimers within the unit cell, and some was 4-fold lower than that of the wild-type enzyme (Table 6). other acyl-CoA synthetases have been reported to function as Under the conditions used, we did not observe methylmalonate dimers in solution (6, 21, 24). However, RpMatB was eluted binding to wild-type RpMatB preequilibrated with ADP or to from a size exclusion column as a single peak with an apparent RpMatBK488A preequilibrated with ATP, suggesting that both the molecular mass of 49.0 kDa (expected molecular mass of 54.5 ␥-phosphate of ATP and the K488 side chain may be involved in kDa), suggesting that under the solution conditions in this methylmalonate binding. study, RpMatB functions as a monomer. Crystal structure of RpMatB. Crystals of native and Se-Met- Within the RpMatB structure without bound ligands, the C- labeled RpMatB were obtained, and although the two types of terminal domain was rotated ϳ20° away from the adenylation crystals showed the same space group and cell dimension, the conformation, such that the catalytic lysine residue (K488) was Se-Met-labeled RpMatB crystals showed significantly greater or- not positioned in the active site (Fig. 4B). This conformation was der and diffraction, and therefore only the Se-Met-labeled struc- seen in all four monomers within the unit cell, suggesting that it

FIG 4 RpMatB crystal structures. (A) Overlay of the RpMatB apo structure (the N-terminal domain is shown in light blue and the C-terminal domain in dark blue), with the structure of the RpMatBK488A/ATP complex (N-terminal domain in light green, C-terminal domain in dark green, and ATP in red). The RpMatBK488A/ATP structure is in the adenylation conformation, whereas in the RpMatB apo structure the C-terminal domain is rotated ϳ20°. (B) Close-up view of the active site, highlighting the position of K488 in the apo structure, compared to the homologous residue A488 in the RpMatBK488A/ATP structure.

September 2012 Volume 78 Number 18 aem.asm.org 6625 Crosby et al. was not a result of crystal packing and might instead be represen- formation were unsuccessful. However, RpMatB is 38% identical tative of a stable apo conformation. Within the structure, each and 53% similar to MatB from Streptomyces coelicolor (ScMatB), monomer contained one sulfate ion that interacted with residue which utilizes malonate and methylmalonate as substrates and K399, which is the conserved hinge residue (often an aspartate in whose structure was recently solved in the thioester-forming con- other acyl-CoA synthetases) that rotates to produce the confor- formation (27). Since the thioester-forming conformation is mational change between the adenylation and thioesterification highly conserved within the acyl-CoA synthetase family, the Sc- states. However, crystals grown without sulfate displayed unit cells MatB structure is an ideal template for modeling the RpMatB and space groups identical to the presented structure, and lower- thioester-forming conformation. As discussed above, comparison resolution structures with the same conformation (data not of the N- and C-terminal domains of RpMatB between the apo and shown) did not contain sulfate ions within the unit cell, suggesting adenylation conformations shows very little change within the that although the sulfate ion may stabilize this conformation domains. Therefore, the N- and C-terminal domains of apo Rp- within the crystal lattice, it is not required. MatB were separately aligned to the ScMatB structure using second- The apo structure appears to represent a novel solution phase ary-structure matching in Coot (19); this gave an RMSD of 1.66 Å for conformation of the C-terminal domain. Structures of other acyl- 414 structurally conserved ␣-carbons. Figure 5A shows an alignment CoA synthetases lacking ligands are highly variable in the position of the N- and C-terminal domains of the RpMatB apo structure with of the C terminus, which is in direct contrast to the adenylation the structure of ScMatB with methylmalonyl-CoA and AMP bound. and thioester-forming conformations, which show high degrees Analysis of the active site shows a high degree of structural and se- of structural conservation. These conformations have been de- scribed as intermediates in the pathway, but we suggest that the quence conservation between RpMatB and ScMatB. An alignment of MatB apo structure represents a stable conformation found before RpMatB and related sequences showed a high degree of conservation substrate binding, as the conformation of the C-terminal domain in the methylmalonate-binding pocket (Fig. 6), further supporting is outside the 140° sweep between the adenylation and thioester- our structural observations. Several RpMatB residues appear to be forming conformations. within 5 Å of the methyl group of methylmalonate, including T208, A structure of methylated RpMatBK488A in complex with ATP M302, T303, and M307. Of these, the closest was T208, whose side was determined to 2.0 Å using molecular replacement with the chain hydroxyl group was located within 3.7 Å of the methyl group of N-terminal domain (residues 1 to 400) of the RpMatB apo struc- methylmalonate. ture as a search model (Fig. 4); density for all residues was ob- Single-amino-acid RpMatB variants with expanded range of served. In order to obtain an ATP-bound structure of RpMatB, it substrates. It was hypothesized that a reduction in the size of the side was necessary to methylate the lysines within RpMatB (51). This is chain of T208 could increase the size of the binding pocket and thus an established method to change the nature of the surface-exposed expand the substrate range of RpMatB. Threonine 208 was mutated residues of a protein, which play a large role in the formation of to alanine or glycine, and the activity of the resulting enzymes was the crystal lattice (18, 45), without changing the intrinsic structure assessed with a variety of substrates. Both enzymes utilized malonate, of the protein, and it has been successfully utilized to crystallize methylmalonate, ethylmalonate, and butylmalonate, and in some numerous recalcitrant proteins (31, 35, 39, 52, 57). Additionally, cases they were significantly more efficient than wild-type RpMatB K488A the RpMatB mutant was utilized, as this protein binds ATP (Table 5). However, like wild-type RpMatB, the variants had unde- ϳ10-fold more tightly than wild-type RpMatB (Table 6), and mu- tectable activity with succinate, 2-methylsuccinate, and the monocar- tation of the active-site lysine to alanine prevented predicted com- boxylic acids acetate, propionate, butyrate, valerate, isobutyrate, ace- plications due to methylated K488 within the active site. toacetate, isovalerate, and ␤-hydroxybutyrate. K488A The methylated RpMatB structure with ATP bound T208A Ϯ ␮ The RpMatB protein had a Km of 144 7 M for ethyl- showed a high degree of similarity to the apo structure in which malonate, which is ϳ9-fold lower than that of wild-type RpMatB, comparison of the N-terminal domains (residues 1 to 400) gave an Ϯ ␮ Ͼ and the Km (349 18 M) for butylmalonate was 50-fold RMSD of 0.92 Å for 1,579 structurally equivalent main-chain at- lower than that of the wild-type enzyme. While RpMatB exhibited oms and comparison of the C-terminal domains (residues 401 to the highest activities with malonate and methylmalonate, 503) gave an RMSD of 0.47 Å for 406 structurally equiv- RpMatBT208A was most active with methylmalonate and ethylma- alent main-chain atoms. This suggested that methylation of ϫ 5 Ϫ1 K488A lonate, having catalytic efficiencies (kcat/Km) of 1.67 10 M RpMatB did not change the intrinsic structure of the protein Ϫ Ϫ Ϫ s 1 and 1.04 ϫ 105 M 1 s 1, respectively. The RpMatBT208G pro- and that very little structural change occurred within the domains Ϯ ␮ between the apo and adenylation conformations. The C-terminal tein had an even lower Km for butylmalonate (282 15 M), and domain of the ATP-bound structure did show an ϳ20° rotation in fact it had the highest catalytic efficiency with butylmalonate (3.90 ϫ 104 MϪ1 sϪ1). Interestingly, the catalytic efficiencies of the relative to the apo structure (Fig. 4A), and this conformation is T208G very similar to adenylation conformations seen with other acyl- RpMatB enzyme with methylmalonate and ethylmalonate ϫ 4 Ϫ1 Ϫ1 ϫ 4 Ϫ1 Ϫ1 CoA synthetases. The ATP-bound methylated RpMatBK488A were similar (3.53 10 M s and 2.97 10 M s , respec- T208A Ϯ structure showed RMSDs of 1.95 Å for 428 structurally equivalent tively) (Table 5). The RpMatB protein had a Km (195 14 ␣-carbons and 2.10 Å for 468 structurally equivalent ␣-carbons ␮M) for ATP similar to that of wild-type RpMatB, whereas the T208G Ϯ ␮ compared to Homo sapiens acyl-CoA synthetase medium-chain RpMatB protein had a Km for ATP (93 8 M) that was family member 2A in the adenylation conformation (PDB acces- ϳ2-fold lower (Table 5), demonstrating that ATP binding was not sion number 3C5E) and Saccharomyces cerevisiae acetyl-CoA syn- adversely affected by changing T208 to an alanine or glycine residue. thetase in the adenylation conformation (PDB accession number These data demonstrated that a reduction in the size of the side chain 1RY2), respectively (29, 36). at position 208 was sufficient to expand the range of substrates used Attempts to crystallize RpMatB in the thioester-forming con- by RpMatB while retaining high catalytic efficiency.

6626 aem.asm.org Applied and Environmental Microbiology Expanded Substrate Range of Methylmalonate-CoA Ligase

FIG 5 Substrate-binding sites in RpMatB. (A) RpMatB (light green and dark green) modeled in the thioester-forming conformation overlaid onto ScMatB/ methylmalonyl-CoA/AMP (PDB accession number 3NYQ, pink and purple); AMP is shown in yellow and methylmalonyl-CoA is shown in gray. Only the phosphopantetheine moiety of CoA is ordered and visible in the ScMatB structure. (B) Close-up view of the active site, with methylmalonyl-CoA shown in gray (AMP is not shown). The side chain of residue T208 of RpMatB is within 3.7 Å of the methyl group of methylmalonyl-CoA.

DISCUSSION dict that methylmalonyl-CoA is converted to succinyl-CoA by RpMatB is the first malonyl-CoA synthetase homologue that has methylmalonyl-CoA mutase (EC 5.4.99.2) and then enters the been shown to function in methylmalonate metabolism in vivo. tricarboxylic acid cycle. The encoding two other MatB homologues that have been In addition to their biological roles, malonyl-CoA and meth- studied within a physiological context (from R. leguminosarum ylmalonyl-CoA are the two most commonly utilized polyketide and S. meliloti) are located within operons containing a malonyl- extender units, and malonyl-CoA synthetases are a common way CoA decarboxylase and dicarboxylate transporter, suggesting that to produce these extender units directly from malonate and meth- their primary roles are in malonate metabolism (3, 13). Although ylmalonate. RlMatB is the most commonly used malonyl-CoA it was reported that an S. meliloti ⌬matB strain grows poorly on synthetase for production of malonyl-CoA and methylmalonyl- malonate, growth on methylmalonate was not assessed (13). In R. CoA and has been reported to use a variety of synthetic C-2 ma- palustris, matB is independently transcribed. In the genome, the lonate derivatives, including isopropyl-, dimethyl-, cyclopropyl-, matB gene is located ϳ1,500 nucleotides away from the sdhCDAB cyclobutyl-, and benzylmalonate, with catalytic efficiencies that operon (encoding succinate dehydrogenase), with a divergently ranged from Ͻ1 to 19% of those seen with malonate as the sub- transcribed hypothetical gene between matB and sdhCDAB. While strate (50). The RpMatB reported here uses malonate more effi- malonyl-CoA is commonly metabolized through decarboxylation ciently than RlMatB (catalytic efficiency of 1.64 ϫ 105 MϪ1 sϪ1 to acetyl-CoA (malonyl-CoA decarboxylase, EC 4.1.1.9), we pre- compared to 1.16 ϫ 105 MϪ1 sϪ1) and methylmalonate signifi-

FIG 6 Alignment of methylmalonate-binding pockets of MatB homologues. Residues indicated by diamonds are located within5Åofthemethyl group of methylmalonate in R. palustris MatB. Sequences are from Rhizobium leguminosarum bv. trifolii, Methylobacterium extorquens AM1, Sinorhizobium meliloti 1021, Nitrobacter winogradskyi, Bradyrhizobium japonicum, Oligotropha carboxidovorans, Rhodopseudomonas palustris CGA009, Streptomyces coelicolor, and the Homo sapiens ACSF3 protein.

September 2012 Volume 78 Number 18 aem.asm.org 6627 Crosby et al. cantly more efficiently, having a catalytic efficiency of 1.48 ϫ 105 LB400: defining the entry point into the novel benzoate oxidation (box) MϪ1 sϪ1 compared to 1.98 ϫ 104 MϪ1 sϪ1, perhaps reflecting pathway. J. Mol. Biol. 373:965–977. adaptation to different physiological roles. 7. Balch WE, Wolfe RS. 1976. New approach to the cultivation of metha- nogenic bacteria: 2-mercaptoethanesulfonic acid (HS-CoM)-dependent Structural studies of RpMatB in the apo and adenylation con- growth of Methanobacterium ruminantium in a pressurized atmosphere. formations combined with a predicted model of RpMatB in the Appl. Environ. Microbiol. 32:781–791. thioester-forming conformation based on a structure of ScMatB 8. Bertani G. 2004. Lysogeny at mid-twentieth century: P1, P2, and other allowed for the identification of T208 in RpMatB, which appears experimental systems. J. Bacteriol. 186:595–600. 9. Bertani G. 1951. Studies on lysogenesis. I. The mode of phage liberation to help define the size of the organic acid-binding pocket. By by lysogenic Escherichia coli. J. Bacteriol. 62:293–300. changing T208 to alanine and glycine, MatB variants were gener- 10. Blommel PG, Fox BG. 2007. A combined approach to improving large- ated that efficiently used malonate derivatives with longer acyl scale production of tobacco etch virus protease. Protein Expr. Purif. 55: chains at the C-2 position. RpMatBT208A was more efficient at 53–68. utilizing methylmalonate than wild-type RpMatB (catalytic effi- 11. CCP4. 1994. The CCP4 suite: programs for protein crystallography. Acta ϫ 5 Ϫ1 Ϫ1 Crystallogr. D Biol. Crystallogr. 50:760–763. ciency of 1.67 10 M s ) and was able to utilize ethylma- 12. Chan YA, Podevels AM, Kevany BM, Thomas MG. 2009. Biosynthesis of lonate at 63% of the catalytic efficiency of malonate by wild-type polyketide synthase extender units. Nat. Prod. Rep. 26:90–114. RpMatB. RpMatBT208G was able to utilize butylmalonate at 23% 13. Chen AM, et al. 2010. Identification of a TRAP transporter for malonate of the catalytic efficiency of malonate by wild-type RpMatB. transport and its expression regulated by GtrA from Sinorhizobium meli- loti. Res. Microbiol. 161:556–564. Additionally, these enzymes had Km values for ethylmalonate 14. Cheng Q, Xiang L, Izumikawa M, Meluzzi D, Moore BS. 2007. Enzy- and butylmalonate of 144 Ϯ 7 and 282 Ϯ 15 ␮M, respectively, matic total synthesis of enterocin polyketides. Nat. Chem. Biol. 3:557–558. and these Km values are low enough for these engineered en- 15. Cowtan K. 2008. Fitting molecular fragments into electron density. Acta zymes to feasibly work under physiological conditions for the Crystallogr. D Biol. Crystallogr. 64:83–89. synthesis of polyketides in heterologous hosts like E. coli. 16. Cowtan K. 2010. Recent developments in classical density modification. Acta Crystallogr. D Biol. Crystallogr. 66:470–478. Interestingly, the four residues that are in close proximity to the 17. Crosby HA, Heiniger EK, Harwood CS, Escalante-Semerena JC. 2010. methyl group of methylmalonate in RpMatB (T208, M302, T303, and Reversible N(epsilon)-lysine acetylation regulates the activity of acyl-CoA M307) are conserved in most malonyl-CoA synthetases (Fig. 6), in- synthetases involved in anaerobic benzoate catabolism in Rhodopseudo- cluding RlMatB. Of the MatB sequences aligned in Fig. 6, only human monas palustris. Mol. Microbiol. 76:874–888. 18. D’Arcy A, Stihle M, Kostrewa D, Dale G. 1999. Crystal engineering: a ACSF3 and ScMatB have any changes in the methylmalonate binding case study using the 24 kDa fragment of the DNA gyrase B subunit from pocket, with a relatively conservative difference of valine instead of Escherichia coli. Acta Crystallogr. D Biol. Crystallogr. 55:1623–1625. threonine at position 208 (based on RpMatB numbering). While the 19. Emsley P, Cowtan K. 2004. Coot: model-building tools for molecular structure of ScMatB is known, kinetic parameters for ScMatB have graphics. Acta Crystallogr. D Biol. Crystallogr. 60:2126–2132. not been reported; thus, we do not yet know its relative activities with 20. Gao X, Wang P, Tang Y. 2010. Engineered polyketide biosynthesis and biocatalysis in Escherichia coli. Appl. Microbiol. Biotechnol. 88:1233– malonate and methylmalonate. A structure is not available for Rl- 1242. MatB, and as residues that contact methylmalonate are highly con- 21. Gibson J, Dispensa M, Fogg GC, Evans DT, Harwood CS. 1994. 4-Hy- served compared to those in RpMatB, it is likely that subtle differences droxybenzoate-coenzymeA ligase from Rhodopseudomonas palustris— in structure outside the methylmalonate- cause RlMatB purification, gene sequence, and role in anaerobic degradation. J. Bacte- to preferentially utilize malonate. riol. 176:634–641. 22. Grosse-Kunstleve RW, Adams PD. 2003. 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