New aminopeptidase from "microbial dark matter" archaeon

The FASEB Journal • Research Communication

New aminopeptidase from “microbial darkmatter” archaeon

Karolina Michalska,*,† Andrew D. Steen,‡,§ Gekleng Chhor,* Michael Endres,*Austen T. Webber,§ Jordan Bird,‡ Karen G. Lloyd,‡ and Andrzej Joachimiak*,†,{,1

*Midwest Center for Structural Genomics and †Structural Biology Center, Biosciences Division, ArgonneNational Laboratory, Argonne, Illinois, USA; Departments of ‡Microbiology and §Earth and PlanetarySciences, University of Tennessee, Knoxville, Tennessee, USA; and {Department of Biochemistry andMolecular Biology, University of Chicago, Chicago, Illinois, USA

ABSTRACT Marine sediments host a large populationofdiverse,heterotrophic, unculturedmicroorganismswithunknown physiologies that control carbon flow throughorganic matter decomposition. Recently, single-cellgenomics uncovered new key players in these processes,such as the miscellaneous crenarchaeotal group. Thesewidespread archaea encode putative intra- and extracellu-lar proteases for the degradation of detrital proteinspresent in sediments. Here, we show that one of theseenzymes is a self-compartmentalizing tetrameric amino-peptidase with a preference for cysteine and hydrophobicresidues at the N terminus of the hydrolyzed peptide. Theability to perform detailed characterizations of enzymesfrom native subsurface microorganisms, without requir-ing that those organisms first be grown in pure culture,holds great promise for understanding key carbon trans-formations in the environment as well as identifying new en-zymes for biomedical and biotechnological applications.—Michalska, K., Steen, A. D., Chhor, G., Endres, M., Webber,A. T., Bird, J., Lloyd, K. G., Joachimiak, A. New amino-peptidase from “microbial dark matter” archaeon.FASEB J. 29, 000–000 (2015). www.fasebj.org

Key Words: carbon cycle • marine sediments • single-cell genomics •

detrital proteins

THE VAST MAJORITY OF MICROORGANISMS in the environmenthave never been grown in the laboratory. They havetherefore been referred to as microbial dark matter(MDM) (1) because it is difficult to study their physiologyand determine their impact on ecosystems and majorglobal elemental cycles. Each of these uncultured micro-organisms harbors uncharacterized enzymes that havenever been studied or tapped for their biotechnologicaland biomedical potential (2). Recently, the sequences ofgenomes fromMDMhave become available, by amplifyingDNA from single cells (3) and assembling MDM genomes

in silico from environmental metagenomes (4). However,the novelty of the information that can be gathered fromthese genomes is restricted because the annotation of in-dividual genes relies on their sequence similarity to proteinsthat have been characterized previously in cultured organ-isms.This is amajordrawback in the studyofMDMgenomesbecause it hinders the discovery of truly unique functionsthat do not exist in the cultured realm studied thus far.

Nevertheless, sequencing genomes fromMDMprojectshas the potential to reveal unexpected functionalities. Forexample, some archaeal species encode predicted pro-teases that may be utilized for degradation of detritalproteins in marine sediments and can contribute to car-bon and nitrogen cycling (5). To uncover the true bio-chemical functions of these enzymes, we have investigatedrecombinant proteins, including here as an examplea putative protease [designated as bathyaminopeptidaseMCG (miscellaneous crenarchaeotal group)-15 (BAP)]from Thaumarchaeota archaeon SCGC AB-539-E09. Thisunculturedmicroorganism is phylogenetically assigned tothe MCG, which is widespread in marine sediments (5)and has recently been given the phylum name CandidatusBathyarchaeota (6). BAP is of particular interest because noclose homologs exist in cultured organisms, making theoriginal annotation ambiguous. As indicated by the BasicLocal Alignment Search Tool (BLAST), it shares se-quence similarity with proteins annotated as 1) X-prolyldipeptidyl aminopeptidase (PepX) from Caulobacter sp.K31 (S15 peptidase family, 51% sequence identity, EC3.4.14.11); 2) cocaine esterase (CocE) from Janthinobacte-rium sp. HH01 (51% sequence identity, EC 3.1.1.84); and3) a-amino acid ester hydrolase (AEH) from Xanthomonascitri (XcAEH; 51% sequence identity, EC 3.1.1.43); someclose homologs are also annotated (most likely in-correctly) as glutaryl-7-ACA acylase, EC 3.5.1.93. Amongthe proteins that have been biochemically character-ized, only the confirmed AEH holds its relatively high se-quence identity. The other homologs with experimentally

Abbreviations: 6-APA, 6-aminopenicillanic acid; 19-ID, 19-In-sertion Device; AEH, a-amino acid ester hydrolase; AMC, 7-amido-4-methylcoumarin; APS, Advanced Photon Source; BAP, bathy-aminopeptidase miscellaneous crenarchaeotal group-15; BLAST,Basic Local Alignment Search Tool; CocE, cocaine esterase; HEPES,4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; IMG, integratedmicrobial genome; KI, inhibition constan; Km, Michaelis constant;

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1 Correspondence: Structural Biology Center, BiosciencesDivision, Argonne National Laboratory, 9700 South Cass Ave.,Building 446, Argonne, IL 60439, USA. E-mail: [email protected]: 10.1096/fj.15-272906This article includes supplemental data. Please visit http://

www.fasebj.org to obtain this information.

0892-6638/15/0029-0001 © FASEB 1

The FASEB Journal article fj.15-272906. Published online June 10, 2015.

validated activities and structural characterization are infact more distant relatives, with sequence identity of 27%for CocE (7) and 16% for PepX (8).

All of the above enzymes belong to the large family ofserine hydrolases that have conserved catalytic resi-dues, and very likely, they share a commonmechanism.Namely, they utilize a classic Ser-His-Asp catalytic triadand a water molecule to carry out acylation and deacy-lation steps with the help of an oxyanion hole, whichstabilizes tetrahedral intermediates (9). These enzymescan carry out both hydrolytic and synthetic reactionsusing a variety of substrates. Aminopeptidases pro-gressively remove N-terminal residues from short pep-tides through peptide bond cleavage. Esterases canhydrolyze ester bonds but also carry out esterificationand transesterification reactions. AEHs catalyze thesynthesis of b-lactam antibiotics using an a-amino acidester [such as D-phenylglycine (Phg) methyl ester] asa donor and a b-lactam as an acceptor (10). They alsoare able to hydrolyze product antibiotics, and someAEHs exhibit a-aminopeptidase activities (11). In thiswork, we collectively refer to themas S15-like family, andwe follow standard nomenclature as used for serineproteases.

MATERIALS AND METHODS

Cloning to vector pMCSG73 and in vitro tobacco veinmottling virus protease cleavage of transcriptiontermination/antitermination protein-BAP fusion

The DNA template for BAP was obtained and amplified froma single cell of T. archaeon SCGC AB-539-E09. The fragmentcoding for theBAPprotein(residuesMet1-Ser623)was amplifiedwith KOD DNA Polymerase using conditions and reagents pro-vided by EMD Millipore (Billerica, MA, USA). The followingprimers were used: 59-TACTTCCAATCCAATGCCATGAAAAA-ACTCAGAGATGATTTCTCCGAAGA-39, and 59-TTATCCACT-TCCAATGTTATGAAATAATATGTATCTCGATCGCTGTCG-39.The PCR product was cloned according to the ligation-independent procedure (12, 13) into vector pMCSG73, which isa derivative of the pMCSG53 vector (14). Proteins expressed fromvectorpMCSG73areproducedas aC-terminal fusion toEscherichiacoli transcription termination/antitermination protein (NusA) inthe following protein construct:NusA-[tobacco veinmottling virus(TVMV) protease recognition site]-His6-Strep tag-[tobacco etch vi-rus (TEV)protease recognition site]-(TARGET) (NusA-ETVRFQ/S-HHHHHH-WSHPQFEK-ENLYFQ/SNA-GENE SEQUENCE).The fusion is cleaved in vitro by TVMVprotease overexpressed ina separate batch of bacteria. An auxiliary plasmid expressingTVMV(pMCSG-TVMV)was createdby cloningof theTVMVgene

into pMCSG7 (15) where a fragment between the START codonand the TEV recognition site had been deleted. The process ofreleasingNusA starts at sonication and is completed by the timeofloading onto the Ni resin. The amount of TVMV protease over-expressed in 1 L growth media is sufficient to cleave an NusA-target fusion from 10 L target-producing bacteria.

Protein expression and purification

The E. coli BL21-Gold(DE3) strain carrying the pMCSG73-BAPplasmid was grown in 1 L enriched M9 medium (16) at 37°C,shaking at 200 rpm until it reached an optical density at a wave-length of 600 nm (OD600) of 1.0. Inhibitory amino acids (25 mgeach of L-valine, L-isoleucine, L-leucine, L-lysine, L-threonine, andL-phenylalanine) and 90 mg selenomethionine (SeMet) (OrionEnterprises,Wheeling, IL,USA)were added to the culture, whichwas then cooled to 4°C for 60min. To induce protein expression,0.5 mM isopropyl-b-D-thiogalactoside was added. The cells wereincubated overnight at 18°C and then centrifuged for 10 min at7880 g. The supernatant was removed, and the cells were resus-pended in 40 ml lysis buffer [500 mMNaCl, 5% glycerol, 50 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH8.0), 20mM imidazole, and 10mM2-ME]. To removeNusA, 3mlTVMV protease-expressing cell suspension (OD600, ;70) wasadded to the target cells, which were then disrupted by 5 min ofsonication. The lysate was centrifuged for 1 h at 29,500 g to sep-arate insoluble fractions, which were discarded. The proteinpresent in the supernatant was purified using Ni-NTA affinitychromatography and the AKTAxpress system (GE HealthcareBiosciences, Pittsburgh, PA, USA) as described previously (17, 18).Subsequently, the N-terminal His6 tag was removed using re-combinant His7-tagged TEV protease. In the next purificationstep, subtractive Ni-NTA affinity chromatography was applied toremove theprotease,affinity tag,andanyuncutprotein.NativeBAPwas also expressed and purified analogously to the SeMet-labeledvariant, with the exception that the growthmedia consisted of bothM9 and Luria-Bertani. The pure proteins were concentrated incrystallization buffer [20mMHEPES (pH 8.0), 250mMNaCl, and2mMDTT]usinganAmiconUltra-15concentrator (EMDMillipore).Protein concentrations were determined based on the absor-bance at 280 nm measured on a NanoDrop 1000 Spectro-photometer (Thermo Scientific,Waltham,MA, USA) and theirtheoretic, sequence-deduced absorption coefficients.

Crystallization

The SeMet-labeled BAP protein was screened for crystallizationconditions with the help of a Mosquito nanoliter liquid handler(TTP Labtech, Cambridge, MA, USA) using the sitting-drop va-por diffusion technique in 96 well CrystalQuick plates (GreinerBio-One,Monroe,NC,USA). The crystallizationwas set up at 4°Cusing the MCSG 1–4 screens from Microlytic (Woburn, MA,USA). Foreachcondition, 0.4ml protein(at50mg/ml)and0.4mlcrystallization formulation were mixed; the mixture was equili-brated against 140 ml of the crystallization solution in each res-ervoir well. For cocrystallization of native BAP (20 mg/ml) withDL-Phe, the protein was incubated with the ligand for 4 h at 4°Cprior to plate setup. The crystals appeared under a number ofconditions, 2 ofwhichwereultimately used.CrystalsX1grew froma solution containing0.2Mpotassium thiocyanate and20%(w/v)polyethylene glycol (PEG) 3350, whereas crystals X2 grew from0.2 M sodium citrate, 20% (w/v) PEG 3350, and 2.8 mM DL-Phe.

Data collection

Prior to data collection, the crystals were briefly soaked inmotherliquors supplemented with 15% glycerol for cryoprotection and

(continued from previous page)MCG, miscellaneous crenarchaeotal group; MDM, microbialdark matter; MR, molecular replacement; NusA, transcriptiontermination/antitermination protein; OD600, optical density ata wavelength of 600 nm; PDB, Protein Data Bank; PEG, poly-ethylene glycol; PepX, X-prolyl dipeptidyl aminopeptidase; Phg,phenylglycine; RAxML, randomized axelerated maximum like-lihood; rmsd, root-mean-SD; SeMet, selenomethionine; TCEP,tris(2-carboxyethyl)phosphine; TEV, tobacco etch virus; TLC,thin-layer chromatography; Topt, optimal temperature; TVMV,tobacco vein mottling virus; Vmax, maximum velocity; XcAEH,a-amino acid ester hydrolase from Xanthomonas citri

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immediately flash cooled in liquid nitrogen. The diffractionexperiments were performed at the 19-Insertion Device (19-ID)beamline of the Structural Biology Center at the Advanced Pho-ton Source (APS) at Argonne National Laboratory using theprogram SBCcollect (19). There were 2 diffraction data sets col-lected at 100 K near the selenium absorption peak. Crystals X1(l = 0.9792 A) diffracted to 2.40 A resolution, whereas the resolu-tionof theX2crystals (l=0.9793A)extendedto2.10A.Thecrystalsare isomorphous andbelong to themonoclinicP21 space group.The diffraction images were processed with the HKL-3000 suite(20). Intensities were converted to structure factor amplitudesin the CTRUNCATE program (21, 22) from the CCP4 package(23). The data collection and processing statistics are given inTable 1 (for X2 only; see below).

Structure solution

Initial attempts to determine the structure of crystal X1 throughthe selenium-based single-wavelength anomalous diffractionmethod failed due to insufficient anomalous signal for successfulexperimental phasing. Therefore, the structure was solved bymolecular replacement (MR) with the XcAEH [Protein DataBank (PDB) entry 1MPX] (24), as a search model. The calcu-lations were performed in the HKL-3000 package (20), which

executes MOLREP (25) for MR search followed by automatedmodel building in ARP/wARP (26). The preliminary model wasfurther rebuilt in Coot (27) and refined in REFMAC (28). Then,the structure was refined against the diffraction data for crystalX2. Because the electron density did not reveal the presence ofthephenylalanine ligand, thefinalmodel of theapo structurewasobtained through alternating manual rebuilding and crystallo-graphic refinement in BUSTER (29) against the X2 crystal data.For the refinement, 4 translation/libration/screw (TLS) groupswere defined, each of them for a single protein chain. In therefined model, the following residues could be reliably modeledin the electron density map: Phe8-Glu391 and Glu397-Ser623 inchains A and D, and Asp7-Glu391 and His399-Ser623 in chains BandC. In addition to theproteinmolecules, 1124watermoleculeshavebeen identified, 15glycerolmolecules, and4PEGchains.Anunassigned positive peak in the DFo-mFc electron density map ispresent near the catalytic Ser158 in chain D. The quality of thefinal structure was verified by the MolProbity server (30). Therefinement statistics are shown in Table 1. The atomic coor-dinates and structure factors have been deposited in the PDBunder accession code 4PF1.

Enzymatic assays

The peptidase activity was determined directly, usingfluorogenicsubstrate analogs bearing 7-amido-4-methylcoumarin (AMC) or4-methylumbelliferone linked to an amino acid or a peptidechain at its C terminus or with dipeptides, or indirectly, viaa competition assay. The AMC-based experiment requires theAMC molecule to be released from the substrate. Thus, for pep-tides containing $2 residues, the detection of AMC would in-dicate that the enzyme is an endopeptidase, a progressiveaminopeptidase that sequentially cleaves off N-terminal residuesuntil AMC is reached, or, much less probably, a nonspecificcarboxypeptidase.

In the AMC-based assays, AMC substrate hydrolysis rates weremeasured at 500 mM substrate concentration (saturating) at25°C. A total of 6.5ml substrate, dissolved in DMSO, was added to245 ml buffer [100 mM sodium citrate, 200 mM sodium phos-phate, and 30 mM KCl (pH 6.85)] containing 4.35 mg enzymeml21. Fluorescence (excitation, 360 nm; emission, 445 nm) wasmonitored over 10 min in a BioTek Cytation 3 plate reader(BioTek Instruments, Winooski, VT, USA) and calibrated withAMC standards. Hydrolysis rates in triplicate samples with theenzyme were compared to no-enzyme controls to assess the im-portance of uncatalyzed substrate hydrolysis.

The competitive inhibition experiment used 2000 mM in-hibitor and 200 mM L-Phe-AMC. Each reaction contained 10 mlsubstrate dissolved in DMSO, 20ml inhibitor dissolved in Q-H2O,and 970 ml citrate/phosphate buffer containing 1.74 mg enzymeml21, mixed in 1 ml methacrylate cuvettes and held at 21°C.Fluorescence was monitored with a Promega QuantiFluor STsolid statefluorimeter (Madison,WI,USA) set to theUVchannel.The inhibition constant KI was calculated as

KI ¼vivuKm ½I ��

12 vivu

�ðKm þ ½S �Þ

where Km is the Michaelis constant, vi is reaction velocity in thepresence of the inhibitor, vu is velocity in theuninhibited control,[I] is inhibitor concentration, and [S] is substrate concentration.

Temperature sensitivity was assessed using L-Leu-AMC at500 mM. The incubation temperature was slowly increased from0 to 46°C. Maximum velocity Vmax was calculated as the hydrolysisrate of fluorescence production between each set of measure-ments, and optimal temperature Topt was calculated based ona nonlinear least-squares fit of a normal function to thetemperature-activity data.

TABLE 1. BAP data collection and refinement statistics

Data processing

Space group P21Cell dimensionsa 118.4 Ab 108.1 Ac 120.4 Ab 95.1°

Temperature 100 KRadiation source 19-IDWavelength 0.9793 AResolutiona 30.00–2.10 A (2.14–2.10)Unique reflections 176,530 (8826)Rmerge

b 0.137 (0.570),I./,sI. 9.0 (1.9)Completeness 99.8% (99.8)Redundancy 3.2 (3.0)

Refinement

Resolution 29.69–2.10 ANo. of reflections work/test set 173,579/2166Rwork/Rfree

c 0.205/0.229No. of atom proteins/ligands/

water19,666/130/1124

Average B factor protein/ligands/water

24.4/36.2/23.8 A2

RmsdBond lengths 0.008 ABond angles 0.73°

Ramachandran plotMost favored 95.9%Outliers 0.25%

MolProbity score 1.26Clashscore 1.39

aValues in parentheses correspond to the highest-resolution shell.bRmerge = ShSj|Ihj 2 ,Ih.|/ShSjIhj, where Ihj is the intensity ofobservation j of reflection h. cRwork = Sh|Fo| 2 |Fc|/Sh|Fo| for allreflections, where Fo and Fc are observed and calculated structurefactors, respectively. Rfree is calculated analogously for the test reflections,randomly selected, and excluded from the refinement.

“MICROBIAL DARK MATTER” AMINOPEPTIDASE 3

For thin-layer chromatography (TLC)-based transferase assay(Supplemental Fig. S1), substrates were dissolved in 50 mMHEPES/Na buffer (pH 6.8 or 7.6; data not shown) and 2.5%DMSO to yield 100mM stock solutions. 6-Aminopenicillanic acid(6-APA) solution also contained 150 mMNaOH. Reactions werecarried out at room temperature for 1.5 h in buffer 1 [50 mMHEPES/Nabuffer (pH6.8) and2.5%DMSO]orbuffer 2 [50mMHEPES/Na buffer (pH 7.6) and 2.5% DMSO] with final con-centrations of substrates 15 mM and enzyme 0.55 mg/ml. Silicagel-coated TLC plates loaded with 1 ml reaction mixture wereeluted in amobile phase consisting of n-butanol:acetic acid:waterin a 3:1:1 ratio. The plates were dried, briefly soaked in ninhydrinstain (0.1 g ninhydrin, 0.5 ml acetic acid, and 100 ml acetone),dried, and heated to develop spots.

Hydrolysis of L-Phe-L-Leu was measured using a distinct TLC-based assay. Here, solutions of the dipeptide (10 mM in thecitrate/phosphate buffer described above and 1% DMSO) wereincubated with 0.435 mg/ml BAP for 3 h at 30°C. Products wereseparated by TLC. Silica gel-coated TLC plates loaded with 0.5mlreaction mixture were eluted in a mobile phase consisting ofn-butanol:acetic acid:water in a 4:1:1 ratio. The plates were dried,sprayed with ninhydrin, and heated to develop.

There were 2 experiments performed to assess the effect ofoxygen on BAP. First, activity of BAP (0.435 mg/ml), which hadbeen stored frozen inoxiccitrate/phosphatebuffer (pH6.85) for;3mo (including;3 h thawed), was assessed in the presence orabsenceof 10mMtris(2-carboxyethyl)phosphine(TCEP). StoredBAP was thawed, mixed with a TCEP solution to a final concen-tration of 10 mM TCEP, and incubated with L-Phe-AMC, L-Leu-AMC, or L-Leu-L-Leu-AMC. Second, fresh BAP [stored with20 mM HEPES (pH 8.0), 250 mM NaCl, and 2 mM DTT] wasthawed, diluted into citrate/phosphate buffer (0.435mg/ml finalconcentration enzyme), and theexperiment described abovewasrepeated.

Phylogenetic analysis

BLAST2.2.28wasused to searchdatabases representing135 largemetagenomes (.109 bp) from integrated microbial genomes(IMGs) (31) and MG-RAST (32) encompassing a wide range of

commensal and environmental samples, for homologs of thecrystallized protein. In addition, all S15 homologs identified byannotations with Pfam PF02129 were obtained from all culturedmicroorganisms in the IMG database. All BLAST hits from envi-ronmental metagenomes with E values , 102100 were alignedalong with the sequence of the crystallized protein and Pfam hitsfrom cultured microorganisms using MUSCLE (33). Thesesequenceswere imported intoARB(www.arb-home.de) andusedto build amaximumlikelihood tree using randomized axeleratedmaximum likelihood (RAxML).Only 4 sequences from amarinesediment metagenome grouped with the sequence of the crys-tallized protein. Metatranscriptomic hits were identified by ap-plying Pfam searches on metatranscriptomes downloaded fromMG-RAST using InterProScan 5.3-46.0 (34) to search the Pfamdatabase with an E value cutoff of 1 3 1025. These were alsoimported intoARBandalignedmanually.Because theseare shortreads, they were not included in the final tree.

RESULTS

BAP is unique to MDM

To investigate genomic BAP abundance, we analyzed 135environmental metagenomes and 15,000 genomes of cul-tured organisms. This search revealed highly similar(84–96% at the amino acid level) sequences only from es-tuarine sediment in North Carolina, United States (Fig. 1).These metagenome sequences share perfect matches tothe residues that appear to define the signaturemotif forBAP-likeenzymes, asdiscussedbelow(SupplementalFig. S2).The existence of these genes in uncultured micro-organisms in such distantly located marine sediments(Denmark and United States) suggests that further ex-ploration of marine sediments will reveal that BAP ispresent inmarine sediments worldwide becauseMCGs arebroadly distributed (5). Many other marine sedimentmetagenomes also contained homologous proteins (Sup-plemental Table S1; BLAST hits of E, 102100), although

Figure 1. Phylogenetic placement of BAP relative to homologs from cultured organisms. RAxML amino acid tree of BAPhomologs in cultured organisms (black), marine sediment metagenomes, and BAP protein characterized functionally andstructurally in this study (red) is presented. Sequences that have been evaluated as proteins are in bold.

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they were not as closely matched as those from the NorthCarolina estuary. These include environments as varied asthe Arctic Ocean, Antarctica, Baltic Sea, and PeruMargin.We also found homologs of BAP in marine sedimentmetatranscriptomes (Supplemental Table S2) (35), sug-gesting that this protein is not just a genomic feature but isactually expressed in the environment.

Enzymatic activity

BAPwas originally annotatedas a putative protease and/oresterase. To validate these predictions, we assessed BAP’sactivity either directly using fluorogenic substrate analogsor a peptide or indirectly through a competition assay (seeSupplementalData). In a standardizedassayusing0.44mg/mlenzyme solution, BAP is an aminopeptidase most effi-ciently hydrolyzing the amide bond of L-configured di-peptideproxieswithcysteineorahydrophobic residueat theN terminus (Table 2). The activity is decreased in thepresence of divalent cations, with Mg2+ having a morepronounced effect than Ca2+, regardless of the substrateused. Among the tested S1 residues, L-Cys is the most pre-ferred (Km

L-Cys-AMC = 806 17mM), followedby L-Ala, L-Phe,L-Leu, L-Tyr, L-Trp, L-Ile, and Gly. L-Arg and L-Phg aremuch poorer substrates. Among longer peptides, the onlytripeptide proxy detectably hydrolyzed under the sameexperimental conditions is L-Leu-L-Leu-AMC. This sub-strate seems tobeprogressively hydrolyzed (first, the L-Leu-L-Leu peptide bond is cut, and then the L-Leu-AMC amidebond). Hydrolysis of Gly-L-Pro-AMC is only detectable withlonger incubations (;3 h) and higher enzyme concen-trations, most likely due to poor performance of L-Pro inthe P1 site, but also, the L-Pro mismatch in the P19 sitecannot be excluded. Similar conditions are necessary toobserve hydrolysis of tetrapeptide proxies Gly-Gly-L-Arg-AMC and L-Ala-L-Ala-L-Phe-AMC, even though all residues

in these substrates are acceptable in the S1 position andshould allow progressive cleavage. Thus, either theseproxies are too large to access BAP’s active site, or theenzyme is an aminopeptidase with specific requirementsfor the P19, P29, and P39 sites. However, the fact that L-Phe-L-Leupeptide is readily hydrolyzed (Supplemental Fig. S2)with efficiency comparable to that against L-Cys-AMC sug-gests that peptide size rather than residue identity at the P1or P19 positions is a discriminating factor.

D-configured substrates are poorly recognized by BAP.The cleavage of D-Phe-AMC could be detected at a muchlower rate (rel v0L

-Cys-AMC = 0.65 6 0.03%). The binding ofother D-configured derivatives of phenylalanine and leucine(esters and amides) was also observed, but affinity of the in-hibitor was considerably lower for D than for L stereoisomers(Supplemental Table S3). Contrary to previous findings forAEHs, D-Phg derivatives are poorly recognized—cleavage ofD-Phg-AMC was not detected under standard conditions;only TLC analysis showed slow hydrolysis of D-Phg-OMe(Supplemental Fig. S2). In addition, no significant dif-ference is observed between D-Phg-OMe and its L enan-tiomer. Therefore, BAP is unlikely to function as anesterase that uses such a donor in a synthetic reaction.

The L-Phe-AMC inhibition assay with 10-fold molar ex-cess of amide (2NH2) and methyl ester (2OMe) deriv-atives of L-Phe shows the importance of the S19 residue,with preference for the more hydrophobic 2AMCmoiety over2OMe (rel v0L

-Phe-AMC = 186 1%) and2NH2(rel v0L

-Phe-AMC = 496 2%). Similarly, stronger binding ofL-Leu methyl ester is observed than L-Leu amide.

Esterase activity was confirmed for BAP with methylesters of L-Leu, L-Cys (data not shown), L-Phe, and D-Phg(Supplemental Fig. S2). Ampicillin was also hydrolyzedwith release of D-Phg (Supplemental Fig. S2).However, notransferase reaction could be observed with either L-Phe-OMe or D-Phg-OMe as a donor and 6-APA as an acceptor.

All these data suggest that the enzyme can hydrolyzeboth amide and ester bonds and is promiscuous inaccepting different substrates. BAP is also the first micro-bial enzyme to showhigh specificity for L-Cys.Thepreferencefor L-Cys and activity toward L-Phe-, L-Tyr-, and L-Trp-containing substrates indicate that BAP might be respon-sible for securing cysteine and aromatic amino acids.Such a function would be highly beneficial for the cellin a nutrient-restricted environment because synthesisof aromatic amino acids requires substantial energyresources, and cysteine is an important source of sulfur.Interestingly, cysteine is a catalytic residue in the ma-jority of archaeal MDM peptidases proposed to processdetrital proteins (5). Because terminal cysteine resi-dues are stable only in anoxic environments, it appearsthat BAP has adapted to life in such an ecosystem; itloses activity on a timescale of days in the presence ofoxygen (Supplemental Fig. S3) and cannot efficientlyutilize oxidized cysteine (Table 2).

Crystal structure

To gain further insight into the molecular basis of BAPfunction, we performed crystallographic studies. The crys-tal structure of BAPwas determined by anMRmethodwithcharacterized XcAEH as a template [PDB entry 1MPX,

TABLE 2. Relative aminopeptidase activity of BAP against a set ofpeptide proxies

Substrate Normalized activity Activity SE

L-Cys-AMC 1.00 3 10+0 3.30 3 1022

L-Ala-AMC 4.50 3 1021 8.28 3 1023

L-Phe-AMC 3.24 3 1021 3.72 3 1023

L-Leu-AMC 1.24 3 1021 2.37 3 1023

L-Tyr-AMC 1.04 3 1021 1.47 3 1023

L-Trp-AMC 4.42 3 1022 4.87 3 1024

L-Ile-AMC 2.46 3 1022 7.12 3 1024

Gly-AMC 2.29 3 1022 6.57 3 1024

L-Leu-L-Leu-AMC 1.17 3 1022 7.62 3 1023

L-Cysteine-AMC 1.11 3 1022 5.80 3 1024

D-Phe-AMC 6.56 3 1023 3.48 3 1024

L-Arg-AMC 3.95 3 1023 1.74 3 1023

L-Phg-AMC 2.07 3 1023 7.04 3 1024

Z-L-Phe-L-Arg-AMC 1.99 3 1023 1.39 3 1023

L-Ala-L-Ala-L-Phe-AMC n.d. n.d.D-Phg-AMC n.d. n.d.Gly-L-Pro-AMC n.d. n.d.Pyr-AMC n.d. n.d.Z-L-Phe-L-Val-L-Arg-AMC n.d. n.d.Gly-Gly-L-Arg-AMC n.d. n.d.

n.d., not determined.

“MICROBIAL DARK MATTER” AMINOPEPTIDASE 5

sequence identity 51% (36)]. The atomic model was re-fined against 2.10 A resolution data (Table 1). The asym-metric unit contains 4 nearly identical protein molecules.Analogously to their structurally closest bacterial cousins,the individual BAP chains associate into a homotetramercomposed of 2 dimers. The assembly adopts a hollowsphere shapewith anexternal diameter of;110 A and222-point group symmetry (Fig. 2).Thechannel runs across thetetramer and can be accessed through 2 identical, elon-gated orifices with approximate dimensions 21 3 9 A.Comparisonswith otherAEHs located 4 independent activesites that face the interior of the homotetramer (see below).This arrangementposes significant restrictionson the sizeofacceptable substrates, which could only reach the catalyticpockets through those relatively narrow openings. Sucharchitecture is consistent with a self-compartmentalizinghydrolase (37). The monomer, nearly identical to 2 other

AEHs available in the PDB [root-mean-SD (rmsd)1.00 A for 597 residues aligned with XcAEH (Fig. 3A),and 0.96 A for 581 residues aligned with Acetobacterturbidans homolog, AtAEH, PDB entry 2B9V (38)], con-sists of 3 domains and anN-terminal protrusion that playsan important role in the formation of the primary dimerunit (Fig. 2). The N-terminal domain, with the classica/b-hydrolase fold, is followed by the cap domain andthe jelly roll-like C-terminal domain.

Figure 3. Comparison of BAP and AEHs. A) Superposition ofthe BAP monomer (green; this work) with XcAEH (pink; PDBentry 1MPX). B) Superposition of active sites: BAP is in green,and XcAEH is shown in pink. C) Superposition of active sites:BAP is shown in green, XcAEH in pink, and AtAEH in blue(PDB entry 2B4K). For reference, the D-Phg ligand from theAtAEH structure is shown in a stick representation.

Figure 2. Structure of BAP. A) A surface representation of theBAP homotetramer. B) A ribbon diagram of the BAP monomer(chain C) with individual domains colored green (N-terminaldomain; “N”), purple (cap domain), and blue (C-terminaldomain; “C”). The key residues in the active site are shown ina stick representation.

6 Vol. 29 September 2015 MICHALSKA ET AL.The FASEB Journal x www.fasebj.org

BAP active site

The active site is formed at the domains’ interface and canbe divided into 2 subcavities. The first one is relatively wideand faces the solvent channel. Deeper inside the pocket,the second subcavity is present. At the boundary betweenthe subcavities, the N-terminal domain carries a catalytictriad consisting of Ser158,His325, andAsp292 (Fig. 3B andSupplemental Fig. S4). By analogy to other AEHs, this re-gion also provides residues that are believed to create anoxyanion hole during catalysis: Tyr159 and Tyr66. In theformer residue, themain-chain amidegroup is presumablyinvolved in the stabilization of the tetrahedral interme-diate, whereas Tyr66 interacts with the substrate moleculethrough its hydroxyl group.

The deep subpocket (acyl binding) closely resembles itsequivalent in AEHs. This pocket has been shown to ac-commodate a D-Phgmolecule in AtAEH [PDB entry 2B4K(38)] and, thus, corresponds to the P1 site.One side of thissurface is lined up with several hydrophobic residues(Met184, Trp193, Tyr207, and Ala203) that interact withthe side chain of D-Phg in AtAEH and very likely with smallside chains of the BAP a-amino acid derivative substrates(estersor amides) (seebelow)(Fig. 3C).The second sideofthe acyl-binding subpocket exposes a constellation ofconserved acidic residues: Asp192, Glu294, and Asp295.This negatively charged cluster is used to anchor ana-amino group of the substrate moiety [as in the structureof AtAEH (PDB entry 2B4K)], and it defines enzymespecificity at the P1 site as well as optimum pH. In fact, thepH optimum for BAP is 6.9. Given that the pKa of the N-terminal amino group is ;7.8 [for phenylalanyl peptide(39)], it implies that the a-amino group of the peptide hasto be protonated for the most efficient binding of the N-terminal residue into the strongly acidic P1 site. Com-pounds that do not bear an a-amino group, such asmethylumbelliferyl-butyrate, are not recognized as sub-strates. These features define BAP as an aminopeptidase.Besides strictly conserved elements in the P1 site, there isalso aprotein fragmentwithin the capdomain (helixaGinXcAEH) that adopts a slightly different main-chain con-formation and has a variable amino acid composition. Asa consequence, in BAP, unlike in XcAEH or AtAEH,a carbonyl group of Ala203 is facing the lumen (Fig. 3C).This feature may be responsible for the shift in substratepreference fromPhg-basedcompounds towardCys,Ala, orPhe derivatives. As the superposition with AtAEH/D-Phgshows, the aromatic ring of Phg would be too close to theC = Omoiety. The longer side chain of the Phe ligand, onthe other hand, most likely moves to avoid an unfavorablecontact with Ala203. An equivalent region of AEHs is pos-sibly responsible for discrimination between Phg-basedsubstrates and its hydroxy-Phg analogs.

The channel-facing subcavity is much larger than theacyl-binding pocket. This space typically accommodatesa leaving fragment of the hydrolyzed peptide (in pepti-dases) or a b-lactam ring (in esterases/transferases). Theavailable data, based on modeling studies with antibiotics,predict very few contacts with the leaving group (24, 39),whichguarantees broad substrate specificitywith respect totheb-lactammoiety. Themajor one appears to be stackinginteractions between the b-lactam ring and the Tyr66equivalent—a dual-function residue that is also proposed

to form an oxyanion hole (see above). The leaving group-binding pocket is more variable in terms of its shape,volume, and amino acid sequence than the acyl-bindingregion. The most striking difference between BAP andAEHs is the presence of Gly157 and Gly326 instead of 2serine residues in the corresponding positions of XcAEH(Ser173 andSer284) andAtAEH(Ser204 andSer371; Fig.3B). Gly326 is neighboring catalytic His325 and intro-duces the peptide bond switch. The altered main-chainconformation is stabilized by another mutation, namelyArg329 substituting Asn344 (inXcAEHnumbering). Thearginine guanidiniumgroup forms a hydrogen bondwiththe carbonyl group of the switched peptide bond. Nota-bly, the Arg329 side chain occupies space used by theserine residues in AEHs. Additional differences betweenBAP and AEHs are observed within the region contrib-uted by the C-terminal domain, which may interact withS1 residues bearing longer side chains. These mod-ifications are sufficiently large to suggest a difference inBAP and AEH function. These residues are conserved inBAPs found in species broadly distributed in marinesediments (Supplemental Fig. S1).

DISCUSSION

BAP is a true example of an enzyme found only in theuncultured MDM because sequenced cultured organismsdo not contain a close homolog. This widespread enzymemay be an important factor in marine biology; yet, itsfunction has been only assigned based on sequence com-parison. Here, we have shown that a hypothesis generatedfrom a single-cell genome of a deeply branching un-cultured archaeon can be experimentally tested, and de-tailed characterizations of enzymes from MDM can beperformed, without requiring that those organisms arefirstgrown in pure culture. Recombinant BAP shows hydrolaseactivities, including that of a self-compartmentalizinga-aminopeptidase. In this capacity, it processes small pep-tides and peptide proxies with preference for cysteine andhydrophobic aminoacids.Thea-aminopeptidase activity isconsistent with the hypothesis that widely distributed Bath-yarchaeota and similar archaea hydrolyze detrital proteins inmarine sediments. They use a set of secreted proteases todegradeproteins to short peptides that are imported acrossthe membrane using peptide transporters. These mole-cules are further degraded into individual amino acids byintracellular peptidases; one of which is BAP. BAP andother peptidases have been identified in all 4 archaealsingle-cell genomes derived from the same sediment sam-ple. Interestingly, the temperature dependence of BAP(optimum, ;28.8 6 0.8°C) is consistent with mild psy-chrotrophy (40). This suggests that it is adapted to thetemperatures of ocean sediments whereMCG functions toprovide an advantage. The a-aminopeptidase may alsoprovide a housekeeping function and, together with otherpeptidases, is involved in recycling of intracellular archaealproteins. The other BAP activities may be relevant to un-identified cellular processes.

Molecular characterization of MDM-encoded pro-teins holds great promise for understanding key nutri-ent transformations in the environment, including veryhard to access and study marine sediments. The com-plex protein/peptide degradation pathway that links

“MICROBIAL DARK MATTER” AMINOPEPTIDASE 7

archaea with carbon, nitrogen, and sulfur cyclingrequires a thorough investigation. These organisms areubiquitous and abundant in marine sediments andtherefore likely influence global geochemical cycles.Small changes in these ocean environmentsmay changethe enzyme activities and affect remineralization pro-cesses. Although sequence analysis can fuel some as-sumptions about microbial life, prediction of specificity,catalysis, turnover rates, thermal stability, and kineticssolely from the primary structure is still out of reach and,in some cases,may lead to false conclusions. For example,recognition of a mesophilic character and a differentsubstrate preference of BAP than that of characterizedbacterial AEHs is a good illustration of discoveries thatcan be easily missed in sequence-based assumptions. Ouranalysis demonstrates that the initial annotation ofMDMgenomes can be further refined and expanded by mo-lecular and structural biology to discover novel functionsthat are not found in the cultured microorganismsstudied thus far. Thorough characterization is also in-escapable in the exploration of the enzymatic space fornovel industrial and biomedical catalysts, includingproteases.

The authors thank Dr. Robert Jedrzejczak (ArgonneNational Laboratory) for discussion of cloning strategy,Dr. Gyorgy Babnigg (Argonne National Laboratory) for help indesigning the cloning construct, Katlyn Fayman (ArgonneNational Laboratory) for help with protein purification,members of the Structural Biology Center at Argonne NationalLaboratory for their help with data collection at the 19-Insertion Device Beamline, Dr. Steven Wilhelm (University ofTennessee Department of Microbiology) for provision of labspace to A.D.S., and Dr. B. B. Jørgensen and the staff of theCenter for Geomicrobiology at Aarhus University (Aarhus,Denmark) for providing amplified genomic deoxyribonucleicacid. This work was supported by the following funds: U.S.National Institutes of Health, National Institute of GeneralMedical Sciences Grant GM094585 (to A.J.); the U.S. De-partment of Energy, Office of Biological and EnvironmentalResearch, under contract DE-AC02-06CH11357 (to A.J.); andCenter for Dark Energy Biosphere Investigations Grants 157595(to K.G.L.) and 36202823 (to A.D.S.). This work is Center forDark Energy Biosphere Investigation Contribution 268. Thesubmitted manuscript has been created by UChicago Argonne,Limited Liability Company, Operator of Argonne NationalLaboratory (“Argonne”). Argonne, a U.S. Department ofEnergy Office of Science laboratory, is operated under ContractNo. DE-AC02-06CH11357. The U.S. Government retains foritself, and others acting on its behalf, a paid-up nonexclusive,irrevocable worldwide license in said article to reproduce,prepare derivative works, distribute copies to the public, andperform publicly and display publicly, by or on behalf of theGovernment.

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Received for publication March 9, 2015.Accepted for publication May 26, 2015.

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