Archaeosortases and exosortases are widely distributed systems linking membrane transit with posttranslational modification

Multiple new prokaryotic C-terminal protein-sorting signals were found that reprise the tripartite architecture shared by LPXTG and PEP-CTERM: motif, TM helix, basic cluster. Defining hidden Markov models were constructed for all. PGF-CTERM occurs in 29 archaeal species, some of which have more than 50 proteins that share the domain. PGF-CTERM proteins include the major cell surface protein in Halobacterium, a glycoprotein with a partially characterized diphytanylglyceryl phosphate linkage near its C terminus. Comparative genomics identifies a distant exosortase homolog, designated archaeosortase A (ArtA), as the likely protein-processing enzyme for PGF-CTERM. Proteomics suggests that the PGF-CTERM region is removed. Additional systems include VPXXXP-CTERM/archeaosortase B in two of the same archaea and PEF-CTERM/archaeosortase C in four others. Bacterial exosortases often fall into subfamilies that partner with very different cohorts of extracellular polymeric substance biosynthesis proteins; several species have multiple systems. Variant systems include the VPDSG-CTERM/exosortase C system unique to certain members of the phylum Verrucomicrobia, VPLPA-CTERM/exosortase D in several alpha- and deltaproteobacterial species, and a dedicated (single-target) VPEID-CTERM/exosortase E system in alphaproteobacteria. Exosortase-related families XrtF in the class Flavobacteria and XrtG in Gram-positive bacteria mark distinctive conserved gene neighborhoods. A picture emerges of an ancient and now well-differentiated superfamily of deeply membrane-embedded protein-processing enzymes. Their target proteins are destined to transit cellular membranes during their biosynthesis, during which most undergo additional posttranslational modifications such as glycosylation.     

Newly discovered Asgard archaea Hermodarchaeota potentially degrade alkanes and aromatics via alkyl/benzyl-succinate synthase and benzoyl-CoA pathway

Asgard archaea are widely distributed in anaerobic environments. Previous studies revealed the potential capability of Asgard archaea to utilize various organic substrates including proteins, carbohydrates, fatty acids, amino acids and hydrocarbons, suggesting that Asgard archaea play an important role in sediment carbon cycling. Here, we describe a previously unrecognized archaeal phylum, Hermodarchaeota, affiliated with the Asgard superphylum. The genomes of these archaea were recovered from metagenomes generated from mangrove sediments, and were found to encode alkyl/benzyl-succinate synthases and their activating enzymes that are similar to those identified in alkane-degrading sulfate-reducing bacteria. Hermodarchaeota also encode enzymes potentially involved in alkyl-coenzyme A and benzoyl-coenzyme A oxidation, the Wood–Ljungdahl pathway and nitrate reduction. These results indicate that members of this phylum have the potential to strictly anaerobically degrade alkanes and aromatic compounds, coupling the reduction of nitrate. By screening Sequence Read Archive, additional genes encoding 16S rRNA and alkyl/benzyl-succinate synthases analogous to those in Hermodarchaeota were identified in metagenomic datasets from a wide range of marine and freshwater sediments. These findings suggest that Asgard archaea capable of degrading alkanes and aromatics via formation of alkyl/benzyl-substituted succinates are ubiquitous in sediments.Subject terms: Microbial ecology, Metagenomics     

FeeM, an N-acyl amino acid synthase from an uncultured soil microbe: structure, mechanism, and acyl carrier protein binding

Attempts to access antibiotics by capturing biosynthetic genes and pathways directly from environmental DNA, which is overwhelmingly derived from uncultured bacteria, have revealed a large and previously unknown family of N-acyl amino acid synthases (NASs). The structure of the NAS FeeM reveals structural similarity to the GCN5-related N-acyl transferases and acylhomoserine lactone synthases. The overall structure has a central beta sheet with alpha helices on both sides. A bound product at a cleft in the beta sheet identifies the active site and the structural basis for catalysis, and sequence conservation in this region indicates a bias for recognition over speed. FeeM interacts with an acyl carrier protein (FeeL), and the structure, mutagenesis, and enzymatic measurements reveal that a small hydrophobic pocket in alpha helix 5 dominates binding of FeeM to FeeL. The structural and mechanistic analyses suggest that the products of FeeM could be bacterial signaling agents.     

Domain loss has independently occurred multiple times in plant terpene synthase evolution

The extensive family of plant terpene synthases (TPSs) generally has a bi-domain structure, yet phylogenetic analyses consistently indicate that these synthases have evolved from larger diterpene synthases. In particular, that duplication of the diterpene synthase genes required for gibberellin phytohormone biosynthesis provided an early predecessor, whose loss of a approximately 220 amino acid 'internal sequence element' (now recognized as the γ domain) gave rise to the precursor of the modern mono- and sesqui-TPSs found in all higher plants. Intriguingly, TPSs are conserved by taxonomic relationships rather than function. This relationship demonstrates that such functional radiation has occurred both repeatedly and relatively recently, yet phylogenetic analyses assume that the 'internal/γ' domain loss represents a single evolutionary event. Here we provide evidence that such a loss was not a singular event, but rather has occurred multiple times. Specifically, we provide an example of a bi-domain diterpene synthase from Salvia miltiorrhiza, along with a sesquiterpene synthase from Triticum aestivum (wheat) that is not only closely related to diterpene synthases, but retains the ent-kaurene synthase activity relevant to the ancestral gibberellin metabolic function. Indeed, while the wheat sesquiterpene synthase clearly no longer contains the 'internal/γ' domain, it is closely related to rice diterpene synthase genes that retain the ancestral tri-domain structure. Thus, these findings provide examples of key evolutionary intermediates that underlie the bi-domain structure observed in the expansive plant TPS gene family, as well as indicating that 'internal/γ' domain loss has occurred independently multiple times, highlighting the complex evolutionary history of this important enzymatic family.     

(E)-beta-ocimene and myrcene synthase genes of floral scent biosynthesis in snapdragon: function and expression of three terpene synthase genes of a new terpene synthase subfamily

Snapdragon flowers emit two monoterpene olefins, myrcene and (E)-beta-ocimene, derived from geranyl diphosphate, in addition to a major phenylpropanoid floral scent component, methylbenzoate. Emission of these monoterpenes is regulated developmentally and follows diurnal rhythms controlled by a circadian clock. Using a functional genomics approach, we have isolated and characterized three closely related cDNAs from a snapdragon petal-specific library that encode two myrcene synthases (ama1e20 and ama0c15) and an (E)-beta-ocimene synthase (ama0a23). Although the two myrcene synthases are almost identical (98%), except for the N-terminal 13 amino acids, and are catalytically active, yielding a single monoterpene product, myrcene, only ama0c15 is expressed at a high level in flowers and contributes to floral myrcene emission. (E)-beta-Ocimene synthase is highly similar to snapdragon myrcene synthases (92% amino acid identity) and produces predominantly (E)-beta-ocimene (97% of total monoterpene olefin product) with small amounts of (Z)-beta-ocimene and myrcene. These newly isolated snapdragon monoterpene synthases, together with Arabidopsis AtTPS14 (At1g61680), define a new subfamily of the terpene synthase (TPS) family designated the Tps-g group. Members of this new Tps-g group lack the RRx(8)W motif, which is a characteristic feature of the Tps-d and Tps-b monoterpene synthases, suggesting that the reaction mechanism of Tps-g monoterpene synthase product formation does not proceed via an RR-dependent isomerization of geranyl diphosphate to 3S-linalyl diphosphate, as shown previously for limonene cyclase. Analyses of tissue-specific, developmental, and rhythmic expression of these monoterpene synthase genes in snapdragon flowers revealed coordinated regulation of phenylpropanoid and isoprenoid scent production.     

Induction of a β-phaseolin promoter by exogenous abscisic acid in tobacco: developmental regulation and modulation by external sucrose and Ca2+ ions

Using degenerate oligonucleotides that correspond to conserved amino acid residues of known 1-aminocyclopropane-1-carboxylic acid (ACC) synthases, we cloned a genomic fragment that encodes ACC synthase in Stellaria longipes. Southern analysis suggests that ACC synthase is encoded by a small gene family comprising about 4 members. We isolated four unique ACC synthase cDNA clones under different growth conditions from alpine and prairie ecotypes of S. longipes. Northern analyses suggest that ACC synthase genes are differentially and synergistically regulated by photoperiod and temperature. Such differential regulation of ACC synthase genes positively correlate with the levels of ACC and ethylene. Since ethylene has previously been shown to partly control the stem elongation plasticity in S. longipes, we propose that differential regulation of ACC synthase genes may represent one of the underlying molecular mechanisms of phenotypic plasticity in S. longipes.     

Widespread occurrence of secondary lipid biosynthesis potential in microbial lineages

Bacterial production of long-chain omega-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), is constrained to a narrow subset of marine γ-proteobacteria. The genes responsible for de novo bacterial PUFA biosynthesis, designated pfaEABCD, encode large, multi-domain protein complexes akin to type I iterative fatty acid and polyketide synthases, herein referred to as "Pfa synthases". In addition to the archetypal Pfa synthase gene products from marine bacteria, we have identified homologous type I FAS/PKS gene clusters in diverse microbial lineages spanning 45 genera representing 10 phyla, presumed to be involved in long-chain fatty acid biosynthesis. In total, 20 distinct types of gene clusters were identified. Collectively, we propose the designation of "secondary lipids" to describe these biosynthetic pathways and products, a proposition consistent with the "secondary metabolite" vernacular. Phylogenomic analysis reveals a high degree of functional conservation within distinct biosynthetic pathways. Incongruence between secondary lipid synthase functional clades and taxonomic group membership combined with the lack of orthologous gene clusters in closely related strains suggests horizontal gene transfer has contributed to the dissemination of specialized lipid biosynthetic activities across disparate microbial lineages.     

Triterpene synthases from the Okinawan mangrove tribe, Rhizophoraceae

Oleanane-type triterpene is one of the most widespread triterpenes found in plants, together with the lupane type, and these two types often occur together in the same plant. Bruguiera gymnorrhiza (L.) Lamk. and Rhizophora stylosa Griff. (Rhizophoraceae) are known to produce both types of triterpenes. Four oxidosqualene cyclase cDNAs were cloned from the leaves of B. gymnorrhiza and R. stylosa by a homology-based PCR method. The ORFs of full-length clones termed BgbAS (2280 bp, coding for 759 amino acids), BgLUS (2286 bp, coding for 761 amino acids), RsM1 (2280 bp, coding for 759 amino acids) and RsM2 (2316 bp coding for 771 amino acids) were ligated into yeast expression plasmid pYES2 under the control of the GAL1 promoter. Expression of BgbAS and BgLUS in GIL77 resulted in the production of beta-amyrin and lupeol, suggesting that these genes encode beta-amyrin and lupeol synthase (LUS), respectively. Furthermore, RsM1 produced germanicol, beta-amyrin, and lupeol in the ratio of 63 : 33 : 4, whereas RsM2 produced taraxerol, beta-amyrin, and lupeol in the proportions 70 : 17 : 13. This result indicates that these are multifunctional triterpene synthases. Phylogenetic analysis and sequence comparisons revealed that BgbAS and RsM1 demonstrated high similarities (78-93%) to beta-amyrin synthases, and were located in the same branch as beta-amyrin synthase. BgLUS formed a new branch for lupeol synthase that was closely related to the beta-amyrin synthase cluster, whereas RsM2 was found in the first branch of the multifunctional triterpene synthase evolved from lupeol to beta-amyrin synthase. Based on these sequence comparisons and product profiles, we discuss the molecular evolution of triterpene synthases and the involvement of these genes in the formation of terpenoids in mangrove leaves.     

Isolation of a gene encoding cysteine synthase from Flavobacterium K3–15

Abstract The cysteine synthase gene ( cysK ) from Flavobacterium K_3–15 was cloned and sequenced. The gene exhibits 30–50% identity to known cysteine synthases on both the DNA and the amino acid levels. The pyridoxal phosphate binding site of the enzyme is part of a conserved motif comprising seven amino acids (SIKDRIA). The lys31 residue of the flavobacterial enzyme is conserved in all known cysteine synthases. The cysK gene from Flavobacterium K_3–15 was heterologously expressed and the gene product identified by immunoblotting and determination of the enzyme activity.     

Cloning and functional expression of cycloartenol synthases from mangrove species Rhizophora stylosa Griff. and Kandelia candel (L.) Druce

To obtain cDNAs encoding oxidosqualene cyclase (OSC), we cloned two cDNAs, KcCAS and RsCAS, from roots of Kandelia candel (L.) Druce and leaves of Rhizophora stylosa Griff. by homology based PCR method respectively. The deduced amino acid sequences of both OSCs showed 82% homology to cycloartenol synthases from Lotus japonicus (OSC5) and Ricinus cummunis (RcCAS), suggesting that these are cycloartenol synthases of K. candel and R. stylosa. The genes obtained were expressed in a lanosterol synthase deficient Saccharomyces cerevisiae (ERG7) strain, GIL77. GC-MS analysis identified the accumulated reaction product in the yeast transformant to be cycloartenol, indicating that both KcCAS and RsCAS encode cycloartenol synthase.     

Metagenome-derived clones encoding two novel lactonase family proteins involved in biofilm inhibition in Pseudomonas aeruginosa

Here we report the isolation and characterization of three metagenome-derived clones that interfere with bacterial quorum sensing and degrade N-(3-oxooctanoyl)-l-homoserine lactone (3-oxo-C(8)-HSL). By using a traI-lacZ gene fusion, the metagenome-derived clones were identified from a soil DNA library and analyzed. The open reading frames linked to the 3-oxo-C(8)-HSL-degrading activities were designated bpiB01, bpiB04, and bpiB07. While the BpiB07 protein was similar to a known lactonase, no significant similarities were observed for the BpiB01 and BpiB04 proteins or the deduced amino acid sequences. High-performance liquid chromatography-mass spectrometry analyses confirmed that the identified genes encode novel lactone-hydrolyzing enzymes. The original metagenome-derived clones were expressed in Pseudomonas aeruginosa and employed in motility and biofilm assays. All clones were able to reproducibly inhibit motility in P. aeruginosa. Furthermore, these genes clearly inhibited biofilm formation in P. aeruginosa when expressed in P. aeruginosa PAO1. Thus, this is the first study in which metagenome-derived proteins have been expressed in P. aeruginosa to successfully inhibit biofilm formation.     

Wide distribution among halophilic archaea of a novel polyhydroxyalkanoate synthase subtype with homology to bacterial type III synthases

Polyhydroxyalkanoates (PHAs) are accumulated as intracellular carbon and energy storage polymers by various bacteria and a few haloarchaea. In this study, 28 strains belonging to 15 genera in the family Halobacteriaceae were investigated with respect to their ability to synthesize PHAs and the types of their PHA synthases. Fermentation results showed that 18 strains from 12 genera could synthesize polyhydroxybutyrate (PHB) or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). For most of these haloarchaea, selected regions of the phaE and phaC genes encoding PHA synthases (type III) were cloned via PCR with consensus-degenerate hybrid oligonucleotide primers (CODEHOPs) and were sequenced. The PHA synthases were also examined by Western blotting using haloarchaeal Haloarcula marismortui PhaC (PhaC(Hm)) antisera. Phylogenetic analysis showed that the type III PHA synthases from species of the Halobacteriaceae and the Bacteria domain clustered separately. Comparison of their amino acid sequences revealed that haloarchaeal PHA synthases differed greatly in both molecular weight and certain conserved motifs. The longer C terminus of haloarchaeal PhaC was found to be indispensable for its enzymatic activity, and two additional amino acid residues (C143 and C190) of PhaC(Hm) were proved to be important for its in vivo function. Thus, we conclude that a novel subtype (IIIA) of type III PHA synthase with unique features that distinguish it from the bacterial subtype (IIIB) is widely distributed in haloarchaea and appears to be involved in PHA biosynthesis.     

GlyGly-CTERM and rhombosortase: a C-terminal protein processing signal in a many-to-one pairing with a rhomboid family intramembrane serine protease

The rhomboid family of serine proteases occurs in all domains of life. Its members contain at least six hydrophobic membrane-spanning helices, with an active site serine located deep within the hydrophobic interior of the plasma membrane. The model member GlpG from Escherichia coli is heavily studied through engineered mutant forms, varied model substrates, and multiple X-ray crystal studies, yet its relationship to endogenous substrates is not well understood. Here we describe an apparent membrane anchoring C-terminal homology domain that appears in numerous genera including Shewanella, Vibrio, Acinetobacter, and Ralstonia, but excluding Escherichia and Haemophilus. Individual genomes encode up to thirteen members, usually homologous to each other only in this C-terminal region. The domain's tripartite architecture consists of motif, transmembrane helix, and cluster of basic residues at the protein C-terminus, as also seen with the LPXTG recognition sequence for sortase A and the PEP-CTERM recognition sequence for exosortase. Partial Phylogenetic Profiling identifies a distinctive rhomboid-like protease subfamily almost perfectly co-distributed with this recognition sequence. This protease subfamily and its putative target domain are hereby renamed rhombosortase and GlyGly-CTERM, respectively. The protease and target are encoded by consecutive genes in most genomes with just a single target, but far apart otherwise. The signature motif of the Rhombo-CTERM domain, often SGGS, only partially resembles known cleavage sites of rhomboid protease family model substrates. Some protein families that have several members with C-terminal GlyGly-CTERM domains also have additional members with LPXTG or PEP-CTERM domains instead, suggesting there may be common themes to the post-translational processing of these proteins by three different membrane protein superfamilies.     

Two anthranilate synthase genes in Arabidopsis: defense-related regulation of the tryptophan pathway.

Arabidopsis thaliana has two genes, ASA1 and ASA2, encoding the alpha subunit of anthranilate synthase, the enzyme catalyzing the first reaction in the tryptophan biosynthetic pathway. As a branchpoint enzyme in aromatic amino acid biosynthesis, anthranilate synthase has an important regulatory role. The sequences of the plant genes are homologous to their microbial counterparts. Both predicted proteins have putative chloroplast transit peptides at their amino termini and conserved amino acids involved in feedback inhibition by tryptophan. ASA1 and ASA2 cDNAs complement anthranilate synthase alpha subunit mutations in the yeast Saccharomyces cerevisiae and in Escherichia coli, confirming that both genes encode functional anthranilate synthase proteins. The distributions of ASA1 and ASA2 mRNAs in various parts of Arabidopsis plants are overlapping but nonidentical, and ASA1 mRNA is approximately 10 times more abundant in whole plants. Whereas ASA2 is expressed at a constitutive basal level, ASA1 is induced by wounding and bacterial pathogen infiltration, suggesting a novel role for ASA1 in the production of tryptophan pathway metabolites as part of an Arabidopsis defense response. Regulation of key steps in aromatic amino acid biosynthesis in Arabidopsis appears to involve differential expression of duplicated genes.     

cDNA cloning, characterization, and functional expression of four new monoterpene synthase members of the Tpsd gene family from grand fir (Abies grandis).

Grand fir (Abies grandis) is a useful model system for studying the biochemistry, molecular genetics, and regulation of defensive oleoresin formation in conifers, a process involving both the constitutive accumulation of resin (pitch) in specialized secretory structures and the induced biosynthesis of monoterpenes and sesquiterpenes (turpentine) and diterpene resin acids (rosin) by nonspecialized cells at the site of injury. A similarity-based cloning strategy, employing primers designed to conserved regions of existing monoterpene synthases and anticipated to amplify a 1000-bp fragment, unexpectedly yielded a 300-bp fragment with sequence reminiscent of a terpenoid synthase. Utilization of this amplicon as a hybridization probe afforded four new, full-length cDNA species from a wounded fir stem cDNA library that appeared to encode four distinct monoterpene synthases. Expression in Escherichia coli, followed by enzyme assay with geranyl diphosphate (C(10)), farnesyl diphosphate (C(15)) and geranylgeranyl diphosphate (C(20)), and analysis of the terpene products by chiral phase gas chromatography and mass spectrometry confirmed that these sequences encoded four new monoterpene synthases, including (-)-camphene synthase, (-)-beta-phellandrene synthase, terpinolene synthase, and an enzyme that produces both (-)-limonene and (-)-alpha-pinene. The deduced amino acid sequences indicated these enzymes to be 618 to 637 residues in length (71 to 73 kDa) and to be translated as preproteins bearing an amino-terminal plastid targeting sequence of 50-60 residues. cDNA truncation to delete the transit peptide allowed functional expression of the "pseudomature" forms of these enzymes, which exhibited no change in product outcome as a result of truncation. Sequence comparison revealed that these new monoterpene synthases from grand fir are members of the Tpsd gene subfamily and resemble sesquiterpene (C(15)) synthases and diterpene (C(20)) synthases from conifers more closely than mechanistically related monoterpene synthases from angiosperm species. The availability of a nearly complete set of constitutive and inducible monoterpene synthases from grand fir (now numbering seven) will allow molecular dissection of the resin-based defense response in this conifer species, and detailed study of structure-function relationships among this large and diverse family of catalysts, all of which exploit the same stereochemistry in the coupled isomerization-cyclization reaction.     

Specificity of Signal-Binding via Non-AHL LuxR-Type Receptors

Quorum sensing is a typical communication system among Gram-negative bacteria used to control group-coordinated behavior via small diffusible molecules dependent on cell number. The key components of a quorum sensing system are a LuxI-type synthase, producing acyl-homoserine lactones (AHLs) as signaling molecules, and a LuxR-type receptor that detects AHLs to control expression of specific target genes. Six conserved amino acids are present in the signal-binding domain of AHL-sensing LuxR-type proteins, which are important for ligand-binding and -specificity as well as shaping the ligand-binding pocket. However, many proteobacteria possess LuxR-type regulators without a cognate LuxI synthase, referred to as LuxR solos. The two LuxR solos PluR and PauR from Photorhabdus luminescens and Photorhabdus asymbiotica, respectively, do not sense AHLs. Instead PluR and PauR sense alpha-pyrones and dialkylresorcinols, respectively, and are part of cell-cell communication systems contributing to the overall virulence of these Photorhabdus species. However, PluR and PauR both harbor substitutions in the conserved amino acid motif compared to that in AHL sensors, which appeared to be important for binding the corresponding signaling molecules. Here we analyze the role of the conserved amino acids in the signal-binding domain of these two non-AHL LuxR-type receptors for their role in signal perception. Our studies reveal that the conserved amino acid motif alone is essential but not solely responsible for ligand-binding.     

Chitin synthase genes in the arbuscular mycorrhizal fungus Glomus versiforme: full sequence of a gene encoding a class IV chitin synthase

Chitin synthase genes of the arbuscular mycorrhizal fungus Glomus versiforme were sought in an investigation of the molecular basis of fungal growth. Three DNA fragments (Gvchs1, Gvchs2 and Gvchs3) corresponding to the conserved regions of distinct chitin synthase (chs) genes were amplified by means of the polymerase chain reaction (PCR) with two sets of degenerate primers. Gvchs1 and Gvchs2 encode two class I chitin synthases, whereas Gvchs3 encodes a class IV chitin synthase. A genomic library was used to obtain the Gvchs3 complete gene (1194 amino acids), which shows a very close similarity to the class IV chitin synthase from Neurospora crassa.     

Analysis of the nucleotide sequence of the Streptomyces glaucescens tcmI genes provides key information about the enzymology of polyketide antibiotic biosynthesis.

Key information about the biosynthesis of polyketide metabolites has been uncovered by sequence analysis of the tetracenomycin C polyketide synthase genes (tcml) from Streptomyces glaucescens GLA.0. The sequence data revealed the presence of three complete open reading frames (ORFs). ORF1 and ORF2 appear to be translationally coupled and would encode proteins containing 426 and 405 amino acids, respectively. The two deduced proteins are homologous to known beta-ketoacyl synthases. ORF3 begins 70 nucleotides after the stop codon of ORF2 and would code for an 83 amino acid protein with a strong resemblance to known bacterial, animal and plant acyl-carrier proteins (ACP). The presence of an ACP gene within the tcm gene cluster suggests that different ACPs are used in fatty acid and polyketide biosynthesis in Streptomyces. We conclude from these data and earlier information that polyketide biosynthesis in S. glaucescens, and most likely in other bacteria, involves a multienzyme complex consisting of at least five types of enzymes: acylCoA transferases that load the acyl and 2-carboxyacyl precursors onto the ACP; a beta-ketoacyl synthase that, along with the acylated ACP, forms the poly-beta-ketoacyl intermediates; a poly-beta-ketone cyclase that forms carbocyclic structures from the latter intermediates; a beta-ketoacyl oxidoreductase that forms beta-hydroxyacyl intermediates or reduces ketone groups in fully formed polyketides; and a thioesterase that releases the assembled polyketide from the enzyme.