Polaina J., MacCabe A.P. (ed.). Industrial Enzymes. Structure, Function and Applications

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534 KAUL ET AL.

mixture) for acid productioncan be developed into a high throughput format for the

screen. A simple, colorimetric, pH responsive method was developed for the rapid

enantioselective screening of nitrile hydrolysing enzyme libraries (Banerjee et al.,

2003b). pH sensitive indicators have been used to monitor various enzyme catalysed

reactions such as carbonic anhydrase (Gibbons and Edsall, 1963), cholinesterase

(Lowry et al., 1954), lipase (Baumann et al., 2000) etc. The method is based on the

drop in pH that occurs as the reaction proceeds due to the formation of acid. The pH

drop is reflected by the colour change of the indicator, provided the colour profile

of the indicator falls within the pH range of the enzyme activity. For the colour

change to be proportional to the number of protons released, both the indicator and

the buffer must have similar affinities for protons (pK

s within 0.1 unit of each

other), such that the relative amounts of protonated buffer to protonated indicator

remain constant during the course of the reaction. This method was utilized to screen

nitrilase producing micro-organisms for the production of R-−-mandelic acid,

a versatile chiral building block (Kaul et al., 2004b). Since only the R-isomer

of mandelonitrile is commercially available, enantioselectivity was determined by

comparing the rate of colour turnover of the R-isomer with that of the racemate.

Conclusive statements regarding the enantioselectivity of the micro-organisms could

not be made in cases where both changed colour simultaneously, reflecting the

importance of using pure enantiomers. The proposed method is simple and, most

importantly, less time consuming. Using this pH responsive strategy a large number

of micro-organisms can be analysed simultaneously in a short time, thus reducing

the number of samples to be analysed in greater quantitative detail (HPLC analysis).

The assay requires very little substrate thus allowing the use of pure enantiomers

which are not always available in large quantities. The disadvantage of the method

is its qualitative nature. The presence of cells interferes with the spectrophotometric

reading. The use of cell free extracts may solve the problem but will also add an

extra step to the screening procedure. Moreover, this may require special instrumen-

tation (e.g. a microplate reader) which can be avoided by visualising the enzyme

catalysed reaction using a suitable indicator. Hence, by compromising quantitative

aspects screen throughput was increased. The goal was to use the method not for

precise quantitation but for screening large numbers of micro-organisms to facilitate

the identification of those having the desired enantioselectivity acceptable for the

development of a biocatalytic resolution process.

2.1.1. Distribution and physiological role of nitrilases

Nitrilases are found relatively infrequently in nature. The existence of the enzyme

activity in 3 out of 21 plant families (Gramineae, Cruciferae and Musaceae)

(Thimann and Mahadevan, 1964) and in a limited number of fungal genera

(Fusarium, Aspergillus, Penicillium) (Harper, 1977) indicates the relative scarcity

of this activity. Nitrile-degrading activity is more frequent in bacteria, though

without extensive screening it is almost impossible to assess the actual distribution

frequency. A number of bacteria (Acinetobacter, Corynebacterium, Arthrobacter,

Pseudomonas, Klebsiella, Nocardia, Rhodococcus etc.) are known to utilize nitriles

NITRILE HYDROLASES 535

as sole sources of carbon and nitrogen. The physiological role of nitrile hydrolysing

enzymes in micro-organisms is not clear. In plants such activities are implicated

in nutrient metabolism, particularly in the degradation of glucosinolates (Bestwick

et al., 1993) and in the synthesis of indole acetic acid (Bartel and Fink, 1994).The

genome sequence of Arabidopsis thaliana revealed four nitrilase-related sequences

(AtNIT 1, 2, 3 and 4) (Bartling et al., 1992). AtNIT1, 2 and 3 are isoenzymes which

are found only in Brassicaceae (Hillebrand et al., 1998). AtNIT4 on the other hand

is not related to the other three nitrilases and its homologues are found in many plant

species such as tobacco (Tsunoda and Yamaguchi, 1995) and rice (Piotrowski et al.,

2001). The major physiological role for these enzymes appears to be glucosinolate

metabolism. AtNIT4 is a -cyano alanine hydratase and plays a role in cyanide

detoxification (Piotrowski et al., 2001). In microbial systems nitrilases probably

form components of a complex pathway controlling the production and degradation

of aldoximes. Nitriles, which are formed by enzyme activities upstream of aldoxime

dehydratases, may further undergo hydrolysis, oxidation, reduction etc. by different

enzymes including nitrilases (Fig. 1).

RCHO + HCN

RCH

Oxygenase

Oxynitrilase

Nitrogenase

Aldoxime

dehydratase

Nitrilase

NHase

Amidase

Metal cyanides

Abiotic

conversion

Accumulated

+ NH

Cyanase

Cyanide

hydratase

Cyanide

dihydratase

HCOOH + N

RCH

COOH

RCH(OH)CN

RCH

CONH

HCN

HCONH

HCN

Figure 1. Different pathways of nitrile metabolism

536 KAUL ET AL.

2.1.2. Molecular structure and genetic analysis

To date many nitrilases have been purified and their subunit molecular mass

and primary structures have been determined. Most nitrilases consist of a single

polypeptide having a molecular mass in the range 30 to 45 kDa, which aggregate

to form the active holoenzyme under different conditions. The preferred form of

the enzyme seems to be a large aggregate of 6 to 26 subunits. Most of the enzymes

show substrate dependent activation, though the presence of elevated concentrations

of salt, organic solvents, pH, temperature or even the enzyme itself may also

trigger subunit association and therefore activation (Nagasawa et al., 2000). The

hydrophobic effect resulting from the above mentioned conditions might change

the conformation of the enzyme thereby exhibiting hydrophobic sites and enabling

subunit assembly and enzyme activation. The nitrilases of Pseudomonas fluorescens

DSM 7155 and Bacillus pallidus Dac 521 were co-purified with chaperonins

(Almatawah et al., 1999; Layh et al., 1998). It was suggested that chaperonins might

play a role in the assembly of the subunits into high molecular weight complexes

and also their stabilisation. When the nitrilase gene of Comamonas testosteroni

was cloned and over-expressed in E. coli, as much as 30% of the total cellular

protein was found to be nitrilase but only 10% of this was soluble in nature. The

solubility of nitrilase was enhanced by co-overexpression of GroEL (chaperonin)

in the same cell and the activity increased five-fold (Schill et al., 1995). This

further confirms the crucial role of chaperonins in the correct folding and subunit

association of nitrilase.

The genes encoding the nitrilases of Klebsiella pneumoniae ssp. ozaenae (Stalker

et al., 1988), R. rhodochrous strain K22 (Kobayashi et al., 1992), A. faecalis JM3

(Kobayashi et al., 1993), C. testosteroni (Schill et al., 1995) and Bacillus sp. strain

OxB-1 (Kato et al., 2000) have been sequenced and over-expressed. The four

nitrilases of A. thaliana have also been over-expressed in E. coli. AtNIT1, 2 and 3 are

clustered in a 13.8 kb region on chromosome 3 and share 80% identity in sequences

(Hillebrand et al., 1998). AtNIT4, not linked to these genes, shows 63% identity

with AtNIT1-3, which reflects their different functionality in A. thaliana (Piotrowski

et al., 2001). A Cys residue has been shown to be essential for the catalytic activity

of the nitrilases (Kobayashi et al., 1992, 1993). This Cys is proposed to be a

part of the catalytic triad Cys-Glu-Lys which has been identified from the crystal

structures of the NitFhit and N-carbamyl-D-amino acid amidohydrolases, members

of nitrilase superfamily (Nakai et al., 2000). The genetic regulation of nitrilases is

largely unknown. A gene (nitR) was identified downstream of the R. rhodochrous

J1 nitrilase gene and encodes a protein that has significant homology to AraC type

transcriptional regulators (Martin and Rosner, 2001). NitR appears to be a positive

regulator of nitrilase gene (nitA) expression (Komeda et al., 1996a). Unlike most

AraC type regulators whose genes are transcribed divergently from the genes they

are regulating, transcriptional analysis of nitR indicates that it is probably expressed

by read through from the nitA promoter (Komeda et al., 1996b). A putative nitrilase

regulator has also been found in Bacillus sp. strain OxB-1 (Kato et al., 2000).

NITRILE HYDROLASES 537

2.1.3. Reaction mechanism

Hydrolysis of a nitrile of the form R -C ≡ N produces the corresponding amide,

R-C =ONH

, with the addition of one molecule of water and the corresponding

acid, R-CO

H, with the addition of second molecule of water. Nitrilases are

interesting enzymes in the sense that the substrates are nitriles but the reaction

does not involve the release of or the reaction with a substantial amount of the

corresponding amide. However, it has been shown that the purified nitrilases of

F. oxysporum ssp. melonis (Goldhust and Bohak, 1989), Pseudomonas fluorescens

DSM7155 (Layh et al., 1998) and the ricinine nitrilase of Pseuodomonas sp. (Hook

and Robinson, 1964) produce a small amount of amide product and therefore

have nitrile hydratase activity. In these cases the amide product is usually < 5%

of the total reaction products. AtNIT1 and AtNIT4 also have NHase activity,

especially AtNIT4 which produces 1.5 times more asparagine than aspartic acid

from -cyano alanine (Piotrowski et al., 2001). Some of the nitrilases use amides,

albeit at a very low rate compared to the nitrile substrate, and therefore have

amidase activity (Kobayashi et al., 1998). A reaction mechanism was proposed

which accounts for all these activities (Hook and Robinson, 1964; Thimann and

Mahadevan, 1964). This mechanism involves a nucleophilic attack on the nitrile

carbon by the sulfhydryl group of the nitrilase, leading to a tetrahedral inter-

mediate via an enzyme thioimidate route (Fig. 2). The tetrahedral intermediate

can frequently break down anomalously to produce an amide instead of the

normal acid product. Nitrilase interaction with its substrates and intermediates

has some geometric constraints: linear substrate (approximately 180



C), planar

ESH

–NH

ESH

Figure 2. Reaction mechanism of nitrilase catalysis

538 KAUL ET AL.

thioimidate and acylenzyme intermediates (approximately 120



C), and tetrahedral

water-bonded intermediates (approximately 1095



C). Since ammonia is a better

leaving group than the enzyme, the first enzyme-dependent water addition does not

produce an acid amide but rather an acylenzyme complex the hydrolysis of which

produces the acid product. Most nitrilases bind strongly to a bulky substrate R group

in a conformation that places the second carbon closer to 120



than to 180



from the

cyano nitrogen. Hence, fitting a distorted nitrile substrate would drive the reaction

towards thioimidation rather than tetrahedral intermediate. In support of this view,

most nitrilases are noted to prefer bulky substrates to non-substituted acetonitrile

(Pace and Brenner, 2001).

2.2. Nitrile Hydratase

Nitrile hydratase (NHase) is a key enzyme in the bienzymatic pathway of the

conversion of nitriles to amides, which are further converted to the corresponding

acid by amidases. A number of micro-organisms having NHase activity have been

isolated and the enzymes have been purified and characterized. These revealed the

wide ranging physiochemical properties and substrate specificities of the NHases,

which are composed of two types of subunits ( and ) complexed in varying

numbers. They are metalloenzymes containing either cobalt or iron. On the basis of

the metal ion present, the NHases can be classified into two broad groups, namely

ferric NHases and cobalt NHases.

2.2.1. Ferric NHases

The NHases from Rhodococcus R312 (formerly known as Brevibacterium R312)

and Pseudomonas chlororaphis B23 are the first examples of non-heme iron

enzymes containing a low spin Fe (III) ion (Sugiura et al., 1987). These have

been well characterized by ESR, extended X-ray absorption (EXFAS), electron

nuclear double resonance (ENDOR) and Raman resonance spectroscopies. These

studies revealed that the NHase from Rhodococcus sp. R312 is a 

tetramer

containing two low spin non-heme ferric ions which exist in a tetragonally distorted

octahedral ligand field of three histidine imidazoles, two cysteine thiolates and a

hydroxide. The activity of NHase has unique features when exposed to light (Endo

et al., 1999). During aerobic incubation in the dark NHase activity in Rhodococcus

sp. N-771 decreases, but this activity is almost completely recovered upon irradi-

ation with visible light. This photo-reactivity of NHase is intrinsic to the enzyme,

and the mechanism has been biochemically elucidated (Endo et al., 1999). The

chromophore involved in the photo-activation is an iron complex in the  subunit,

and light irradiation of the complex induces a conformational change of the subunit.

Moreover, activity is regulated by nitric oxide (NO). An endogenous NO molecule

is bound to the non-heme iron (III) centre in the inactive NHase and is released

upon photo-activation, resulting in recovery of the original NHase activity. Three-

dimensional analysis of Rhodococcus R312 NHase showed that it contains a novel

iron centre (Huang et al., 1997). All the metal ion protein ligands are contained

NITRILE HYDROLASES 539

within the  subunit. Three cysteine thilotes and two main chain nitrogen atoms are

ligands. These five iron ligands (Cys 110, Cys113, Cys115, Ser 114 and Cys 115)

are located on five vertices of an octahedron, the sixth position being unoccupied.

Considering the experimental data for hydrogen and oxygen ENDOR resonances,

a hydroxide ion is likely to occupy this position. Furthermore, Cys112 and Cys114

(corresponding to Cys113 and Cys115 in the Rhodococus R312 NHase) are post-

translationaly oxidised to cysteine-sulfinic and sulfenic acids, respectively, in the

Rhodococcus N-771 NHase.

2.2.2. Cobalt NHase

In the presence of cobalt ions the actinomycete R. rhodochrous J1 (Komeda et al.,

1996b) produces two Nhases, depending on the inducer. When cultured in a medium

containing urea and cyclohexane carboxamide, high and low molecular weight

NHases (H- and L-NHases) are selectively induced (Yamada and Kobayashi, 1996).

H-NHases (Nagasawa et al., 1991) act preferentially on aliphatic nitriles, whereas

L-NHases (Komeda et al., 1996b) exhibit higher affinity for aromatic nitriles. H- and

L-NHases have been used for the industrial production of acrylamide and nicoti-

namide from acrylonitrile and 3-cyanopyridine, respectively. Both purified NHases

contain cobalt as co-factor. The cobalt in H-NHase exists as a low-spin Co ion in

a tetragonally distorted octahedral ligand field. The similarities of the pre-edge and

extended X-ray absorption fine structure (EXAFS) spectra suggests that the ligand

environments of the metal ions in the cobalt and iron containing NHases are similar.

Recently, a NHase from Pseudomonas putida with stereoselectivity toward 2-(S)-

(4-chlorophenyl)-3-methylobutyronitrile was found to contain noncorrin low-spin

Co (Payne et al., 1997). The three dimensional structures of both the Rhodococcus

rhodochrous J1 and Pseudomonas putida NHase are probably similar to that of

the Rhodococcus R 312 NHase because of their sequence similarities. The cobalt

NHases have threonine in the –V-C-(T/S)-L-C-S-C-sequence as the active site,

(Payne et al., 1997) whereas the ferric NHase has serine. The difference in the metal

co-factors may be ascribed to the different amino acid residues at this position.

Spectroscopic and structural analyses of novel NHases in Agrobacterium tumifa-

ciens which contain cobalt and iron should improve our understanding of the

functions of these metals. There may be two main reasons for having Co in the

active site: 1) Cobalt is a very good catalyst for CN-triple-bond hydration, and 2)

it is required for the folding of the enzyme. In addition to the function of the active

centre, cobalt ions may play a role in enhancing the folding or stabilization of the

subunit polypeptides of the enzyme.

2.2.3. Reaction mechanism

One possible reaction model is that catalysis proceeds without direct coordination

of the substrate to the metal ion, where the metal ion plays a role as a lewis

acid activating a water molecule. The next step could be either of the following:

(1) the nitrile substrate approaches a metal-bound hydroxide ion which can act as a

nucleophile attacking the nitrile carbon atom (mechanism I), or (2) a metal bound

540 KAUL ET AL.

Figure 3. Reaction mechanism of nitrile hydratase catalysed hydrolysis of nitriles

hydroxide ion as a general base activates a water molecule which then attacks the

nitrile carbon (mechanism II) resulting in the formation of an imidate. The imidate

finally tautomerizes to form an amide (Fig. 3).

2.3. Amidase

Amidases catalyse the hydrolysis of carboxylic acid amides to free carboxylic acids

and ammonia. These enzymes are involved in nitrogen metabolism in cells and

are widely distributed in nature. They have been found both in prokaryotic and

eukaryotic cells. Unlike nitrile hydratases, the association of amidases with metals

such as cobalt or iron is reported only in case of K. pneumoniae (Nawaz et al., 1996).

Using site-directed mutagenesis it was confirmed that these are sulfhydryl enzymes.

The other amidases from Pseudomonas chlororaphis B23, R. erythropolis MP50, R.

rhodochrous J1 etc. belong to a group of amidases containing the Gly-Gly-Ser-Ser

NITRILE HYDROLASES 541

Figure 4. Reaction mechanism of amidase

signature (Chebrou et al., 1996). Surprisingly the amidase of R. rhodochrous J1

is found to catalyse the hydrolytic cleavage of the nitrile functional group to an

acid and ammonia stoichiometrically. The amidase exhibited a K

of 3.26 mM for

benzonitrile in contrast to that of 0.15 mM for benzamide as the original substrate

(Kobayashi et al., 1998).Thus the reaction mechanisms of both the nitrilase- and

amidase-catalysed reactions are analogous, but the active nucleophile present in

both enzymes differs. Fig. 4 shows the reaction mechanism of amidase, which

also involves nitrile hydrolysis. The carbonyl group of the amide undergoes a

nucleophilic attack resulting in the formation of a tetrahedral intermediate which

is converted to an acyl-enzyme with the removal of ammonia and subsequently

hydrolysed to acid. Although there is no homology between the amidase and nitrilase

families, comparison of their reaction mechanisms can provide novel insights for

the construction of catalysts required for the hydrolysis of nitriles as well as amides.

All of the different amidases also exhibit acyl transfer activity in the presence

of hydroxylamine (Fournand et al., 1998). Hydroxamic acids thus produced are

known to possess good chelating properties and can act as potent inhibitors of

matrix metalloproteases (Cawston, 1996). They also have the potential to be used

as anti-HIV agents or antimalarial agents (Gao et al., 1995).

3. APPLICATIONS

Biocatalysis offers a new dimension, innovative approaches and enormous

opportunity to prepare useful chiral compounds (Patel, 2001). Research over the

past decade has shown that there are very few barriers to the use of enzymes

and/or whole cells as biocatalysts in organic synthesis (Faber, 1997). Enzymes

are remarkable catalysts capable of accepting a wide array of complex molecules

as substrates and are exquisitely selective, catalysing reactions with unparallel

enantio- and regio-selectivities (Schmid et al., 2001; Kaul et al., 2004a). Enzymatic

transformation of nitriles in particular provides great potential for synthetic chemists.

The ability of the enzyme system to convert cyano functionality to either an acid

542 KAUL ET AL.

or an amide is, in itself, of great use. Traditional chemical methods for conversion

of nitriles to acids or amides have several drawbacks: (1) reactions need to be

carried out either in strongly acidic (6 M HCl) or basic (2 M NaOH) reflux condi-

tions, (2) a requirement for high reaction temperatures, (3) the formation of by-

products such as toxic HCN or large amounts of salts, etc. Biocatalytic hydrolysis

of nitriles is attractive due to its ability to effect reactions in a more ‘green’ manner

and because of the potential for carrying out chemo-, regio-, and enantio-selectiv

transformations.

3.1. Synthetic Biocatalysis

Industrial scale processes for the production of acrylamide (Mitsubishi Rayon

Co. Japan) (Nagasawa, 1993; Yamada and Nagasawa, 1994) and nicotinamide

(Lonza AG, Switzerland) (Chassin, 1996) involving nitrile converting enzymes

are the two most prominent examples of biocatalytic reactions being implemented

at larger scale and pave the way for the development of other processes

involving this class of enzymes. Nitrilases can also selectively convert a single

cyano group of a polynitrile which is virtually impossible using conventional

chemical methods. R. rhodochrous K22 catalyses the conversion of adiponitrile

to 5-cyanovaleric acid, which is an intermediate for the synthesis of nylon-6

(Godtfredsen et al., 1985) and Tranexamic acid, a homeostatic drug, is obtained

by selective mono-hydrolysis of trans 1,4-dicyano cyclohexane by Acremonium

sp. (Nishise et al., 1987). Nitrilases also offer the possibility of stereoselective

transformation which includes the production of several important fine chemicals

and pharmaceutical intermediates, for example S-phenyllactic acid (Hashimoto

et al., 1996), L--amino acids (Bhalla et al., 1992), R −-hydroxy acids (Wu

and Li, 2003), S-ibuprofen (Yamamoto et al., 1990) etc. Although in recent

years several applications of nitrilases have been recognized (Kobayashi and

Shimizu, 1994; Sugai et al., 1997) to date there exist only two industrial scale

processes for the production of 1,5-dimethyl 2-piperidone (DuPont, USA) and

R-−-mandelic acid (BASF, Germany; Mitsubishi Rayon, Japan) employing

these enzymes as biocatalysts. 1,5-dimethyl 2-piperidone (Xolvone

™

) which has

desirable properties for electronics, coatings and solvent applications, is produced

from 4-cyanopentanoic acid, which in turn is generated from regio-selective

nitrilase-mediated transformation of 2-methyl glutaronitrile (MGN) employing

Acidovorax facilis 72W cells (DiCosimo et al., 2000). When compared to existing

chemical process in which MGN is directly converted via hydrogenation to a mixture

of 1,3-DMPD and 1,5-DMPD, the chemoenzymatic method generates less waste and

produces a single lactam isomer at significantly higher yield. BASF and Mitsubishi

Rayon use nitrilase-mediated processes for the production of R-−-mandelic

acid and its derivatives at multiton scale (Endo and Tamura, 1993; Ress-Loschke

et al., 1998). The advantage of this process over its chemical counterpart is that

no organic solvent is required and a high degree of stereoselectivity is achieved

because of the presence of the biocatalyst.

NITRILE HYDROLASES 543

3.2. Bioremediation

Synthetic nitrile compounds are widespread in the environment in the form of

industrial wastewaters. Most of these are toxic, carcinogenic and mutagenic in

nature (Pollak et al., 1991), thus there is a need to control their release into the

environment. A mixed culture of bacteria containing different nitrile hydrolyzing

enzymes (including NHase, amidase and nitrilase) able to metabolise effluents

containing acrylonitrile, fumaronitrile, succinonitrile, etc. were grown in batch

and continuous cultures on these components of industrial waste. A reduction

in COD (75%) and significant removal (99%) of detectable toxic components

was achieved by biodegradation of the effluent from acrylonitrile manufacturing

industries using mixed cultures of bacteria (Wyatt and Knowles, 1995). The use of

specialized consortia of micro-organisms to degrade toxic wastes therefore could

be a viable alternative approach to the classical activated sludge system. Prolonged

exposure to nitrile herbicides [dichlobenil (2,6-dichlorobenzonitrile), bromoxynil

(3,5-dibromo-4-hydroxybenzonitrile) etc] results in symptoms of weight loss,

fever, vomiting, headache and urinary problems (Freyssinet et al., 1996). Nitrile-

metabolising enzymes efficiently degrade these cyano group-containing herbi-

cides and prevent them from entering the food chain. Agrobacterium radiobacter,

a bromoxynil-degrading soil bacterium, was used for the degradation of the

herbicide under non-sterile batch and continuous conditions yielding a 65%

reduction in the bromoxynil concentration in a column reactor after 5 days. The

efficacy of degradation is enhanced by the addition of ferrous, cobaltous or cupric

ions (Muller and Gabriel, 1999). A gene encoding the nitrilase has been cloned

from Klebsiella pneumoniae ssp. ozaenae (McBride et al., 1986) and was used

to raise herbicide resistant plants (Stalker et al., 1988). Bromoxynil resistant

transgenic plants resulting from the introduction of microbial bromoxynil-specific

nitrilase genes into tomato or tobacco are already approved for commercial use

(Freyssinet et al., 1996). Similarly, other nitrile-degrading enzymes could also be

potential candidates for molecular manipulation of bio-degradative systems in plant

biotechnology.

4. CONCLUSIONS

The versatile biocatalytic nature and applications of nitrile converting enzymes

are now increasingly recognized for the production of several pharmaceutically

important compounds and fine chemicals. This relatively new biocatalytic technique

also shows promise for use as a method for the preparation of enantio-, regio-,

chemo-selective amides or acids, the synthesis of which is not feasible by chemical

routes. Commercial scale processes involving kiloton syntheses of acrylamide,

nicotinamide and nicotinic acid using these enzymes are excellent examples of this

methodology. By virtue of their ability to eliminate highly toxic nitriles, these nitrile-

degrading enzymes can also play a significant role in protecting the environment.

Advances in our understanding of biosynthetic regulation, genetics and the structure