Studies On The Regioselectivity And Stereoselectivity Of...

Biocatalysis, 1987, Vol. 1, pp. 23-36 Photocopying permitted by license only

0 1987 hanvood academic publishers GmbH Printed in the United Kingdom

STUDIES ON THE REGIOSELECTIVITY AND STEREOSELECTIVITY OF THE SOLUBLE

METHANE MONOOXYGENASE FROM METHYLOCOCCUS CAPSULATUS (BATH)

DAVID J. LEAK* and HOWARD DALTON Department of Biological Sciences, University of Warwick, Coventry CV4 7A L,

England, UK (Received 4 July 1986; in final form 12 September 1986)

Alkyl substituted derivatives of cyclohexane and cyclohexene have been used as active site probes of the soluble methane monooxygenase (MMO) from Methylococcus capsulam (Bath). It is proposed that the products obtained are those that would be predicted on the grounds of chemical reactivity modulated by two enzymic constraints (i) steric hindrance favouring hydroxylation at positions distal to bulky substituents and (ii) limited penetration of the substrate beyond the active site of oxygen insertion. Evidence for inversion of stereochemistry during the hydroxylation of ck- dimethylcyclohexane and rearrangement during the hydroxylation of 3-methyl-1-cyclohexene supports the suggestion that a stepwise mechanism (hydrogen abstraction and hydroxylation) operates in the hydroxylation of aliphatic carbons.

KEY WORDS Monooxygenase, Methylococcus capsulatus, hydroxylation selectivity, active site

INTRODUCTION

The soluble methane monooxygenase from Methylococcus capsulatus (Bath) is comprised of three components. Component C (Lund and Dalton, 1985) is the NADH: acceptor reductase that transfers electrons to component A (Woodland and Dalton, 1984), the oxygenase that interacts with the hydrocarbon substrate. Component B is involved in coupling substrate oxidation to electron transfer (Green and Dalton, 1985). Component A is a non-heme iron protein of relative molecular mass 210000 comprising three subunits of Mr = 54 OOO, 42 000 and 17 OOO, suggesting an cu,, p2, y2 arrangtment containing approximately 2 mol/mol iron and OSmol/mol zinc (Woodland and Dalton, 1984). Studies with cell extracts and whole cells have demonstrated that this enzyme has low substrate specificity, being capable of hydroxylation of aliphatic, alicyclic and aromatic hydrocarbons (Dalton, 1980). However, some of the whole cell studies must be treated with caution following the demonstration that two forms of methane monooxygenase with different substrate specificities may co-exist in some methanotrophs (Stanley et al., 1983; Burrows et al., 1984).

In recent years several patents have been issued indicating the potential of methane-oxidizing bacteria in the bioconversion of several of the above- mentioned substrates (Higgins et al., 1980). If the full capabilities of this system

* Present address: Centre for Biotechnology, Imperial College of Science and Technology, London SW7 2AB, England, UK.

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24 D. J . LEAK AND H. DALTON

are to be realized in practice, a detailed understanding of the mechanism of action of the enzyme will be essential. There are basically three approaches that can be adopted to reach this goal. These are: physico-chemical characterization of the enzyme active site; kinetic studies on the interaction of the individual components and their prosthetic groups; and evaluation of active site chemistry through observation of products formed from substrate hydroxylations.

Hydroxylation of short chain alkanes gives rise to primary and secondary alcohols (Dalton, 1980). However, this observation provides little information on preferential sites of hydroxylation due to the flexibility and capacity for carbon-carbon bond rotation in these substrates. The semi-rigid structure of cyclohexane derivatives, however, allows differentiation of regioselectivity and stereoselectivity of hydroxylation as well as an assessment of steric constraints on substrate utilization. This capability has previously been exploited in probing the active site of the paraffin hydroxylase from Pseudomonas aeruginosu (van Ravenswaay Claasen and van der Linden, 1971), and a similar approach has been utilized in comparing the major constraints in hydroxylation by cytochrome P-450 isoenzymes P-450,,, and P-450LM2 (White et a f . , 1984). In this paper we report on the use of alicyclic compounds as methane monooxygenase substrates to help determine the nature of the active site.

MATERIALS AND METHODS

Crude soluble extract was prepared from 1001 batch cultures of M. cupsufutus (Bath) as previously described (Stanley et ul., 1983; Colby and Dalton, 1978). All experiments were done using extract from a single batch, which was stored as frozen pellets at -80°C. The protein concentration was 66 mg ml-' determined by the method of Bradford (1976) with bovine serum albumen as standard.

Bioconversions were done at 45°C in 5 ml McCartney bottles with neoprene stoppers. Incubation mixtures (1 ml) contained 5 mM magnesium chloride, 2 pl of hydrocarbon substrate and 6.6 or 13.2 mg protein in 20 mM Tris-chloride buffer pH 7.0. After one minute pre-incubation, the bioconversion was initiated by the addition of 5 mM NADH (ethanol free) plus 10 mM potassium formate. For rate determination, duplicate bottles containing 6.6 mg protein ml-' were incubated for three, six and nine minutes. For analysis of total products, bottles containing 13.2 mg protein ml-' were incubated for 3 minutes. Assays were terminated by plunging the bottle into ice, followed by extraction of products into dichloro- methane. Control experiments to demonstrate the involvement of MMO in the bioconversion contained 0.2 ml acetylene, a potent inhibitor (Dalton and Whit- tenbury, 1976).

Products were analysed by gas chromatography (Pye Unicam GCV) on Carbowax 20M or Carbowax 20M TPA (10% on chromosorb W 80/100 mesh) and on SP2100 (3% on chromosorb W 80/100 mesh). All analyses were isothermal with temperatures in the range 100-140°C depending on the products analysed. Nitrogen carrier gas was maintained at 30 ml min-'. Quantitation was by internal standardization using a Hewlett Packard 3390A integrator, and identification by co-retention with authentic standards, chemical reactivity and electron impact mass spectrometry (Carlo Erba GC-MS 80 Kratos) with separation on Carbowax 20M. For initial assay optimization, products were

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HYDROXYLATION SELECTIVITY OF MMO 25

analyzed directly (without extraction) on Tenax GC (isothermal 190°C with N2 carrier gas at 20 ml min-’).

Chemicals Nicotinamide adenine dinucleotide, reduced form (NADH) was obtained from BDH Chemicals Ltd, Atherstone, Warwickshire, UK; the Bradford protein reagent was obtained from Bio-Rad Labs Ltd, Watford, Herts, UK.

Cyclohexane, cyclohexanol, cyclohexanone, cyclohexene, methylcyclohexane, and 2-methylcyclohexanol were obtained from BDH Chemicals Ltd, Atherstone, Warwickshire, UK.

2-cyclohexen-l-ol, cyclohexene oxide, 1-, 3- and 4-methylcyclohexanol, cyclohexylmethanol, cyclohexanecarboxaldehyde, cyclohexanecarboxylic acid, ethylcyclohexane, 2- and 4-ethylcyclohexanol, 1-cyclohexylethanol, t-butylcyclohexane, 3-methyl-l-cyclohexene, and 3-methyl-2-cyclohexen-1-01 were obtained from Aldrich Chemical Co Ltd, Gillingham, Dorset, UK.

2-cyclohexylethanol, cis- and trans-l,4-dimethylcyclohexane (separate and mixed isomers), 2,5-dimethylcyclohexanol (mixed isomers), l-methyl-l-cyclohexene, and 4-methyl-1-cyclohexene were obtained from Fluorochem Ltd, Glossop, Derbyshire, UK.

RESULTS AND DISCUSSION

Preliminary Studies Preliminary experiments with cyclohexane as substrate demonstrated that the maximum rate of hydroxylation was obtained with the assay technique described. With the formate + formate dehydrogenase (present in the soluble extract) NADH regeneration system, the rate of product formation was linear for at least nine minutes. Doubling the substrate concentration or halving the protein concentration reduced the specific activity. A requirement for a minimum protein concentration for maximum specific activity has previously been observed using this system.

Cyclohexanol was found to be the sole product in these assays, judged by co-retention with an authentic standard. No cyclohexanone was detected, although this has been observed in long-term whole cell assays.

Methylcyclohexane The products from hydroxylation of methylcyclohexane were separated into three peaks on Carbowax 20M TPA. Using authentic standards of 1-, 2-, 3- and 4-methylcyclohexanol, cyclohexylmethanol, cyclohexylcarboxaldehyde and cyclo- hexylcarboxylic acid, the major product co-chromatographed with cis-3- and trans-4-methylcyclohexano1, which could not be differentiated on either of the columns utilized. Pairs of stereoisomers in each standard were separated on Carbowax 20M P A and were assigned according to the criteria of Paris and Alexandre (1972) (this assignment has since been confirmed with “pure” isomers). Although cis-3- and trans-4-methylcyclohexanols had identical retention times, they were distinguishable by their fragmentation patterns obtained by mass spectrometry (Figures l(a) and (b)) notably in their major fragmentation ion.

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Inspection of the major enzyme product indicated that it was predominantly cis-3-methylcyclohexanol (Figure l(c)) although there was a small contribution from the trans-derivative. On the basis of the major peaks M/Z 57 and M/Z 71, the contribution of trans-4-methylcyclohexano1 was estimated at 12%. The dominance of cis-3- and trans-4-methylcyclohexanols over trans-3- and cis-4- derivatives (not detected in this assay, but see the analysis of ethylcylcohexanols) indicates that, while the MMO is regioselective rather than regiospecific for the 3 position, an overriding feature is the formation of planar products, cis-3- and trans-4- positions being equatorial with respect to an equatorial methyl sub- stituent. While this could reflect a strict enzymic control on hydroxylation, equatorial products would also be the major products based on chemical criteria if one assumed a stepwise mechanism involving “random” hydroxylation of an intermediate formed by hydrogen abstraction, the stereoselectivity of hydroxylation being determined solely by steric interactions with the substrate.

Of the two minor product peaks, that with the longest retention time co-chromatographed with cyclohexylmethanol, and this was supported by mass spectral analysis. The early peak co-chromatographed with 1-methylcylcohexanol, although previous studies suggested that this might have been difficult to separate from 2-methy1cyc10hexanone7 which was not available for comparison. A molecular ion at M/Z 114 in the mass spectral analysis, however, indicated the former. The total composition of products from the hydroxylation of methylcyclohexane is presented in Table 1.

Higher A lkylcylcohexanes The major product from ethylcyclohexane co-chromatographed with the later of the two peaks obtained with a standard of 4-ethylcyclohexanol (cis and trans) on Carbowax 20M TPA. On the basis of criteria already outlined this was assigned as trans-4- and/or c~-3-ethylcyclohexanol. In the absence of an authentic standard of 3-ethylcyclohexanol it was impossible to assess the contribution made by each isomer. A small peak that co-chromatographed with the early isomer, assumed to be cis-4- or truns-3-ethylcyclohexanol, was also evident. It is not known whether this reflected a genuine difference between the types of products obtained with methyl- and ethylcyclohexane, as the separation of ethylcyclohexanols was better than for methylcyclohexanols on this systeni. A small amount of cis-4- or trans-3-methylcyclohexanols may therefore have been masked in the previous analyses.

Both of the side chain hydroxylated products 1- and 2-cyclohexylethanol, identified using authentic standards, were present as minor products in similar amounts. Two further peaks were present comprising approximately 2% of total products. Neither co-chromatographed with cis- and trans-2-ethylcyclohexanol and in the absence of a standard of 1-ethylcyclohexanol were not identified. The rate of hydroxylation of ethylcyclohexane, based on total product accumulation, was about half that with methylcyclohexane.

The effect of steric hindrance of the alkyl side chain on hydroxylation was examined using t-butylcyclohexane as substrate. Even in extended assays no products were obtained, indicating that a bulky side chain restricts access of the substrate to the active site, prohibiting hydroxylation even at a distal point on the cyclohexane ring. This is consistent with previous evidence for lack of hydroxylation of t-butylbenzene.

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28 D. J. LEAK AND H. DALTON

Table 1 Products formed from cyclohexane, cyclohexene and alkyl substituted derivatives by the soluble methane monooxygenase (MMO) from Methylococcus capsulatus (Bath).

Substrate Prod u c t.i Amount(%) Maximum activity t

Cyclohexane cyclohexanol

Methylcyclo hexane

Ethylcyclohexane

cis-3-methylcyclohexanol rram-4-methylcyclohexanoI cyclohexylmethanol 1-methylcyclohexanol

cis-3-/tram-4-ethylcyclohexanol cis-4-/fruans-3-ethylcyclohexanol 1-cyclohexylethanol 2-cyclohexylethanol 1-ethylcyclohexanol (?)

t-butylcyclohexane - cis -1,4-dimethyIcyclohexane 1 ,tram-4-dimethylcyclohexanol

1 ,cis-4-dimethylcyclohexanol cis-4-rnethylcyclohexylmethanol r-1, c-2,c-5-dimethylcyclohexanol

1 ,-cis-4-dimethylcyclohexanol r-1, r-2,~-5-dirnethylcyclohexanol (1 ,tram-4-dimethylcyclohexanol?)

mm.s-l,4-dimethylcyclohexane nam-4-methylcyclohexylmethanol

Cyclohexene

1 methyl- 1-cyclohexene

3-methyl-1-cyclohexene

4-me thylcyclohexene

2-cyclohexenol cyclohexene oxide cyclohexanone

3-methyl-2-cyclohexen- 1-01 l-cyclohexene carboxaldehyde 1-methyl-2-cyclohexen- 1-01 2-methyl-2-cyclohexen-1-01 cis-6-methyl-2-cyclohexen-l-ol traans-6-methyl-2-cyclohexen-l-ol 3-methyl-1-epoxycyclohexane [saturated ketone]

[secondary ring alcohol]' [secondary ring a l c o h ~ l ] ~ 4 methyl-1-epoxycyclohexane [saturated ketone]

100 8.2

83.9 11.4 5.0 2.8 1.8

76.8 17.9 2.1 1.5 0.5

2.25

0

2.5

1.6

27.6

57.3 35.3 4.2 3.5

86.9 10.9 1.9 0.3

85.5 11.5 3 .O

36.8 33.4 ND 15.5 14.3

78.4 12.3 10.4 4.7 4.6

43.3 38.8 6.7 15.7 2.2

t Maximum activity (nmollminlmg protein) was that measured with 2 pllml substrate and 6.6 mglml protein in all cases I]? some produa identifications are tentativelinfompletethew are discussed in the text. ND = not determined.

and signify the order of elution from Carbowax 20M TPA. i.e. =early = late

Dimethylcyclohexane The effect of a second ring substitutent was analysed using cis- and trans-1,4- dimethylcyclohexane as substrates. Although only a single standard of 2,5- dimethylcyclohexanol (mixed isomers) was available, all four isomers were separated on Carbowax 20MTPA and were assigned on the basis of retention indices according to the findings of Paris and Alexandre (1972). Due to the

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inherent symmetry of the substrate, 1,4-dimethylcyclohexane, several potential hydroxylation points are equivalent. Thus, of singly hydroxylated products, only three positional isomers were possible, i.e. 2,5-dimethylcyclohexanol (two epime- ric pairs a-d), 1,4-dimethylcyclohexanol (cis and trans e, f) and 4-methylcyclohexanols would be expected to have the shortest retention time followed by the 2,5-dimethylcyclohexanols and finally the 4-methylcyclohexylmethanols. Possible products are illustrated in Figure 2.

The major products from cis-1,4-dimethylcyclohexane were identified as 1,4-dimethylcyclohexanols (both isomers e and f) on the basis of retention time and mass spectrometry. The major fragmentation ions of these two products were identical, and the presence of a molecular ion at M/Z 128 but no peak at M/Z 110 (M-18) was indicative of a tertiary alcohol. The assignment was supported by the lack of oxidation with Jones reagent (0.1 M sodium dichromate in 2.5 M sulphuric acid). The presence of both isomers indicated that hydroxylation had proceeded with partial inversion of stereochemistry, supporting the suggestion that hydroxylation of aliphatic carbons involves a stepwise mechanism. By comparison with the products obtained from trans-l,4-dimethylcyclohexane and reference to previous studies, the product with the shortest retention time was assigned as 1 ,cis-4-dimethylcyclohexanol (f). Two minor products were also evident, one correlating with r-1, c-2, c-5-dimethylcyclohexanol (d) and a late running peak presumed to be cis-4-methylcyclohexylmethanol (h). Relative amounts of each product (based on peak area) are presented in Table 1.

The products from trans-l,4-dimethylcyclohexane differed both quantitatively and qualitatively. The major product has a long retention time on Carbowax 20M TPA, was oxidizable with Jones reagent and had a mass spectrum consistent with its assignment as trans-4-methylcyclohexylmethanol (8) (peaks at M-18 and M-31 were prominent but the molecular ion was absent). Two early peaks with identical retention times to those produced from cis-1,4-dimethylcyclohexane were also evident and were assigned as 1,4-methylcyclohexanols. The earlier of the two peaks, assumed to be 1 ,cis-4-dimethylcyclohexanol (f), was prediminant, while the later peak was also evident in similar proportions in the starting material, indicating that it was probably not a genuine product. A minor product was also evident which co-chromatographed with r-1, t-2, c-5-dimethylcyclohexanol (a). Relative amounts and rates of hydroxylation are presented in Table 1. It is interesting to note that all of the products mentioned were obtained in similar proportions when the substrate was a mixture of cis and trans isomers, indicating that the affinity of MMO for the two isomers does not differ greatly.

Cy clohexene Three products obtained with cyclohexene as substrate were identified as 2-cyclohexene-1-01, cyclohexene oxide and cyclohexanone (in order of decreasing abundance) by correlation (GC-MS) with authentic standards. The rate of formation of the major product 2-cyclohexen-1-01 was almost three times as high as cyclohexane oxidation, suggesting that the allylic position is more amenable to attack than a fully saturated hydrocarbon. However, the greater solubility of the substrate (cyclohexene us cyclohexane) may also have contributed to the increased rate.

It is useful to compare the types of products obtained with cyclohexene as

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a

b

C

d

e

f

h

doH HoH OH

CH20H

CH20H

Figure 2 Potential products from the hydroxylation of cis- and rrans-dimethycyclohexane: (a) r-l,t-2,c-S-dimethylcyclohexanol; (b) r-l,c-2,t-S-dimethylcyclohexanol; (c) r - 1 , c-2, f-5-dimethylcyclohexanol; (d) r- 1 , c-2, t- 5-dimethylcyclohexanol; (e) 1 ,trans-4-dimethylcyclohexanol (f) 1 ,cis-4-dimethylcyclohexanol; (g) lrum-4- methylcyclohexylmethanol; (h) cis-4-methylcyclohexylmethanol.

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a b C r------ - - - -7 ,- - - - - - - - - - --_I

Figure 3 Proposed active site geometry of MMO illustrat- ing substrate orientation and the site of oxygen insertion (A) for alkylcyclohexanes (a), trans-2-butene (b) and cis-2- butene (c).

substrate and those from cis- and trans-Zbutenes. Although one might expect a similarity in the active site configuration for cis-2-butene and cyclohexene, the epoxide was the major product from the former. However, the allylic hydroxylation product (crotyl alcohol) predominated with the trans isomer. (Statistical weighting to take account of the equivalence of 1 and 4 positions would, in fact, alter this order but the argument remains qualitatively the same.) This gives us some insight into the active site geometry (Figure 3).

Visualizing the substrate fairly tightly enclosed within the active site of the enzyme (broken line), the results from alkylcyclohexane oxidation suggest a preference for hydroxylation at a point delineated A (Figure 3). Assuming the substrate can penetrate sufficiently, this gives rise predominatly to cis-3- alkylcyclohexanols. When the structures of cis- and trans-Zbutene are placed into this framework it is evident that the double bond in the cis is much more amenable to attack from position A than the trans isomer, which would explain the higher levels of epoxide formation in the former. (A preference for epoxidation over terminal allylic hydroxylation is already evident from the oxidation of propylene, where propylene oxide is the sole product.) On the basis of this model, however, one would predict that the epoxide should also comprise the major product in the case of cyclohexene oxidation. Two possible explana- tions for this discrepancy may be entertained.

1. Allylic positions in cyclohexene are secondary and would therefore form charged intermediates more readily than primary allylic carbons. Combined with their resonance stabilization a stepwise mechanism by this route could become more favourable than expoxidation.

2. 2-cyclohexen-1-01 could arise from the rearrangement of cyclohexene oxide in the active site. (An alternative possibility, that rearrangement occurs after release of the product from the enzyme or by subsequent attack, was discounted by demonstrating the stability of cyclohexene oxide under the assay conditions described.) This would be envisaged as epoxide formation followed by hydrogen abstraction.

It should be noted that there was no evidence for such a rearrangement with cis- and trans-2-butene as substrates, indicating that the propensity for this proposed rearrangement reflected the greater reactivity of secondary over primary aliphatic carbons. Both possiblities could involve double bond shifting but in mechanism 2 this would be obligatory (i.e. loo%), whereas this need not

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be so in mechanism 1. Analysis of these two possibilities will require specifically labelled ("C) cyclohexene.

Alkyl Cyclohexenes

Although only a single standard for potential products was available, namely 3-methy1-2-cyc1ohexeno1, it was possible to obtain positive or tentative identification of most products by a combination of gas chromatography, mass spectrometry and chemical reactivity. Quantification was based on the amount/area ratio for 3-methyl-2-cyclohexenol.

Some commercial samples of 1-methyl-1-cyclohexene appeared to react spon- taneously in the assay buffer giving a complex mixture of products by a process that has not been further investigated. However, enzyme dependent assays were achieved with one batch, which yielded four major products. On the basis of previous analyses of products from alkylcyclohexanes and cyclohexene, it was apparent that the order of elution from Carbowax 20M TPA for products derived from a single substrate is 1) epoxides, 2) ring ketones, 3) ring alcohols and 4) side chain products. While the earliest product from 1-methyl-1-cyclohexene was stable to acid treatment, its mass spectrum was inconsistent with assignment as 2-methylcyclohexanone. A peak at M/Z 79 (M-33, 24% abundance) is charac- teristic of methylcyclohexenols, arising from loss of H 2 0 and CH3 and is not a feature of ketones. This product was therefore assigned to 1-methyl-Zcyclo- hexenol on the basis of its short retention time. Major fragmentation ions were also consistent with the predicted retro-Diels-Alder degradation product (M-28) and subsequent loss of CH3 (M-43). The most likely route to this product is considered to be via epoxidation (transient?) and rearrangement, the latter step possibly being purely chemical (Figure 4).

Of two GC peaks evident in the secondary ring alcohol region, the later, major peak was identical in all respects to the standard 3-methyl-2-cyclohexenol. The mass spectrum of the earlier peak differed in the relative abundance of fragmentation ions, notably that of the molecular ion M/Z 112 and the major fragment M/Z 97 (M-15). Both spectra, however, contained peaks at M-28 indicative of a retro-Diels-Alder elimination of GH, (Figure 5). Retention of the hydroxyl group in the larger fragment indicated that this product was probably 2-methyl-2-cyclohexenol. (3-methyl-3-cyclohexenol was also excluded on the basis of its 8 peak index.)

On the basis of earlier results the final peak was suspected of being 1-cyclohexenemethanol. However, the mass spectrum of this product revealed a

J

Figure 4 Possible mechanism for formation of 1- methyl-2-cyclohexen-1-01 from l-methyl-l-cyclohexene catalyzed by MMO.

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Figure 5 Retro-Diels-Alder elimination of C,H, from the molecular ion of 3-methyl-2-cyclohexen-1-01.

molecular ion at M/Z 110 and major fragment at M/Z = 82 (M-28) ascribable to retro-Diels-Alder elimination as previously outlined. The third most abundant fragment at M/Z = 54 could be [HC=C-CHO]+, which would be consistent with the proposed assignment as 1-cyclohexene carboxaldehyde. If correct, this is an interesting observation, as methanol dehydrogenase (a non-specific primary alcohol dehydrogenase) would be inactive under these assay conditions. This suggests that both the initial hydroxylation and further oxidation of the methyl group were catalyzed by the MMO. With 3- and 4-methylcyclohexenes as substrates no comparable peak was observed, suggesting that hydroxylation of the methyl group was dependent on its allylic position. Furthermore, the absence of side chain hydroxylation with these substrates indicates that unsaturation has a considerable orientating effect determining hydroxylation specificity.

With 3-methyl-1-cyclohexene as substrate, two minor products were identified as the epoxide (acid sensitive) and a saturated ketone. Although positive identification of the latter was not made, its mass spectrum differed from that ascribed to 1-methyl-2-cyclohexen-1-01. In fact, from the three possible methyl- cyclohexanones the spectrum was closest to that of 4-methylcyclohexanone (data from “Eight Peak Index”). However, the possibility of mixed , methylcyclo- hexanones has not been examined. The two major products from 3-methyl-l- cyclohexene had retention times consistent with assignment as ring alcohols (mass fragment ions at M-18 supported this) and almost identical mass spectra suggesting that they were epimers. The major fragment at M/Z70 (M-42) was indicative of a neutral elimination process. It is difficult to envisage an alternative to retro-Diels-Alder elimination of C H 4 H 2 - C H 3 but this would require that the product be either 5-methyl-2-cyclohexen-1-01 or 6-methyl-2-cyclohexen-l-ol, neither of which would have been expected. Indeed, 4-methyl-2-cyclohexen-l-ol, the expected product from allylic hydroxylation of 3-methyl-1-cyclohexene would be expected to yield a fairly abundant fragment at M-28 by the same process. This was completely absent. On this basis it is evident that some rearrangement process must have occurred. Although further analysis will be required for confirmation, the most likely rearrangement is considered to be that yielding 6-methyl-2-cyclohexen-1-01 from a transient epoxide. According to the work of Paris and Alexandre (1972), the major product would then be the cis isomer.

The epoxide (acid sensitive) was a considerably more abundant product from 4-methyl-1-cyclohexene than the previous two substrates, indicating that the relative position of the methyl group is important in determining the extent of epoxidation. Unfortunately, identification of the remaining products was ham- pered by the lack of mass spectral data. However, some tentative suggestions may

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be made. A minor product with similar retention time to the ketone identified with the previous substrate was probably also a ketone, 3- or 4-methylcyclohexanone being the likely candidates. The two major products had retention times in the range ascribed to secondary ring alcohols. Given the inherent symmetry of the allylic intermediate formed by hydrogen removal at the 6 position, it is probable that at least one of these products is 5-methyl-2- cyclohexen-1-01. (A mechanism involving epoxide rearrangement would give the same product.)

Active Site Modelling and Predictions of Reactivity The soluble MMO from both M. capsulatus (Bath) (Dalton et al. , 1981) and Methylosinus trichosporium OB3b (Jezequel and Higgins, 1983) produce phenols from aromatic substrates via epoxide intermediates, which subsequently re- arrange with concomitant hydride shift. However, contradictory evidence has been presented for the mechanism of hydroxylation at aliphatic carbons. The partial inversion of stereochemistry observed in this study during the hydroxylation of cis-1,4-dimethylcyclohexane at the 1 position supports the conclusion that a stepwise mechanism is involved (Jezequel and Higgins, 1983). Partial epimeriza- tion is also a feature of some cytochrome P450 systems (Groves et al., 1978), with which parallels may be drawn.

It is evident from the products obtained from monoalkylcyclohexanes that alkyl groups have an orientating effect on substrate approach to the active site, the site of attack being distal to the alkyl group. Additionally, the evidence for steric hindrance with t-butylcyclohexane suggests that during hydroxylation a substrate the size of methylcyclohexane is completely enclosed within the enzyme and that this binding site is too narrow to accommodate a t-butyl group. Thus, in the case of alkylcyclohexanes the regioselectivity of hydroxylation is determined primarily by the steric constraints on substrate binding. However, given this constraint, the evidence for a stepwise mechanism indicates that the stereoselectivity of hydroxylation results primarily from chemical criteria; hydroxyl group addition occurs from the least hindered direction, giving rise to predominantly diequatorial products. A more detailed analysis will be necessary to ascertain any enzymic contribution in this respect.

The requirement for substrate burial within the active site provides a possible explanation for the hydroxylation products from the dimethylcyclohexanes. Trarzs-l,4-dimethylcyclohexane is naturally diequatorial in the energetically favourable cyclohexane ring “chair” conformation and this is presumably the structure present in the active site. However, cis-l,4-dimethylcyclohexane can only be diequatorial in the “boat” conformation (Figure 6 (b)). From the evidence for a preferential site of attack at the 3 position of methylcyclohexane, the products from cis- and trans-l,4-dimethylcyclohexane can be readily under- stood if it is assumed that:

1. penetration of the substrate beyond the active site is limited, resulting in predominantly subterminal products where steric constraints allow:

2. steric constraints in the active site favour the substrate orientations depicted in Figure 6 (a) and (b) as opposed to those in 6 (c) and (d) relative to an active site A; (energetically a “skew-boat’’ conformation would be more likely than the full boat of Figure 6 (b), but the outcome would be the same);

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a

z3a A A C d

A A Figure 6 (a-d) Possible binding conformations of trans- (a and c) and cis- (b and d), 1,4-dimethylcyclohexanes relative to the active site 'A' of methane monooxygenase.

3. the position of the active site A is primarily the site of hydrogen abstraction; subsequent hydroxylation is less stereospecific, giving rise to the sterically most favourable products.

Inversion of stereochemistry during hydroxylation at the 1 position with cis- but not truns-l,4-dimethylcyclohexane probably reflects the tendency of the proposed planar intermediate to flip to the energetically more favourable chair conformation.

The authors appreciate that this explanation can be no more than a working model at this stage and that the energetic barrier to substrates binding in the skew-boat or boat conformation is considerable, particularly in the face of the low substrate specificity of the MMO.

While the low percentage of hydroxylation at the 1 position with the trans substrate may reflect the proportion of substrate bound in conformation c (Figure 6) exposing the C1 hydrogen to the active site (the major product presumably arises with substrate bound in conformation a but incompletely buried), the greater relief of steric strain in conversion of the cis isomer to a tertiary planar intermediate should also be taken into account. The failure of Dalton et ul. (1981) to observe ring opening during the hydroxylation of cyclopropane and methylcy- clopropane, which led to the proposal of direct oxygen insertion, suggests that the intermediates formed during aliphatic hydroxylation may be stabilized within the active site.

Alkyl substitution also has an orientating effect in determining the products from methylcyclohexenes, except with 1-methyl-1-cyclohexene where the allylic position of the methyl group is evidently both sterically and electronically more favourable for hydroxylation. Given the models proposed in Figures 3 and 6, the greater proportion of epoxide formed from 4-methyl-compared to 3-methyl-l- cyclohexene would be consistent with the proposal of Jezequel and Higgins (1983) that the same active oxygen species could be responsible for hydrogen abstraction or epoxidation depending on the nature of the substrate. However, further work will be required on the nature of some of the rearrangements taking place before firm conclusions can be drawn.

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Acknowledgements The authors would like to thank Donna Balaam for technical assistance and Mr I. Kaytal for running the mass spectra. DJL is grateful to Allelix Inc., Mississauga, Ontario, Canada, for financial support.

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