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The crystal structure of the reduced form of the enzyme p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens, complexed with its substrate p-hydroxybenzoate, has been obtained by protein X-ray crystallography. Crystals of the reduced form were prepared by soaking crystals of the oxidized enzyme-substrate complex in deaerated mother liquor containing 300-400 mM NADPH. A rapid bleaching of the crystals indicated the reduction of the enzyme-bound FAD by NADPH. This was confirmed by single crystal spectroscopy. X-ray data to 2.3 A were collected on oscillation films using a rotating anode generator as an X-ray source. After data processing and reduction, restrained least squares refinement using the 1.9 A structure of the oxidized enzyme-substrate complex as a starting model, yielded a crystallographic R-factor of 14.8% for 11,394 reflections. The final model of the reduced complex contains 3,098 protein atoms, the FAD molecule, the substrate p-hydroxybenzoate and 322 solvent molecules. The structures of the oxidized and reduced forms of the enzyme-substrate complex were found to be very similar. The root-mean-square discrepancy for all atoms between both structures was 0.38 A. The flavin ring is almost completely planar in the final model, although it was allowed to bend or twist during refinement. The observed angle between the benzene and the pyrimidine ring is 2 degrees. This value should be compared with observed values of 10 degrees for the oxidized enzyme-substrate complex and 19 degrees for the enzyme-product complex. The position of the substrate is virtually unaltered with respect to its position in the oxidized enzyme. No trace of a bound NADP+ or NADPH molecule was found.

The enzyme methylamine dehydrogenase or primary-amine:(acceptor) oxidoreductase (deaminating) (EC I .4.99.3) was purified from the bacterium Thiobacillus versutus to homogeneity, as judged by polyacrylamide gel electrophoresis. The native enzyme has a M , of 123500 and contains four subunits arranged in a c12j2 configuration, the light and heavy subunits having a M , of 12900 and 47500 respectively. The isoelectric point is 3.9. The purified enzyme was crystallized from 37-42% saturated ammonium sulphate in 0.1 M sodium acetate buffer, pH 5.0. The space group is P3121 or P3221, with one a2P2 molecule in the asymmetric unit. The cell dimensions are: a = h = 13.01 nm; c = 10.40 nm. The X-ray diffraction pattern extends to at least 0.25-nm resolution.When certain methylotrophic bacteria are grown on methylamine as the sole source of carbon and energy, this compound is degraded via methylamine dehydrogenase to formaldehyde and ammonia [ l , 21 according to: CH,NH: + H 2 0 --f HCHO + NHZ + 2H+ + 2e-Thus, methylamine dehydrogenase is the first enzyme involved in the carbon dissimilation pathway of these bacteria.The enzyme was first isolated and purified from the bacterium Pseudomonas AM 1 by Eady and Large [l]. Afterwards, similar enzymes were isolated from Methylomonas methylovora [3] and from Methylomonas J [4]. The special feature of methylamine dehydrogenase is that it contains a prosthetic group which is covalently linked to the protein have proposed that this prosthetic group is similar to the recently discovered cofactor pyrroloquinoline quinone (PQQ; Fig. 1). The natural primary acceptor for methylamine dehydrogenase was found to be a copper-containing protein, amicyanin [9], which is coupled to the respiratory chain at the level of a c-type cytochrome [9]. A similar amicyanin has been isolated and characterized from Thiobacillus versutus [lo].Correspondence to W.

The structure of the enzyme p-hydroxybenzoate hydroxylase was determined to a resolution of 0.25 nm wierenga et al. (1979) J. Mol. Biol. 131, 53-73] with crystals belonging to space group C2221. Subsequently it was impossible to repeat the growth of this crystal form and only poor quality tetragonal crystals could be obtained. We have thoroughly investigated this problem and found that Cibacron-blue-purified enzyme appears to be heterogeneous with respect to aggregation state and Cys-116 oxidation. Most importantly, it could be firmly established that C2221 crystals can only be grown from purely dimeric p-hydroxybenzoate hydroxylase possessing an intact SH group. Ion-exchange chromatography on DEAE-Sepharose can successfully remove those forms of the enzyme which impede successful crystallization. Sulfite and dithiothreitol improve crystallization by dissociating the enzyme oligomers into dimers; sulfite especially gives excellent results.