NORMAL GENOMIC

• Zheng YJ, Xia Zx, Chen Zw, Mathews FS, Bruice TC
• Catalytic mechanism of quinoprotein methanol dehydrogenase: A theoretical and x-ray crystallographic investigation.
• Proc Natl Acad Sci U S A. 2001; 98: 432-4
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• The catalytic mechanism of the reductive half reaction of the quinoprotein methanol dehydrogenase (MDH) is believed to proceed either through a hemiketal intermediate or by direct transfer of a hydride ion from the substrate methyl group to the cofactor, pyrroloquinoline quinone (PQQ). A crystal structure of the enzyme-substrate complex of a similar quinoprotein, glucose dehydrogenase, has recently been reported that strongly favors the hydride transfer mechanism in that enzyme. A theoretical analysis and an improved refinement of the 1.9-A resolution crystal structure of MDH from Methylophilus methylotrophus W3A1 in the presence of methanol, reported earlier, indicates that the observed tetrahedral configuration of the C-5 atom of PQQ in that study represents the C-5-reduced form of the cofactor and lends support for a hydride transfer mechanism for MDH.

• Afolabi PR et al.
• Site-Directed Mutagenesis and X-ray Crystallography of the PQQ-Containing Quinoprotein Methanol Dehydrogenase and Its Electron Acceptor, Cytochrome c(L)(,).
• Biochemistry. 2001; 40: 9799-809
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• Two proteins specifically involved in methanol oxidation in the methylotrophic bacterium Methylobacterium extorquens have been modified by site-directed mutagenesis. Mutation of the proposed active site base (Asp303) to glutamate in methanol dehydrogenase (MDH) gave an active enzyme (D303E-MDH) with a greatly reduced affinity for substrate and with a lower activation energy. Results of kinetic and deuterium isotope studies showed that the essential mechanism in the mutant protein was unchanged, and that the step requiring activation by ammonia remained rate limiting. No spectrally detectable intermediates could be observed during the reaction. The X-ray structure, determined to 3 A resolution, of D303E-MDH showed that the position and coordination geometry of the Ca(2+) ion in the active site was altered; the larger Glu303 side chain was coordinated to the Ca(2+) ion and also hydrogen bonded to the O5 atom of pyrroloquinoline quinone (PQQ). The properties and structure of the D303E-MDH are consistent with the previous proposal that the reaction in MDH is initiated by proton abstraction involving Asp303, and that the mechanism involves a direct hydride transfer reaction. Mutation of the two adjacent cysteine residues that make up the novel disulfide ring in the active site of MDH led to an inactive enzyme, confirming the essential role of this remarkable ring structure. Mutations of cytochrome c(L), which is the electron acceptor from MDH was used to identify Met109 as the sixth ligand to the heme.

• Haeseleer F, Palczewski K
• Short-chain dehydrogenases/reductases in retina.
• Methods Enzymol. 2000; 316: 372-83

• Anthony C
• Methanol dehydrogenase, a PQQ-containing quinoprotein dehydrogenase.
• Subcell Biochem. 2000; 35: 73-117

• Hayashi-Iwasaki Y, Oshima T
• Purification and characterization of recombinant 3-isopropylmalate dehydrogenases from Thermus thermophilus and other microorganisms.
• Methods Enzymol. 2000; 324: 301-22

• Keitel T, Diehl A, Knaute T, Stezowski JJ, Hohne W, Gorisch H
• X-ray structure of the quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa: basis of substrate specificity.
• J Mol Biol. 2000; 297: 961-74
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• The homodimeric enzyme form of quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa ATCC 17933 crystallizes readily with the space group R3. The X-ray structure was solved at 2.6 A resolution by molecular replacement.Aside from differences in some loops, the folding of the enzyme is very similar to the large subunit of the quinoprotein methanol dehydrogenases from Methylobacterium extorquens or Methylophilus W3A1. Eight W-shaped beta-sheet motifs are arranged circularly in a propeller-like fashion forming a disk-shaped superbarrel. No electron density for a small subunit like that in methanol dehydrogenase could be found. The prosthetic group is located in the centre of the superbarrel and is coordinated to a calcium ion. Most amino acid residues found in close contact with the prosthetic group pyrroloquinoline quinone and the Ca(2+) are conserved between the quinoprotein ethanol dehydrogenase structure and that of the methanol dehydrogenases. The main differences in the active-site region are a bulky tryptophan residue in the active-site cavity of methanol dehydrogenase, which is replaced by a phenylalanine and a leucine side-chain in the ethanol dehydrogenase structure and a leucine residue right above the pyrrolquinoline quinone group in methanol dehydrogenase which is replaced by a tryptophan side-chain. Both amino acid exchanges appear to have an important influence, causing different substrate specificities of these otherwise very similar enzymes. In addition to the Ca(2+) in the active-site cavity found also in methanol dehydrogenase, ethanol dehydrogenase contains a second Ca(2+)-binding site at the N terminus, which contributes to the stability of the native enzyme.

• Xia ZX, He YN, Dai WW, White SA, Boyd GD, Mathews FS
• Detailed active site configuration of a new crystal form of methanol dehydrogenase from Methylophilus W3A1 at 1.9 A resolution.
• Biochemistry. 1999; 38: 1214-20
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• The three-dimensional structure of a new crystal form of methanol dehydrogenase from Methylophilus W3A1 has been obtained in the presence of substrate using data recorded at a synchrotron. The structure of this approximately 140 kDa heterotetramer, refined at 1. 9 A resolution, reveals the detailed configuration of its redox cofactor, pyrroloquinoline quinone (PQQ). C4, one of the oxygen-bearing atoms of this orthoquinone is in a planar configuration while C5, which bears the other quinone oxygen, is tetrahedral, suggesting that the PQQ is in the semiquinone redox state. The substrate binding site has been identified close to PQQ and to the side chain of Asp297, the putative active site base. The proximity of the hydroxyl of methanol to C5 of PQQ compared to the greater separation of the substrate methyl group from C5 supports the addition-elimination reaction mechanism involving a hemiketal intermediate.

• Anthony C
• An unusual role of tryptophan in PQQ-containing quinoproteins.
• Adv Exp Med Biol. 1999; 467: 597-602
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• Methanol dehydrogenase is a bacterial quinoprotein containing PQQ at its active site, which is in the centre of a superbarrel structure made up of eight beta-sheets arranged radially as the blades of a propeller. A series of novel tryptophan-docking interactions between the beta-sheets make planar, stabilizing girdles around the periphery of the protein. The tryptophan residues form stacking interactions, and hydrogen bonds through their indole ring NH groups.

• Diehl A, von Wintzingerode F, Gorisch H
• Quinoprotein ethanol dehydrogenase of Pseudomonas aeruginosa is a homodimer--sequence of the gene and deduced structural properties of the enzyme.
• Eur J Biochem. 1998; 257: 409-19
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• The gene coding for the periplasmic quinoprotein ethanol dehydrogenase of Pseudomonas aeruginosa ATCC 17933 was cloned and sequenced. The deduced amino acid sequence contained a signal peptide of 34 residues and the major protein of 589 amino acids showed high similarities to pyrroloquinoline-quinone-dependent periplasmic and membrane-bound dehydrogenases acting on alcohols, glucose and quinate or shikimate. It was demonstrated by alignment with the amino acid sequence of the large subunit of the quinoprotein methanol dehydrogenase from Methylobacterium extorquens, whose X-ray structure is known, that the amino acid residues involved in the binding of pyrroloquinoline quinone and Ca2+ at the active site are conserved in the quinoprotein ethanol dehydrogenase of P. aeruginosa. Also, the glycine/tryptophan docking motifs involved in stabilizing the superbarrel structure of the quinoprotein methanol dehydrogenase of M. extorquens were conserved. The known sequences of pyrroloquinoline-quinone-dependent dehydrogenases were used to derive new, more specific sequence motifs for detecting members of this family of enzymes. Despite the sequence similarity between the large a subunit of quinoprotein methanol dehydrogenase from M. extorquens and the quinoprotein ethanol dehydrogenase from P. aeruginosa, the two enzyme systems were quite different. In the presence of the prosthetic group, pyrroloquinoline quinone expression of the Pseudomonas gene encoding the 60-kDa subunit of quinoprotein ethanol dehydrogenase in Escherichia coli resulted in formation of active enzyme. The formation of active quinoprotein methanol dehydrogenase, however, is known to require, in addition to the large alpha subunit, the expression of a small beta subunit, and helper proteins [Lidstrom, M. E. (1995) Genetics of bacterial quinoproteins, Methods Enzymol. 258, 217-227].

• Anthony C, Ghosh M
• The structure and function of the PQQ-containing quinoprotein dehydrogenases.
• Prog Biophys Mol Biol. 1998; 69: 1-21
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• Bacterial methanol and glucose dehydrogenases containing a novel type of prosthetic group, subsequently identified as pyrrolo-quinoline quinone (PQQ), were first described about 30 years ago. Quinoproteins were originally defined as proteins containing PQQ but this definition has since been broadened to include those proteins containing other types of quinone-containing prosthetic groups, and the X-ray structures of representatives of each type of quinoprotein have recently been published. This review is mainly concerned with the structure and function of the PQQ-containing methanol dehydrogenase, whose structure has been determined at high resolution, and related proteins. Their basic structure consists of a 'propeller' fold superbarrel made up of 8-sheet 'propeller blades' which are held together by novel tryptophan-docking motifs. In methanol dehydrogenase the PQQ in the active site is coordinated to a Ca2+ ion and is maintained in position by a stacked tryptophan and a novel 8-membered ring structure made up of a disulphide bridge between adjacent cysteine residues. This review describes these features and discusses them in relation to previously proposed mechanisms for this enzyme.

• Dengler U, Niefind K, Kiess M, Schomburg D
• Crystal structure of a ternary complex of D-2-hydroxyisocaproate dehydrogenase from Lactobacillus casei, NAD+ and 2-oxoisocaproate at 1.9 A resolution.
• J Mol Biol. 1997; 267: 640-60
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• D-2-hydroxyisocaproate dehydrogenase (D-HicDH) from Lactobacillus casei is a homodimer with 333 amino acids and a molecular mass of 37 kDa per subunit. The enzyme belongs to the protein family of NAD+-dependent D-2-hydroxycarboxylate dehydrogenases and within this family to the subgroup of D-lactate dehydrogenases (D-LDHs). Compared with other D-LDHs D-HicDH is characterized by a very low specificity regarding size and chemical constitution of the accepted D-2-hydroxycarboxylates. Hexagonal crystals of recombinant D-HicDH in the presence of NAD+ and 2-oxoisocaproate (4-methyl-2-oxopentanoate) were grown with ammonium sulphate as precipitating agent. The structure of these crystals was solved by molecular replacement and refined to a final R-factor of 19.6% for all measured X-ray reflections in the resolution range (infinity to 1.86 A). Both NAD+ and 2-oxoisocaproate were identified in the electron density map; binding of the latter in the active site, however, competes with a sulphate ion, which is also defined by electron density. Additionally the final model contains 182 water molecules and a second sulphate ion. The binding of both an in vitro substrate and the natural cosubstrate in the active site provides substantial insight into the catalytic mechanism and allows us to assess previously published active site models for this enzyme family, in particular the two most controversial points, the role of the conserved Arg234 and substrate binding. Furthermore the overall topology and details of the D-HicDH structure are described, discussed against the background of homologous structures and compared with one closely and one distantly related protein.

• McDonald IR, Murrell JC
• The methanol dehydrogenase structural gene mxaF and its use as a functional gene probe for methanotrophs and methylotrophs.
• Appl Environ Microbiol. 1997; 63: 3218-24
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• The methanol dehydrogenase gene mxaF, encoding the large subunit of the enzyme, was amplified from the DNA of a number of representative methanotrophs, methyletrophs, and environmental samples by PCR using primers designed from regions of conserved amino acid sequence identified by comparison of three known sequences of the large subunit of methanol dehydrogenase. The resulting 550-bp PCR products were cloned and sequenced. Analysis of the predicted amino acid sequences corresponding to these mxaF genes revealed strong sequence conservation. Of the 172 amino acid residues, 47% were conserved among all 22 sequences obtained in this study. Phylogenetic analysis of these MxaF sequences showed that those from type I and type II methanotrophs form two distinct clusters and are separate from MxaF sequences of other gram-negative methylotrophs. MxaF sequences retrieved by PCR from DNA isolated from a blanket bog peat core sample formed a distinct phylogenetic cluster within the MxaF sequences of type II methanotrophs and may originate from a novel group of acidophilic methanotrophs which have yet to be cultured from this environment.

• Nakanishi M et al.
• Site-directed mutagenesis of residues in coenzyme-binding domain and active site of mouse lung carbonyl reductase.
• Adv Exp Med Biol. 1997; 414: 555-61

• Zheng YJ, Bruice TC
• Conformation of coenzyme pyrroloquinoline quinone and role of Ca2+ in the catalytic mechanism of quinoprotein methanol dehydrogenase.
• Proc Natl Acad Sci U S A. 1997; 94: 11881-6
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• The ab initio structures of 2,7,9-tricarboxypyrroloquinoline quinone (PQQ), semiquinone (PQQH), and dihydroquinone (PQQH2) have been determined and compared with ab initio structures of the (PQQ)Ca2+, (PQQH)Ca2+, and (PQQH2)Ca2+ complexes as well as the x-ray structure of (PQQ)Ca2+ bound at the active site of the methanol dehydrogenase (MDH) of methyltropic bacteria. Plausible mechanisms for the MDH oxidation of methanol involving the (PQQ)Ca2+ complex are explored via ab initio computations and discussed. Considering the reaction of methanol with PQQ in the absence of Ca2+, nucleophilic addition of methanol to the PQQ C-5 carbonyl followed by a retro-ene elimination is deemed unlikely due to large energy barrier. A much more favorable disposition of the methanol C-5 adduct to provide formaldehyde involves proton ionization of the intermediate followed by elimination of methoxide concerted with hydride transfer to the oxygen of the C-4 carbonyl. Much the same transition state is reached if one searches for the transition state beginning with Asp-303-CO2-general-base removal of the methanol proton of the (PQQ)Ca2+O(H)CH3 complex concerted with hydride transfer to the oxygen at C-4. For such a mechanism the role of the Ca2+ moiety would be to (i) contribute to the formation of the ES complex (ii) provide a modest decrease in the pKa of methanol substrate,; and (iii) polarize the oxygen at C-5.

• Stoorvogel J, Kraayveld DE, Van Sluis CA, Jongejan JA, De Vries S, Duine JA
• Characterization of the gene encoding quinohaemoprotein ethanol dehydrogenase of Comamonas testosteroni.
• Eur J Biochem. 1996; 235: 690-8
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• The gene encoding quinohaemoprotein ethanol dehydrogenase type I (QH-EDHI) from Comamonas testosteroni has been cloned and sequenced. Comparison of the amino acid sequence deduced from this with that determined for the N-terminal amino acid stretch of purified QH-EDHI, suggests that the gene also contains a leader sequence of 31 residues. Based on this information, the molecular mass of the apo-enzyme, i.e. the enzyme without the cofactors pyrroloquinoline quinone (PQQ) and haem c, and without the Ca2+, appears to be 73 200 Da. Alignment of the deduced amino acid sequence to that of other PQQ-containing dehydrogenases showed that good similarity (up to 43% identity) exists with most of them. This also showed that the amino acid residues presumed to be involved in PQQ and Ca2+ binding and in the typical features of structure and catalysis of methanol dehydrogenase, are conserved at the same positions in QH-EDHI. The C-terminal part of the protein, containing the haem c, exhibited some similarity to cytochromes C553 from cyanobacteria and algae. Correct processing of the qhedh gene appeared to occur in Escherichia coli strain JM 109 in which the gene was placed under control of the lac promoter, as judged from a positive reaction with antibodies raised against authentic QH-EDHI, the size of the protein, the presence of haem c in it, and the specific activity value obtained after reconstitution with PQQ. The qhedh gene seems to form part of an operon which is organized in a way different from that of the genes required for methanol oxidation in methylotrophic bacteria.

• Sakon J, Adney WS, Himmel ME, Thomas SR, Karplus PA
• Crystal structure of thermostable family 5 endocellulase E1 from Acidothermus cellulolyticus in complex with cellotetraose.
• Biochemistry. 1996; 35: 10648-60
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• The crystal structure of the catalytic domain of the thermostable endocellulase E1 from Acidothermus cellulolyticus in complex with cellotetraose has been solved by multiple isomorphous replacement and refined at 2.4 A resolution to an R-factor of 0.18 (Rfree = 0.24). E1cd is a member of the 4/7 superfamily of hydrolases, and as expected, its structure is an (alpha/beta)8 barrel, which constitutes a prototype for family 5-subfamily 1 cellulases. The cellotetraose molecule binds in a manner consistent with the expected Michaelis complex for the glycosylation half-reaction and reveals that all eight residues conserved in family 5 enzymes are involved in recognition of the glycosyl group attacked during cleavage. Whereas only three residues are conserved in the whole 4/7 superfamily (the Asn/Glu duo and the Glu from which the name is derived), structural comparisons show that all eight residues conserved in family 5 have functional equivalents in the other 4/7 superfamily members, strengthening the case that mechanistic details are conserved throughout the superfamily. On the basis of the structure, a detailed sequence of physical steps of the cleavage mechanism is proposed. A close approach of two key glutamate residues provides an elegant mechanism for the shift in the pKa of the acid/base for the glycosylation and deglycosylation half-reactions. Finally, purely structural based comparisons are used to show that significant differences exist in structural similarity scores resulting from different methods and suggest that caution should be exercised in interpreting such results in terms of implied evolutional relationships.

• Ramamoorthi R, Lidstrom ME
• Transcriptional analysis of pqqD and study of the regulation of pyrroloquinoline quinone biosynthesis in Methylobacterium extorquens AM1.
• J Bacteriol. 1995; 177: 206-11
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• Methanol dehydrogenase, the enzyme that oxidizes methanol to formaldehyde in gram-negative methylotrophs, contains the prosthetic group pyrroloquinoline quinone (PQQ). To begin to analyze how the synthesis of PQQ is coordinated with the production of other methanol dehydrogenase components, the transcription of one of the key PQQ synthesis genes has been studied. This gene (pqqD) encodes a 29-amino-acid peptide that is thought to be the precursor for PQQ biosynthesis. A unique transcription start site was mapped to a guanidine nucleotide 95 bp upstream of the pqqD initiator codon. RNA blot analysis identified two transcripts, a major one of 240 bases encoding pqqD and a minor one of 1,300 bases encoding pqqD and the gene immediately downstream, pqqG. Both transcripts are present at similar levels in cells grown on methanol and on succinate, but the levels of PQQ are about fivefold higher in cells grown on methanol than in cells grown on succinate. These results suggest that PQQ production is regulated at a level different from the transcription of pqqD. The genes mxbM, mxbD, mxcQ, mxcE, and mxaB are required for transcription of the genes encoding the methanol dehydrogenase subunits and were assessed for their role in PQQ production. PQQ levels were measured in mutants defective in each of these regulatory genes and compared with levels of pqqD transcription, measured with a transcriptional fusion between the pqqD promoter and xylE. The results showed that only a subset of these regulatory genes (mxbM, mxbD, and mxaB) is required for transcription of pqqD, and only mxbM and mxbD mutants affected the final levels of PQQ significantly.

• Ghosh M, Anthony C, Harlos K, Goodwin MG, Blake C
• The refined structure of the quinoprotein methanol dehydrogenase from Methylobacterium extorquens at 1.94 A.
• Structure. 1995; 3: 177-87
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• BACKGROUND: Methanol dehydrogenase (MDH) is a bacterial periplasmic quinoprotein; it has pyrrolo-quinoline quinone (PQQ) as its prosthetic group, requires Ca2+ for activity and uses cytochrome cL as its electron acceptor. Low-resolution structures of MDH have already been determined. RESULTS: The structure of the alpha 2 beta 2 tetramer of MDH from Methylobacterium extorquens has now been determined at 1.94 A with an R-factor of 19.85%. CONCLUSIONS: The alpha-subunit of MDH has an eight-fold radial symmetry, with its eight beta-sheets stabilized by a novel tryptophan docking motif. The PQQ in the active site is held in place by a coplanar tryptophan and by a novel disulphide ring formed between adjacent cysteines which are bonded by an unusual non-planar trans peptide bond. One of the carbonyl oxygens of PQQ is bonded to the Ca2+, probably facilitating attack on the substrate, and the other carbonyl oxygen is out of the plane of the ring, confirming the presence of the predicted free-radical semiquinone form of the prosthetic group.

• de Jong GA, Geerlof A, Stoorvogel J, Jongejan JA, de Vries S, Duine JA
• Quinohaemoprotein ethanol dehydrogenase from Comamonas testosteroni. Purification, characterization, and reconstitution of the apoenzyme with pyrroloquinoline quinone analogues.
• Eur J Biochem. 1995; 230: 899-905
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• Pyrroloquinoline-quinone(PQQ)-free quinohaemoprotein ethanol dehydrogenase (QH-EDH) apoenzyme was isolated from ethanol-grown Comamonas testosteroni. The purified apoenzyme, showing a single band of 71 kDa on native gel electrophoresis, could be only partially converted into active holoenzyme by addition of PQQ in the presence of calcium ions. In addition to a band with a molecular mass of 71 kDa, additional bands of 51 kDa and 25 kDa were observed with SDS/PAGE. Analysis of the N-terminal sequences of the bands and comparison with the DNA sequence of the gene, suggested that the latter two originate from the former one, due to scission occurring at a specific site between two vicinal residues in the protein chain. The extent of scission appeared to increase during growth of the organism. After addition of PQQ to apoenzyme, holoenzyme and nicked, inactive enzyme could be separated. Holoenzyme prepared in this way was found to contain equimolar amounts of PQQ, Ca2+ and covalently bound haem. EPR spectra of fully oxidized apo-QH-EDH and holo-QH-EDH showed g values typical for low-spin haem c proteins. In partially oxidized holo-QH-EDH an organic radical signal attributed to the semiquinone form of PQQ was observed. Binding of PQQ leads to conformational changes, as reflected by changes of spectral and chromatographic properties. Reconstitution of apoenzyme with PQQ analogues resulted in a decreased activity and enantioselectivity for the oxidation of chiral alcohols. Compared with PQQ, analogues with a large substituent had a lower affinity for the apoenzyme. Results with other analogues indicated that possession of the o-quinone/o-quinol moiety is not essential for binding but it is for activity.

• Ghosh M, Avezoux A, Anthony C, Harlos K, Blake CC
• X-ray structure of PQQ-dependent methanol dehydrogenase.
• EXS. 1994; 71: 251-60
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• The three-dimensional structure of the PQQ-dependent quinoprotein, methanol dehydrogenase from Methylobacterium extorquens AM1, has been determined at 3A resolution. The a2b2 tetrameric enzyme has a large a-chain of almost spherical form with a chain fold in which eight 4-stranded antiparallel b-sheets segments are arranged radially around a pseudo 8-fold molecular symmetry axis. The much smaller b-chain is surprisingly not globular, but has an extended conformation running across the surface of the alpha-subunit. The PQQ prosthetic group is buried within the large a-subunit located on the pseudo 8-fold molecular symmetry axis. It is surrounded by protein side-chains but not covalently bound. Associated with the PQQ are two unexpected features: a vicinal disulphide bridge formed between Cys103 and Cys104, and a calcium ion bound between the protein and the PQQ. Vicinal disulphide bridges forming highly distorted structures containing a cis peptide bond, have been proposed to be present in one or two enzymes but have not previously been available for detailed structural investigation. Activity studies have indicated that the ability of the enzyme to transfer electrons derived from the reduction of the alcohols to the specific cytochrome CL receptor is lost when the vicinal disulphide bridge is reduced. The roles of the calcium ions and the b-chain in the enzyme's activity remain to be determined.

• White S et al.
• The active site structure of the calcium-containing quinoprotein methanol dehydrogenase.
• Biochemistry. 1993; 32: 12955-8
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• Pyrroloquinoline quinone (PQQ), widely found in nature, serves as the redox cofactor in bacterial methanol dehydrogenase (MEDH), a heterotetrameric enzyme that oxidizes methanol to formaldehyde. The refined structure of MEDH at 2.4-A resolution, based on recently obtained amino acid sequence data, reveals that the PQQ, located in a central channel of the disk-shaped protein, is sandwiched between a Trp side chain and a very unusual vicinal disulfide. A Ca2+ ion forms a bridge between PQQ and the protein molecule, very close to a putative substrate binding pocket. The vicinal disulfide may form during PQQ incorporation and possibly act to hold the latter in place.

• Krook M, Ghosh D, Stromberg R, Carlquist M, Jornvall H
• Carboxyethyllysine in a protein: native carbonyl reductase/NADP(+)-dependent prostaglandin dehydrogenase.
• Proc Natl Acad Sci U S A. 1993; 90: 502-6
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• Two different forms of the monomeric NADP(+)-linked prostaglandin dehydrogenase/carbonyl reductase were purified from human placenta and shown to differ by the modification of a lysine residue. The modified and the unmodified proteins were reproducibly recovered in a ratio of approximately 1:3, and both were chemically stable. The modified form was more acidic (pI approximately 7.4 versus pI approximately 7.7) but indistinguishable from the unmodified form in specificity and activity. Amino acid analysis, sequence analysis, mass spectrometry, and chemical synthesis identified the modified residue as N6-(1-carboxyethyl)lysine with C-2 of propionic acid attached to the side-chain N of Lys-238. This compound can be formed from the lysine residue and pyruvate via a Schiff base and subsequent reduction. The enzyme and its NAD(+)-dependent counterpart are distantly related (23% residue identity) and have the same family assignment to short-chain dehydrogenases. Alignments and model-building into the tertiary structure of 3 alpha/20 beta-hydroxysteroid dehydrogenase show that carbonyl reductase has an extra loop (positions 149-189) that forms a separate extension and replaces a backbone C-terminal beta-strand. This change affects the substrate pocket, explaining the different substrate specificities but conserves residues of known functional importance. Carboxyethyllysine at position 238 corresponds to a proteolysis-sensitive position in several short-chain dehydrogenases, less well-defined in the model but close to a surface, and is compatible with the accessibility and enzyme properties observed.

• Verschueren KH, Franken SM, Rozeboom HJ, Kalk KH, Dijkstra BW
• Non-covalent binding of the heavy atom compound [Au(CN)2]- at the halide binding site of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10.
• FEBS Lett. 1993; 323: 267-70
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• The Na[Au(CN)2] heavy atom derivative contributed considerably to the successful elucidation of the crystal structure of haloalkane dehalogenase isolated from Xanthobacter autotrophicus GJ10. The gold cyanide was located in an internal cavity of the enzyme, which also contains the catalytic residues. Refinement of the dehalogenase-gold cyanide complex at 0.25 nm to an R-factor of 16.7% demonstrates that the heavy atom molecule binds non-covalently between two tryptophan residues pointing into the active site cavity. At this same site also chloride ions can be bound. Therefore, inhibition of dehalogenase activity by the Au(CN)-2 presumably occurs by competition for the same binding site as substrates.

• Cox JM, Day DJ, Anthony C
• The interaction of methanol dehydrogenase and its electron acceptor, cytochrome cL in methylotrophic bacteria.
• Biochim Biophys Acta. 1992; 1119: 97-106
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• The interactions of methanol dehydrogenase (MDH, EC1.1.99.8) with its specific electron acceptor cytochrome cL has been investigated in Methylobacterium extorquens and Methylophilus methylotrophus. The MDHs of these two very different methylotrophs have the same alpha 2 beta 2 structure; the interaction of these MDHs with their specific electron acceptor, cytochrome cL, has been studied using a novel assay system. Electrostatic reactions are involved in 'docking' of the two proteins. EDTA inhibits the reaction by a process involving neither metal chelation nor the 'docking' process. Chemical modification studies showed that the two proteins interact by a 'docking' process involving interactions of lysyl residues on MDH and carboxyl residues on cytochrome cL. When 'zero length', two stage cross-linking was done (with proteins from both bacteria), the alpha-subunits of MDH cross-linked with cytochrome cL by way of lysyl groups on MDH and carboxyl groups on the cytochrome. Tuna mitochondrial cytochrome c provided a model for cytochrome cH which is the electron acceptor for cytochrome cL in the 'methanol oxidase' electron transport chain. Tuna cytochrome c was shown to form crosslinked products with carboxyl-modified cytochrome cL. MDH and tuna cytochrome c competed for the same domain on cytochrome cL. It was concluded that MDH reacts with cytochrome cL by an electrostatic reaction which involves carboxyl groups on cytochrome cL and amino groups on the alpha-subunit of MDH. The same domain on cytochrome cL is involved in subsequent 'docking' with its electron acceptor.

• Xia ZX et al.
• The three-dimensional structures of methanol dehydrogenase from two methylotrophic bacteria at 2.6-A resolution.
• J Biol Chem. 1992; 267: 22289-97
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• The structures of methanol dehydrogenase (MEDH) from two closely related methylotrophic bacteria, Methylophilus methylotrophus and W3A1, have been determined at 2.6-A resolution. The molecule, a quinoprotein of molecular mass of about 138 kDa, contains two heavy (H) and two light (L) subunits of unknown sequence and two molecules of noncovalently associated pyrroloquinoline quinone. The two enzymes crystallize isomorphously in space group P2(1) with one H2L2 heterotetramer in the asymmetric unit. The electron density map of the M. methylophilus enzyme was obtained by multiple isomorphous replacement with anomalous scattering and improved by solvent leveling and electron density averaging. For model building, the amino acid sequence of MEDH from Paracoccus denitrificans for the H subunit and from Methylobacterium extorquens AM1 for the L subunit were used to represent the unknown amino acid sequence. At the present time, 579 and 57 amino acid residues for the large and small subunits, respectively, have been fitted into the map. The phases for MEDH from M. methylophilus were used directly to analyze the W3A1 structure, and both structures were refined to R-factors (where R = sigma[Fo-Fc[/sigma Fo) of 0.277 and 0.266, respectively. The L subunit contains a long alpha-helix and an extended N-terminal segment, both lying on the molecular surface of the H subunit. The H subunit contains eight antiparallel beta-sheets, each consisting of four strands arranged topologically like the letter W. The eight Ws are arranged circularly, forming the main disc-shaped body of the subunit, with some short helices and loops connecting the consecutive Ws, as well as some excursions within and between some of the Ws. The pyrroloquinoline quinone prosthetic group is located in the central channel of the large subunit near the surface of the molecule. The topology of the eight-W folding unit is similar to those of the six- and seven-W folding units previously reported for three other proteins, neuraminidase, methylamine dehydrogenase, and galactose oxidase.

• McIntire WS, Wemmer DE, Chistoserdov A, Lidstrom ME
• A new cofactor in a prokaryotic enzyme: tryptophan tryptophylquinone as the redox prosthetic group in methylamine dehydrogenase.
• Science. 1991; 252: 817-24
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• Methylamine dehydrogenase (MADH), an alpha 2 beta 2 enzyme from numerous methylotrophic soil bacteria, contains a novel quinonoid redox prosthetic group that is covalently bound to its small beta subunit through two amino acyl residues. A comparison of the amino acid sequence deduced from the gene sequence of the small subunit for the enzyme from Methylobacterium extorquens AM1 with the published amino acid sequence obtained by the Edman degradation method, allowed the identification of the amino acyl constituents of the cofactor as two tryptophyl residues. This information was crucial for interpreting 1H and 13C nuclear magnetic resonance, and mass spectral data collected for the semicarbazide- and carboxymethyl-derivatized bis(tripeptidyl)-cofactor of MADH from bacterium W3A1. The cofactor is composed of two cross-linked tryptophyl residues. Although there are many possible isomers, only one is consistent with all the data: The first tryptophyl residue in the peptide sequence exists as an indole-6,7-dione, and is attached at its 4 position to the 2 position of the second, otherwise unmodified, indole side group. Contrary to earlier reports, the cofactor of MADH is not 2,7,9-tricarboxypyrroloquinoline quinone (PQQ), a derivative thereof, or pro-PQQ. This appears to be the only example of two cross-linked, modified amino acyl residues having a functional role in the active site of an enzyme, in the absence of other cofactors or metal ions.

• Nunn DN, Day D, Anthony C
• The second subunit of methanol dehydrogenase of Methylobacterium extorquens AM1.
• Biochem J. 1989; 260: 857-62
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• The nucleotide and deduced amino acid sequence of a novel small (beta) subunit of methanol dehydrogenase of Methylobacterium extorquens AM1 (previously Pseudomonas AM1) has been determined. Work with the whole protein has shown that is has an alpha 2 beta 2 configuration.

• Lindqvist Y, Branden CI
• The active site of spinach glycolate oxidase.
• J Biol Chem. 1989; 264: 3624-8
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• The amino acid sequence of glycolate oxidase from spinach has been fitted to an electron density map at 2.2 A resolution. From a refined model we give a detailed description of the flavin mononucleotide-binding site and the residues which might be involved in the catalytic action of the enzyme. The cofactor is bound to the enzyme at the carboxy end of the beta strands in the alpha/beta barrel domain and forms a number of hydrogen bonds both to residues at the end of the beta strands and in the loop regions after the strands. In particular Lys-230 interacts with atoms N1 and O2 of the isoalloxazine ring, which produces an inductive effect that could enhance the nucleophilicity of the electron acceptor N5 of the flavin ring. Almost the entire coenzyme is buried in the interior of the enzyme. The exceptions are one phosphate oxygen atom and a region around the N5 position on the si-face of the isoalloxazine ring, which are accessible to solution. Based on a model of bound glycolate to our structure we propose that the following residues are important in the catalytic reaction: Arg-257, Tyr-24, and Tyr-129 for binding the substrate, and His-254 for abstracting a proton from the C2 atom of the substrate.

• Vellieux FM et al.
• Structure of quinoprotein methylamine dehydrogenase at 2.25 A resolution.
• EMBO J. 1989; 8: 2171-8
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• The three-dimensional structure of quinoprotein methylamine dehydrogenase from Thiobacillus versutus has been determined at 2.25 A resolution by a combination of multiple isomorphous replacement, phase extension by solvent flattening and partial structure phasing using molecular dynamics refinement. In the resulting map, the polypeptide chain for both subunits could be followed and an X-ray sequence was established. The tetrameric enzyme, made up of two heavy (H) and two light (L) subunits, is a flat parallellepiped with overall dimensions of approximately 76 x 61 x 45 A. The H subunit, comprising 370 residues, is made up of two distinct segments: the first 31 residues form an extension which embraces one of the L subunits; the remaining residues are found in a disc-shaped domain. This domain is formed by a circular arrangement of seven topologically identical four-stranded antiparallel beta-sheets, with approximately 7-fold symmetry. In spite of distinct differences, this arrangement is reminiscent of the structure found in influenza virus neuraminidase. The L subunit consists of 121 residues, out of which 53 form a beta-sheet scaffold of a central three-stranded antiparallel sheet flanked by two shorter two-stranded antiparallel sheets. The remaining residues are found in segments of irregular structure. This subunit is stabilized by six disulphide bridges, plus two covalent bridges involving the quinone co-factor and residues 57 and 107 of this subunit. The active site is located in a channel at the interface region between the H and L subunits, and the electron density in this part of the molecule suggests that the co-factor of this enzyme is not pyrrolo quinoline quinone (PQQ) itself, but might be instead a precursor of PQQ.

• Davidson VL, Neher JW, Cecchini G
• The biosynthesis and assembly of methanol dehydrogenase in bacterium W3A1.
• J Biol Chem. 1985; 260: 9642-7
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• Bacterium W3A1, a restricted facultative methylotroph, produces a periplasmic methanol dehydrogenase composed of two identical subunits of Mr = 57,300, and two noncovalently bound methoxatin prosthetic groups. A precursor form of Mr = 1,500 larger than the mature subunit was identified among the products of an in vitro translation of total RNA isolated from bacterium W3A1. The precursor form of the protein could not be detected in cells during in vivo pulse-labeling studies, suggesting that the processing of this precursor occurs entirely co-translationally. Whereas the holoenzyme was detectable only as a dimer, removal of the prosthetic group yielded an apoenzyme that could be detected as either a dimeric or monomeric species. After readdition of the purified prosthetic group to the apoenzyme, only the dimeric form of the protein, bearing the cofactor and exhibiting an absorption spectrum similar to that of the holoenzyme, was detected. Neither the mature apoprotein nor the holoenzyme demonstrated any affinity for phospholipid membranes, as assayed by their inability to bind to liposomes. Taken together, these data suggest a scheme of co-translational processing and export of the apoprotein subunits, followed by assembly of the subunits and prosthetic groups in the periplasmic space to form the mature holoenzyme. The suitability of bacterium W3A1, and other methylotrophic bacteria, for use in studies of protein biosynthesis and export, is also discussed.