Abstract
A strictly conserved active site arginine residue (αR157) and two histidine residues (αH80 and αH81) located near the active site of the Fe-type nitrile hydratase from Comamonas testosteroni Ni1 (CtNHase), were mutated. These mutant enzymes were examined for their ability to bind iron and hydrate acrylonitrile. For the αR157A mutant, the residual activity (k cat = 10 ± 2 s−1) accounts for less than 1 % of the wild-type activity (k cat = 1100 ± 30 s−1) while the K m value is nearly unchanged at 205 ± 10 mM. On the other hand, mutation of the active site pocket αH80 and αH81 residues to alanine resulted in enzymes with k cat values of 220 ± 40 and 77 ± 13 s−1, respectively, and K m values of 187 ± 11 and 179 ± 18 mM. The double mutant (αH80A/αH81A) was also prepared and provided an enzyme with a k cat value of 132 ± 3 s−1 and a K m value of 213 ± 61 mM. These data indicate that all three residues are catalytically important, but not essential. X-ray crystal structures of the αH80A/αH81A, αH80W/αH81W, and αR157A mutant CtNHase enzymes were solved to 2.0, 2.8, and 2.5 Å resolutions, respectively. In each mutant enzyme, hydrogen-bonding interactions crucial for the catalytic function of the αCys104-SOH ligand are disrupted. Disruption of these hydrogen bonding interactions likely alters the nucleophilicity of the sulfenic acid oxygen and the Lewis acidity of the active site Fe(III) ion.
References
Kovacs JA (2004) Chem Rev 104:825–848
Yamada H, Kobayashi M (1996) Biosci Biotechnol Biochem 60:1391–1400
Brady D, Beeton A, Zeevaart J, Kgaje C, Rantwijk F, Sheldon RA (2004) Appl Microbiol Biotechnol 64:76–85
Wang M-X (2005) Top Catal 35:117–130
Velankar H, Clarke KG, Preez RD, Cowan DA, Burton SG (2010) Trends Biotechnol 28:561–569
Prasad S, Bhalla TC (2010) Biotechnol Adv 28:725–741
Harrop TC, Mascharak PK (2004) Acc Chem Res 37:253–260
Nagashima S, Nakasako M, Dohmae N, Tsujimura M, Takio K, Odaka M, Yohda M, Kamiya N, Endo I (1998) Nat Struct Mol Biol 5:347–351
Miyanaga A, Fushinobu S, Ito K, Wakagi T (2001) Biochem Biophys Res Commun 288:1169–1174
Hourai S, Miki M, Takashima Y, Mitsuda S, Yanagi K (2003) Biochem Biophys Res Commun 312:340–345
Huang W, Jia J, Cummings J, Nelson M, Schneider G, Lindqvist Y (1997) Structure 15:691–699
Martinez S, Wu R, Sanishvili R, Liu D, Holz R (2014) J Am Chem Soc 136:1186–1189
Hopmann KH (2014) Inorg Chem 53:2760–2762
Kayanuma M, Hanaoka K, Shoji M, Shigeta Y (2015) Chem Phys Lett 623:8–13
Nishiyama M, Horinouchi S, Kobayashi M, Nagasawa T, Yamada H, Beppu T (1991) J Bacteriol 173:2465–2472
Hashimoto Y, Nishiyamaa M, Horinouchia S, Beppuab T (1994) Biosci Biotechnol Biochem 58:1859–1869
Nojiri M, Yohda M, Odaka M, Matsushita Y, Tsujimura M, Yoshida T, Dohmae N, Takio K, Endo I (1999) J Biochem 125:696–704
Petrillo KL, Wu S, Hann EC, Cooling FB, Ben-Bassat A, Gavagan JE, DiCosimo R, Payne MS (2005) Appl Microbiol Biotechnol 67:664–670
Wu S, Fallon RD, Payne MS (1997) Appl Microbiol Biotechnol 48:704–708
Kuhn ML, Martinez S, Gumataotao N, Bornscheuer U, Liu D, Holz RC (2012) Biochem Biophys Res Commun 424:365–370
Z. Otwinowski and W. Minor (1997) In: Charles W. Carter, Jr. (ed) Methods enzymol. Academic Press, pp. 307–326
McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ (2007) J Appl Crystallogr 40:658–674
N. Collaborative Computational Project (1994) Acta Crystallogr Sect D Biol Crystallogr 50:760–763
Emsley P, Cowtan K (2004) Acta Crystallogr Sect D: Biol Crystallogr 60:2126–2132
Murshudov GN, Vagin AA, Dodson EJ (1997) Acta Crystallogr Sect D: Biol Crystallogr 53:240–255
Adams PD, Grosse-Kunstleve RW, Hung L-W, Ioerger TR, McCoy AJ, Moriarty NW, Read RJ, Sacchettini JC, Sauter NK, Terwilliger TC (2002) Acta Crystallogr Sect D: Biol Crystallogr 58:1948–1954
Murakami T, Nojiri M, Nakayama H, Dohmae N, Takio K, Odaka M, Endo I, Nagamune T, Yohda M (2000) Protein Sci 9:1024–1030
Arakawa T, Kawano Y, Katayama Y, Nakayama H, Dohmae N, Yohda M, Odaka M (2009) J Am Chem Soc 131:14838–14843
Piersma SR, Nojiri M, Tsujimura M, Noguchi T, Odaka M, Yohda M, Inoue Y, Endo I (2000) J Inorg Biochem 80:283–288
Endo I, Nojiri M, Tsujimura M, Nakasako M, Nagashima S, Yohda M, Odaka M (2001) J Inorg Biochem 83:247–253
Yamanaka Y, Arakawa T, Watanabe T, Namima S, Sato M, Hori S, Ohtaki A, Noguchi K, Katayama Y, Yohda M, Odaka M (2013) J Biosci Bioeng 116:22–27
Nelp MT, Astashkin AV, Breci LA, McCarty RM, Bandarian V (2014) Biochemistry 53:3990–3994
Dey A, Chow M, Taniguchi K, Lugo-Mas P, Davin S, Maeda M, Kovacs JA, Odaka M, Hodgson KO, Hedman B, Solomon EI (2006) J Am Chem Soc 128:533–541
Odaka M, Noguchi T, Nagashima S, Yohda M, Yabuki S, Hoshino M, Inoue Y, Endo I (1996) Biochem Biophys Res Commun 221:146–150
Tsujimura M, Dohmae N, Odaka M, Chijimatsu M, Takio K, Yohda M, Hoshino M, Nagashima S, Endo I (1997) J Biol Chem 272:29454–29459
Yamanaka Y, Hashimoto K, Ohtaki A, Noguchi K, Yohda M, Odaka M (2010) J Biol Inorg Chem 15:655–665
Tsujimura M, Odaka M, Nakayama H, Dohmae N, Koshino H, Asami T, Hoshino M, Takio K, Yoshida S, Maeda M, Endo I (2003) J Am Chem Soc 125:11532–11538
Sugiura Y, Kuwahara J, Nagasawa T, Yamada H (1987) J Am Chem Soc 109:5848–5850
Miyanaga A, Fushinobu S, Ito K, Shoun H, Wakagi T (2004) Eur J Biochem 271:429–438
Gumataotao N, Kuhn ML, Hajnas N, Holz RC (2013) J Biol Chem 288:15532–15536
Lugo-Mas P, Dey A, Xu L, Davin SD, Benedict J, Kaminsky W, Hodgson KO, Hedman B, Solomon EI, Kovacs JA (2006) J Am Chem Soc 128:11211–11221
Kennepohl P, Neese F, Schweitzer D, Jackson HL, Kovacs JA, Solomon EI (2005) Inorg Chem 44:1826–1836
Brodkin HR, Novak WRP, Milne AC, D’Aquino JA, Karabacak NM, Goldberg IG, Agar JN, Payne MS, Petsko GA, Ondrechen MJ, Ringe D (2011) Biochemistry 50:4923–4935
Acknowledgments
This work was supported by the National Science Foundation (CHE-1412443, RCH and CHE-1308672, DL). HC gratefully acknowledges the ACS Project SEED for funding a summer internship. GM/CA @ APS has been funded in whole or in part with federal funds from the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006).
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Martinez, S., Wu, R., Krzywda, K. et al. Analyzing the catalytic role of active site residues in the Fe-type nitrile hydratase from Comamonas testosteroni Ni1. J Biol Inorg Chem 20, 885–894 (2015). https://doi.org/10.1007/s00775-015-1273-3
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DOI: https://doi.org/10.1007/s00775-015-1273-3