Structural Comparison of Creatinases for Investigating Substrate Binding

International Journal of Analytical Bio-Science Int J Anal Bio-Sci Vol. 2, No 4 (2014) estimate of EE. An important advantage of using HR 〈Original Article〉 over motion sensors is that HR monitoring provides an References index of both the relative (%VO2R), as well as the １ Janz KF: Validation of the CSA accelerometer for absolute intensity (METS) of the physical activity assessing childrenﾕs physical activity. Med Sci Sports Exerc, 26: 369-375, 1994. performed. The importance of relative intensity can be ２ Montoye HJ, Kemper HCG, Saris WHM, et al.: Structural comparison of creatinases for seen when classifying different individuals on the Measuring Physical Activity and Energy Expenditure. investigating substrate binding basis of exercise intensity. Champaign, IL: Human kinetics, pp3-102, 1996. The Karvonen formula determines target HR as ３ Eston RG, Rowlands AV, Ingledew DK: Validity of follows: target HR = (maximum HR － resting HR) heart rate, pedometry, and accelerometry for predicting Yoshiaki Nishiya × ％HRR ＋ resting HR. The American College of the energy cost of children's activities. J Appl Physiol, Sports Medicine categorizes exercise intensity as 84: 362-37, 1998. ４ Rodahl K, Vokac Z, Fugelli P, et al.: Circulatory strain, Summary For further understanding the substrate binding of creatinase, the kinetic parameters of follows based on %VO2R or %HR: very light, <20%; estimated energy output and catecholamine excretion in seven known enzymes and the tertiary structures of more significant ones of those were compared. light, 20-39%; moderate, 40-59%; hard, 60-84%; very Norwegian coastal fishermen. Ergonomics, 17: 585- Used structures were constructed by homology modeling based on the X-ray structure of the enzyme hard, ≧85%; maximum, 100%. 602, 1974. from Pseudomonas putida. The structural comparison of these creatinases showed that the specific In the preset study, there was a tendency that ５ Treiber FA, Musante L, Hartdagan S, et al.: Validation residues in the N-terminal domain, rather than the C-terminal catalytic domain, had influenced on in measured EE was greater than estimated EE calculated of a heart rate monitor with children in laboratory and using the estimation formula, although, a favorable field setting. Med Sci Sports Exerc, 21: 338-342, 1989. the substrate affinity. This result would be assistance in the improvement of creatinase for creatinine correlation was found between measured EE and ６ Washburn RA, Montoye HJ: Reliability of the heart rate and creatine determinations. estimated EE (R2=0.99), supporting the study result of as a measure of mean daily energy expenditure. Exerc Scott et al.12 If the regression equation is employed as Physiol, 2: 161-172, 1986. ７ Goldsmith R, Miller DS, Mumford P, et al.: The use of Key words: Creatinase, Tertiary structure, Homology modeling, Substrate affinity, a correction method, physical activity intensity can be long-term measurements of heart rate to assess energy Creatinine assay calculated from HR and METs; the assessments based expenditure. J Physiol, 189(2): 61-62, 1967. on expensive calculation apparatus and the complexity ８ Washburn RA, Montoye HJ: Reliability of the heart rate of the technique required for accurate measurements of response to submaximal upper and lower body exercise.

VO2 are not necessary. The result of this study Res Q Exerc Sports, 56: 166-169, 1985. suggested that the estimation formula would provide ９ Swain DP, Leutholtz BC: Heart rate reserve is equiva- 1. Introduction enzymes for creatinine determination, creatininase and sarcosine oxidase have been already studied and appropriate EE in individual physical activity. lent to %VO2 reserve, not to %VO2max. Med Sci Sports Exsec, 29: 410-414, 1997. Creatinase (creatine amidinohydrolase, EC 3.5.3.3) improved by using protein engineering techniques 10 Swain DP, Leutholtz BC, King ME, et al.: Relationship 14-16 Conclusion catalyses the hydrolyzation of creatine to sarcosine and on the basis of tertiary-structural analysis . In between % heart rate reserve and % VO2 reserve in Actual EE and estimated EE were derived from urea. It is involved in the bacterial metabolism of contrast, the structure-based improvement of creatinase treadmill exercise. Med Sci Sports Exerc, 30: 318-321, creatinine with the related enzymes1. The enzyme is still has not progressed. HR and VO2 while healthy and young subjects 1998 commercially used in the diagnostic analysis of creati- This report shows the tertiary structures of creati- performing various physical activities. Further inves- 11 Davis JA, Convertino VA: A comparison of heart rate nine and creatine with coupling of related enzymes2, 3. nases constructed by homology modeling. Comparison tigation and data collection including larger study methods for predicting endurance training intensity. population are required for the more detailed and Med Sci Sports, 7: 295-298, 1975. Several creatinases have been found, produced, and of these structures indicated that the N-terminal 4-10 clinical evaluation. 12 Strath SJ, Swartz AM, Bassett DR Jr. et al.: Evaluation proved useful in the enzymatic assays . However, the domain, rather than the C-terminal catalytic domain, of heart rate as a method for assessing moderate enzymatic properties of these enzymes are not yet was important for the substrate affinity. This provides intensity physical activity. Med Sci Sports Exerc, 32 (9 Acknowledgement discussed from the viewpoint of tertiary-structural a reasonable starting point for analyzing the structure- suppl): S465-470, 2000. The author thanks Shohei Ichikawa, Takuya comparison, although the X-ray structures of the function relationships and improving the enzyme 13 Karvonen J, Kertala K, Muatala O: The effects of enzymes from Pseudomonas putida and Actinobacillus properties. Katayama, Naoko Shimakura, and Mami Nakazawa in training heart rate: a longitudinal study. Ann Med Exp sp. have already been solved11-13. With respect to the the Department of Health Sciences, Saitama Biochem, 35: 307-315, 1957. Prefectural University, who volunteered to partic- 14 Jackson AS, Blair SN, Mahar MT, et al.: Prediction of Department of Life Science, Faculty of Science and Received for Publication August 27, 2014 ipate in this study. This study was supported by a functional aerobic capacity without exersice testing. Engineering, Setsunan University, Accepted for Publication October 15, 2014 grant from Saitama Prefectural University. Med Sci Sports Exerc, 22: 863-870, 1990. 17-8 Ikeda-Nakamachi, Neyagawa, Osaka 572-8508, Japan E-mail: [email protected]

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2. Materials and methods used for molecular visualization.

Homology search analysis of the amino acid 3. Results and discussion sequences of several creatinases was performed using the software GENETYX (Software Development Co., In previous studies, several creatinases from seven Ltd., Tokyo, Japan). The sequences used in this study different genera have been reported with their are in the DDBJ database under accession numbers enzymatic properties4-10. The enzymes from E17219, D00656, D14463, E16405, and BD342394, Arthrobacter, Flavobacterium, Bacillus, Actinobacillus, and in the UniProt ID: P38488. Homology modeling Alcaligenes, Erwinia, and Pseudomonas were desig- was used to build the models of creatinases. The nated CreA, CreF, CreB, CreC, CreL, CreE, and CreP, three-dimensional protein models were generated by respectively. The kinetic parameters and assay condi- the software MODELLER17, based on the structure of tions of these creatinases are compared in Table 1. The P. putida (PDB ID: 1chm). The program Pymol was parameters of CreP and CreE could not be compared

Table 1 Comparison of kinetic parameters and assay conditions

Creatinase Km (mmol/L) Vmax (U/mg) Assay codition Reference CreA 46 21 37℃ pH 7.6 4 CreF 40 11.3 37℃ pH 7.7 5 CreB NF 10.3 37℃ pH 7.5 6 CreC 19 18.1 37℃ pH 7.5 7 CreL 15.2 19.5 37℃ pH 7.6 8 CreP 14.3 15.0 25℃ pH 7.8 9 CreE 4.2 7.8 25℃ pH 7.8 10

CreA, CreF, CreB, CreC, CreL, CreP, and CreE are from Arthrobacter, Flavobacterium, Bacillus, Actinobacillus, Alcaligenes, Pseudomonas, and Erwinia, respectively. NF; Data not found.

Fig. 1 Homology search of amino acid sequences of creatinases. Total sequences and N-terminal domains were compared, and each identity was indicated by a percentage. The CreC sequence was not compared because it was incomplete.

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2. Materials and methods used for molecular visualization. with those of others because they were assayed at mean square deviation (RMSD) of atomic Cα

the different reaction temperature. Generally, Km and positions of 0.14-0.34Å. Creatinase molecule is

Homology search analysis of the amino acid 3. Results and discussion Vmax values are decreased at low temperatures. The Km composed of the two identical subunits, which are sequences of several creatinases was performed using value of CreL was smaller than those of CreA, CreF, designated subunit A and B. Two active sites are the software GENETYX (Software Development Co., In previous studies, several creatinases from seven and CreC, i. e., its binding affinity was higher for formed from the C-terminal catalytic domain of

Ltd., Tokyo, Japan). The sequences used in this study different genera have been reported with their creatine. On the other hand, the Km of CreE was much subunit A (or subunit B) and the N-terminal domain of are in the DDBJ database under accession numbers enzymatic properties4-10. The enzymes from smaller than that of CreP. Of all known creatinases, subunit B (or subunit A). For the purpose of discussing E17219, D00656, D14463, E16405, and BD342394, Arthrobacter, Flavobacterium, Bacillus, Actinobacillus, CreL and CreE are practical for diagnostic use because the interaction of creatinase with substrate, the active and in the UniProt ID: P38488. Homology modeling Alcaligenes, Erwinia, and Pseudomonas were desig- of their superior substrate-binding abilities. The sites of CreL and CreE were structurally compared was used to build the models of creatinases. The nated CreA, CreF, CreB, CreC, CreL, CreE, and CreP, different substrate affinities appeared from the important with those of CreF and CreP, respectively. three-dimensional protein models were generated by respectively. The kinetic parameters and assay condi- viewpoint of tertiary-structural comparison, although the After superimpositions of CreL-CreF and CreE- 17 the software MODELLER , based on the structure of tions of these creatinases are compared in Table 1. The Vmax values also differed from each other (Table 1). CreP, the selected amino acid residues that are within P. putida (PDB ID: 1chm). The program Pymol was parameters of CreP and CreE could not be compared The creatinase molecule forms a dimer of identical 6Å of the substrate analog, N-carbamoylsarcosine, are subunits. Each subunit is characterized by two clearly shown in Figure 2. N-Carbamoylsarcosine is a separated domains which are the smaller N-terminal competitive inhibitor that binds to the active site of domain with approximately 160 amino acid residues creatinase. Each active site of the structure model Table 1 Comparison of kinetic parameters and assay conditions and the C-terminal catalytic domain with approxi- including the substrate analog was obtained by super- Creatinase Km (mmol/L) Vmax (U/mg) Assay codition Reference mately 240 residues. Many active site residues exist on posing the coordinates on that of CreP11. CreA 46 21 37℃ pH 7.6 4 the C-terminal domain, including His232 of CreP In the case of structural comparison between CreL CreF 40 11.3 37℃ pH 7.7 5 that is the general acid and base involved in the and CreF, the residues belonging to the C-terminal CreB NF 10.3 37℃ pH 7.5 6 hydrolysis of the guanidinium group of creatine. All domains were highly conserved (Fig. 2A). However, CreC 19 18.1 37℃ pH 7.5 7 active site residues were confirmed to be perfectly the residues belonging to the N-terminal domains CreL 15.2 19.5 37℃ pH 7.6 8 conserved in all enzymes (data not shown). could recognize some different states between both CreP 14.3 15.0 25℃ pH 7.8 9 Homology search of total and N-terminal amino enzymes. In particular, an aspartate residue in the N- CreE 4.2 7.8 25℃ pH 7.8 10 acid sequences are summarized in Figure 1. It shows terminal domain shifted to quite different positions that these amino acid sequences are highly similar between CreL and CreF. The side chain of D101 in CreA, CreF, CreB, CreC, CreL, CreP, and CreE are from Arthrobacter, Flavobacterium, Bacillus, though belonging to different genera. The total CreF was located at a distance of 4.8Å from the Actinobacillus, Alcaligenes, Pseudomonas, and Erwinia, respectively. sequence homologies were extremely higher (62-95% NF; Data not found. carboxyl group of the substrate analog, whereas that of identities). In contrast, the homologies of N-terminal D102 in CreL was 6.3Å away. The carboxyl group of domains indicated relatively lower tendencies (49- creatine can interact with R64 of subunit B and R335 94% identities). Two practical enzymes, CreL and of subunit A in CreF, or R66 of subunit B and R336 of CreE, had similar sequences, and the total and N- subunit A in CreL. The substrate binding would be terminal sequence identities were 94 and 91%, respec- likely to be disturbed by the negative charge of D101 tively (Fig. 1). On the other hand, CreF and CreP in CreF. also had similar sequences, and the total and N- In the case of structural comparison between CreE terminal sequence identities were 95% (Fig. 1). These and CreP, the residues belonging to the C-terminal results suggest that the structural comparison between domains were slightly altered, although those CreL and CreF or that between CreE and CreP would belonging to the N-terminal domains showed clear

be helpful for investigating the substrate binding of differences (Fig. 2B). As shown in Table 1, the Km,

creatinase. Vmax, and catalytic efficiency (Vmax/Km) of CreE were Therefore, three-dimensional structure models of 0.29, 0.52, and 1.8 times those of CreP, respectively.

the dimer molecules of CreF, CreL, and CreE, were In contrast, the Km, Vmax, and Vmax/Km of CreL were built by computer analysis based on the CreP sequence 0.38, 1.7, and 4.5 times those of CreF, respectively. Fig. 1 Homology search of amino acid sequences of creatinases. Total sequences and N-terminal domains were compared, and its X-ray structure11. Overall subunit structures of The different situation of the active sites may reflect and each identity was indicated by a percentage. The CreC sequence was not compared because it was incomplete. all four enzymes could be well superimposed with root the different Vmax values.

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Fig. 2 Close-up stereo view of the active site regions. These are one of two active sites in the creatinase structures, that are composed of the C-terminal catalytic domain from the subunit A and the N-terminal domain from the subunit B. The side chains of amino acid residues within 6 Å of the substrate analog, N-carbamoylsarcosine, are labeled and shown by stick drawings. The subunit to which each residue belongs (A or B) are also labeled. N-Carbamoylsarcosine is represented by ball and sticks. (A) Superimposition of CreL model (orange) over CreF (green). (B) Superimposition of CreE model (orange) over CreP (green).

These results would be of assistance in the present study provide a reliable basis to predict improvement of the enzyme using protein engineering mutations for desired effects on the substrate binding. techniques. In comparison with the C-terminal catalytic domain, changes in amino acid residues of the Acknowledgment N-terminal domain are expected to improve the This work was supported by JSPS KAKENHI substrate binding with a smaller influence on the Grant Number 13251655. enzyme activity. The creatinase structures of the

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